Use of Streptomycin-Dependence as Selection/Counterselection for Sequential Oligonucleotide Mediated Recombineering in Mycobacteria

Use of Streptomycin-Dependence as Selection/Counterselection for Sequential Oligonucleotide Mediated Recombineering in Mycobacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Use of Streptomycin-Dependence as Selection/Counterselection for Sequential Oligonucleotide Mediated Recombineering in Mycobacteria Clifton Barry, III, Peter Finin, Nicholaus Mnyambwa, Helena Boshoff This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5639512/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Oligonucleotide mediated recombineering is a powerful technique for genome editing in bacteria. In mycobacteria, researchers usually transform bacteria with two oligonucleotides: one conferring the mutation of interest, and a second conferring selectable antibiotic resistance. Multiple genetic modifications may be performed sequentially, however, this requires either the introduction of multiple antibiotic resistances or a tedious process of reversing antibiotic resistance between steps. Rather than using antibiotic resistance for selection, we instead develop a system that uses streptomycin-dependence. Because streptomycin-dependence can easily be selected both for and against, this allows a theoretically unlimited number of recombineering edits to be sequentially selected for, by toggling between streptomycin-sensitivity and dependence. After an even number of editing cycles, strains are automatically unmarked. Strains which have undergone an odd number of edits can be unmarked with a single additional recombineering step. This allows significantly easier exploration of combinatorial interactions between multiple mutations in mycobacteria. Biological sciences/Microbiology/Pathogens Biological sciences/Genetics/Microbial genetics INTRODUCTION Please have a look at courier new font provided for text in article. Oligonucleotide mediated recombineering was first developed for E. coli using the bacteriophage λ Red system 1 and subsequently adapted for mycobacteria using the mycobacteriophage Che9c ssDNA annealing protein gp61 2 . A plasmid encoding Che9c gp61 under an inducible promoter is first transformed into mycobacterial cells, expression is induced, and mycobacteria are then electroporated with ssDNA oligonucleotides generally 50–100 bp in length. Che9c gp61 anneals the ssDNA oligonucleotides to the lagging strand at the replication fork of the target bacterial DNA. If the recombineering oligonucleotide is mostly identical to the host cell DNA, but contains a small number of mutations, daughter cells can inherit these mutations, resulting in a targeted genetic edit. If this edit confers a selectable phenotype, it is simple to isolate clones which carries the desired mutation. If the edit does not confer a known selectable phenotype, one can electroporate cells with both the oligonucleotide conferring the desired edit, as well as additional DNA which does confer a selectable phenotype. This can be a plasmid encoding a hygromycin resistance cassette 2 , an ssDNA oligonucleotide resulting in an rpsL K43R mutation conferring streptomycin resistance (Sm R ) 3 , or a ssDNA oligonucleotide resulting in a aph(4)-1A *190W mutation repairing a defective hygromycin resistance cassette 4 . After selecting onto appropriate antibiotic-containing media, up to 10% of colonies may carry the desired edit. By screening a manageable number of colonies, successful edits can often be found. Although this system works well for single edits, it is much more cumbersome to make multiple edits. The system is not efficient enough to practically introduce multiple edits simultaneously in mycobacteria, as can be done in E. coli 5 . Multiple edits can be made sequentially, however once resistance to a given antibiotic is used as a selection marker, it cannot be reused without first reversing it somehow. Streptomycin-dependence (Sm D ) is a phenotype first described in Neisseria meningitidis in 1947 6 , whereby cells are only able to grow in the presence of streptomycin, and are unable to grow without it. It has since been described in many other bacteria, including the mycobacterial species Mycobacterium fortuitum 7 , Mycobacterium tuberculosis ( Mtb ) 8 , 9 , and Mycobacterium smegmatis 10 . We considered whether streptomycin-dependence might not make a better selectable marker for sequential genetic editing. Whereas antibiotic resistance can be easily selected for (by plating onto antibiotic-containing media), it cannot generally be easily selected against. Antibiotic dependence, on the other hand, is easy to select either for (by plating onto antibiotic containing media) or against (by plating onto drug-free media). We thus hypothesized that by using ssDNA oligonucleotides that either introduce or reverse mutations conferring a Sm D phenotype as a selection system, an unlimited number of sequential edits could be made without requiring additional intermediate steps to remove resistance markers. A genetic basis for streptomycin-dependence has been well characterized in a variety of organisms. Mutations in the highly conserved proline 91 (mycobacterial numbering) residue of rpsL have been described to result in streptomycin-dependence in a wide variety of organisms across the tree of life, including the gram positive prokaryote Deinococcus geothermalis 11 , gram negative prokaryotes E. coli 12 and Thermus thermophilus 13 , and the chloroplast of the eukaryote Chlamydomonas reinhardtii 14 . The only streptomycin-dependent mycobacterial strain which has undergone genetic sequencing is Mtb strain 18b. This is a derivative of the lab strain Mtb H2 9 , which unfortunately seems to have been lost, and for which no sequence is available for comparison. The mutations responsible for 18b’s streptomycin-dependent phenotype have been proposed to be insertions in both the 16S rRNA and in infC 15 , although this has not been experimentally verified. When deprived of streptomycin, Mtb 18b can grow for several generations before entering a state of non-replicating persistence, in which it neither grows nor experiences substantial cell death 16 . In this work, we attempt to create a streptomycin-dependent phenotype in both M. smegmatis and Mtb . Multiple rpsL P91 mutations cause a streptomycin-dependent phenotype in both Mtb and M. smegmatis , although rpsL P91W results in the best growth characteristics in both organisms. The number of colonies obtained on streptomycin containing agar after these recombineering steps is substantially less than those obtained using rpsL K43R or aph(4)-1a *190W selection systems. In M. smegmatis , introduction of an rpsL P91W mutation can be used to select for a desired mutation using streptomycin selection, and introduction of an rpsL W91P mutation can be used to select for a second desired mutation using growth on drug-free media as selection. In Mtb , the total number of streptomycin dependent colonies was very low, and despite multiple efforts we were unfortunately not able to identify our desired mutations among these. RESULTS Multiple rpsL P91 mutations result in streptomycin-dependence in M. smegmatis We first tested whether a streptomycin-dependent phenotype could be introduced to M. smegmatis by oligonucleotide mediated recombineering. M. smegmatis carrying the recombineering plasmids pKM461 and pKM427 was transformed with oligonucleotides designed to introduce multiple candidate mutations that we expected might result in a streptomycin-dependent phenotype. These included multiple rpsL P91 mutations, which have been associated with streptomycin dependence in other organisms. We also tested oligonucleotides designed to introduce 16S rRNA 512_513insC and infC 23_24insE mutations, which have been proposed to explain Mtb 18b’s streptomycin-dependent phenotype. Transformants were plated onto streptomycin containing agar. The negative control rpsL P91P oligonucleotide gave rise to no colonies on agar plates with streptomycin, and the positive control rpsL K43R oligonucleotide (a mutation known to confer high-level streptomycin-resistance) resulted in numerous colonies. All tested rpsL P91 mutating oligonucleotides resulted in colonies on streptomycin plates, although these were present at 1-2.5 logs reduced frequency as compared to rpsL K43R colonies, and grew at markedly different rates (Table 1 ). rpsL P91W colonies grew the fastest, being visible after 4 days of incubation, and being similar in size to the rpsL K43R colonies at that time. rpsL P91K colonies grew the slowest, becoming visible only after 11 days of incubation, at which time they were still very small; after additional time they slowly grew and were noted to additionally be quite dysmorphic. The 16S rRNA and infC mutating oligonucleotides did not result in any colonies on streptomycin plates, either alone or in combination. Colonies were patched onto both fresh streptomycin-containing and drug-free agar plates to assess the streptomycin phenotype. As expected, rpsL K43R colonies grew well with or without streptomycin, indicating streptomycin-resistance (Sm R ). rpsL P91H colonies grew on both streptomycin-containing and drug-free agar plates, however, grew faster on streptomycin-containing plates; this phenotype is sometimes described as streptomycin-enhanced (Sm E ). All other tested colonies grew only on streptomycin-containing plates, indicating an Sm D phenotype. Table 1 Recombineering oligonucleotides thought likely to result in a streptomycin dependent phenotype in M. smegmatis . Attempted Mutation ssDNA recombineering oligo(s) 10 − 1 plate CFU 10 − 2 plate CFU Day visible Note Patching results rpsL P91P oPMF170 0 0 NA NA rpsL K43R oPMF184 TMTC 122 4 20/20 Sm R rpsL P91W oPMF180 17 0 4 Similar in size to rpsL 43R 17/17 Sm D rpsL P91E oPMF183 TMTC 11 4 Smaller than rpsL 43R or 91W 20/20 Sm D rpsL P91Q oPMF167 70 6 6 20/20 Sm D rpsL P91R oPMF168 4 1 6 5/5 Sm D rpsL P91L oPMF182 15 2 6 17/17 Sm D rpsL P91H oPMF181 11 1 8 Patched colonies grew faster on streptomycin plates than drug free plates 12/12 Sm E rpsL P91K oPMF169 11 0 11 Very small, dysmorphic colonies 11/11 Sm D infC 23_24insE oPMF178 0 0 NA NA NA 16S rRNA 512_513insC oPMF179 0 0 NA NA NA infC 23_24insE and 16S rRNA 512_513insC oPMF178 and oPMF179 0 0 NA NA NA Results of attempt at recombineering streptomycin-dependence in M. smegmatis . TMTC: Too many to count. NA: Not applicable. After plating appropriate dilutions of outgrowth onto streptomycin-containing agar plates, colonies were patched onto streptomycin-containing and drug-free plates to assess the streptomycin phenotype. Multiple rpsL P91 mutations produced Sm D colonies. Most of these had a noticeably slower growth rate than positive control Sm R rpsL K43R colonies. rpsL P91W had the closest growth rate to rpsL K43R. Streptomycin-dependence can be used as a selection marker to introduce a desired mutation into M. smegmatis Given the good growth rate of rpsL P91W mutants, we decided to attempt to use this as a selection method to introduce a desired mutation. We transformed M. smegmatis (carrying the recombineering plasmids pKM461 and pKM427) with a reversible rpsL P91W oligonucleotide (oPMF348), as well as a nucleotide designed to introduce an arr P5* K6* mutation (oNP07), and plated onto streptomycin-containing agar. We screened 24 colonies using MAMA PCR, identified two candidate successes, and confirmed them both via sequencing. This demonstrates that the Sm D phenotype can be used as a selection marker for introducing another desired mutation. Reversion of streptomycin-dependence can be used as a selection marker to introduce another desired mutation into M. smegmatis We next tested whether we could use reversion from Sm D rpsL 91W to Sm S rpsL 91P as a selection method to introduce a second desired mutation. We started with the M. smegmatis rpsL P91W arr P5* K6* strain generated in the previous step, and transformed with an rpsL W91P oligonucleotide (oPMF349) alone or in combination with oligonucleotides designed to introduce either an rpoC G494E mutation (oNP01) or an rpoB S450L mutation (oNP13). These were plated onto drug-free agar plates. After MAMA screening and confirmatory sequencing, we found successfully recombineered colonies for all three. We had thus created Sm S strains of M. smegmatis with the arr mutation alone, as well as two double mutants carrying arr + rpoB and arr + rpoC mutations. This demonstrated that reverting the rpsL 91 residue from tryptophan to proline can restore an Sm S phenotype, and that this can be used select for additional edits. Sequentially toggling between streptomycin-dependence and -sensitivity can be used to introduce further mutations We next tested whether we could make further edits by continuing to switch between Sm D and Sm S phenotypes. Starting with the Sm S arr + rpoC double mutant, we transformed it with the rpsL P91W (oPMF348) and the rpoB S450L oligonucleotide (oNP13) and then plated onto streptomycin plates. After MAMA PCR screening and sequencing, we found successfully recombineered streptomycin-dependent colonies carrying arr + rpoC + rpoB mutations. We had thus obtained, in an arr P5* K6* background, Sm D strains with either no additional edits, rpoB S450L, rpoC G494E, or both. We wanted to have a Sm S strain with the arr , rpoB , and rpoC mutations, so we transformed this with the reversion oligo oPMF349, resulting in a Sm S strain carrying the set of three desired mutations but with otherwise unmarked chromosomal DNA. This demonstrated that multiple rounds of switching between Sm S and Sm D could be used to generate unmarked mutants carrying multiple other mutations, suitable for exploring combinatorial effects of mutations of interest. Rate of additional spontaneous mutations during serial recombination is low We wanted to assess how many other mutations spontaneously arose in other locations in the genome over the course of multiple rounds of recombineering. We obtained whole genome sequences for M. smegmatis before recombineering and after multiple rounds of recombineering via toggling Sm D . Genome sequencing confirmed the presence of all the mutations intended to be introduced during recombineering and identified 1–2 additional new SNPs from the generated mutants (Table 2 ). The initial recombineering strain had 42 SNPs compared to the reference genome, of which only one SNP was not detected in all generated mutants. One more SNP was not detected in the third-generation mutant with oNP07 + 01 + 13. Table 2 SNPs and indels identified from each recombineered M. smegmatis mutant Strain ID Characteristics No of edits introduced New SNPs identified No of indels detected Recombineering strain Carrying plasmid pKM461 and pKM427 None 42 112 Mutant with oNP07 STR dependent P91W: 5 edits oNP07: 4 edits 50 − 9 = 41 112 Mutant with oNP07 + oNP01 STR susceptible oNP01: 4 edits oNP07: 4 edits 51 − 8 = 42 114 Mutant with oNP07 + 01 + 13 STR dependent P91W: 5 edits oNP01: 4 edits oNP07: 4 edits oNP13: 3 edits 57 − 16 = 41 111 Number of mutations introduced by each oligonucleotide: P91W = 5, oNP01 = 4, oNP07 = 4, and oNP13 = 3 mutations. Number of SNPs and small indels between each isolate and the M. smegmatis mc 2 155 reference genome are listed. SNPs due to deliberately introduced edits are subtracted, giving the net number of unexplained differences to the reference genome. Multiple rpsL P91 mutations result in streptomycin dependence in Mtb , although with low efficiency We next sought to repeat our creation of an Sm D phenotype in Mtb . Mtb carrying the recombineering plasmids pKM461 and pKM427 was transformed with oligonucleotides designed to introduce several rpsL P91 mutations, a 16S rRNA insertion, an infC insertion, and the 16S rRNA + infC combination, and then plated onto streptomycin plates. As with M. smegmatis , the total number of colonies obtained with Sm D conferring oligonucleotides was several orders of magnitude lower than with traditional Sm R ones. The number of colonies appeared to be slightly above the background rate of the negative control for several rpsL P91 mutating oligonucleotides. Intriguingly, we also obtained numerous very slow-growing colonies with the 16S rRNA oligonucleotide – these were barely visible pinpoint colonies after more than 8 weeks of incubation, however technical difficulties relating to their growth rate prevented us from further analyzing these. The infC insertion oligonucleotide yielded around the background number of colonies. The combination of the 16S rRNA and infC oligonucleotides yielded a small number of colonies (we plated an additional plate with 10X concentrated outgrowth, to increase the chances of catching potentially rare double recombinants) with roughly wild-type growth rate. We additionally saw a larger number of small pinpoint colonies on these plates after 8 weeks of incubation, which we though likely represented 16S rRNA single mutations. Table 3 R ecombineering oligonucleotides thought likely to result in an Sm D phenotype in Mtb Mutation ssDNA recombineering oligo(s) 10 1 plate CFU 10 0 plate CFU 10 − 1 plate CFU Week visible Note Patching results rpsL P91P oPMF253 ND 4 0 3 1/4 Sm D , 3/4 Sm R rpsL K43R oPMF259 ND Lawn TMTC 3 20/20 Sm R rpsL P91W oPMF255 ND 19 2 3 20/20 Sm D rpsL P91E oPMF257 ND 3 0 3 3/3 Sm R rpsL P91Q oPMF252 ND 10 1 4 11/11 Sm D rpsL P91L oPMF256 ND 4 0 3–4 2/4 Sm D rpsL P91H oPMF254 ND 3 2 3 Several patched colonies grew faster on streptomycin plates than drug free plates 1/5 Sm D 3/5 Sm E 1/5 Sm R rpsL P91K oPMF258 ND 1 0 3 1/1 Sm R 16S rRNA insertion oPMF177 ND ~ 20 few 8–10 Very slow growth rate prevented further analysis ND infC insertion oPMF176 ND 4 0 3 2/4 Sm D , 2/4 Sm R 16S rRNA + infC oPMF 176 + oPMF177 14 0 3–4 Additional very slow colonies became apparent after approximately 8 weeks. 10/14 Sm D , 4/14 Sm R Results of attempt at recombineering streptomycin-dependence in Mtb . ND: Not done. TMTC: Too many to count. After plating appropriate dilutions of outgrowth onto streptomycin-containing agar plates, colonies were patched onto streptomycin-containing and drug-free plates to assess the streptomycin phenotype. rpsL P91W and P91Q oligonucleotides gave rise to consistently Sm D colonies. Negative control ( rpsL P91P) and other oligonucleotides gave rise to a mix of Sm R and Sm D colonies. Sequencing Mtb streptomycin-dependent strains confirms successful rpsL P91W recombineering The number of colonies obtained by attempts at recombineering streptomycin-dependence into Mtb was generally not much more than the negative control, raising the question of whether these represented successful recombineering at a very low efficiency or spontaneous mutants unrelated to the recombineering process. We noted that some oligos seemed to result in a higher rate of streptomycin-dependence than the negative control, suggesting that at least some of them might represent successful recombineering. To clarify this question, we performed whole-genome sequencing on several Sm D strains of MTB isolated after recombineering. Both sequenced rpsL P91W strains carried the precise edit we had attempted to make, suggesting that these were bona fide successful recombinants. Both rpsL P91Q strains did carry the desired edit, however both also carried an additional mutation in the rpsL gene; one carried an rpsL G85A, and another an rpsL R94G mutation. This suggests that we had successfully recombineered an rpsL P91Q mutation, but that additional spontaneous compensatory mutations may have been additionally selected for, perhaps by rescuing a slower growth rate. These results suggest that recombineering rpsL P91 mutations into Mtb can be accomplished, that it can result in an Sm D phenotype, and that rpsL P91W is the most promising mutation, as we previously found to be the case in M. smegmatis. For the Sm D strains obtained after attempting rpsL P91L or infC mutation, or 16S + infC double mutation, we found no evidence of the desired mutation. In 5 cases, we found a 16S 515G > C mutation, and in one case we found an 16S 515 G > T mutation. This location is intriguingly close to the 16S 513_514insC mutation we had attempted to create, and is within the footprint of expected annealing of our recombineering ssDNA oligonucleotide to the genome, raising the possibility that it might have been a consequence of misannealing of the recombineering oligonucleotide to the genome. However, the presence of these same mutations in the colonies isolated after attempting rpsL P91L or infC mutations suggests that these more likely represented spontaneous Sm D mutations not derived from recombineering. The consistency with which mutations were found at this location suggests that this may be the location in the genome at which spontaneous mutations resulting in a Sm D phenotype are most likely to arise. Streptomycin-dependence may not be an effective selection marker for introducing another desired mutation into Mtb Next, we attempted to replicate our previous successes using streptomycin-dependence as a selection method for recombineering in Mtb . We transformed Mtb with a reversible rpsL P91W oligonucleotide (oPMF389), as well as a nucleotide designed to introduce Mtb whiB6 E8D (oNPM01), Mtb rpoB S450L (oNPM07) or Mtb rpoC G594E (oNPM13) mutations, and plated the outgrowth onto streptomycin plates. Despite multiple attempts, we consistently obtained only a small number of colonies, typically on the order of 10, and we were not able to identify our desired mutations in any of these. We have had recombineering success in Mtb using oNPM01, oNPM07 and oNPM13 oligonucleotides with the more established aph(4)-1A *190W hygromycin-based selection system, which typically yields many more colonies. This suggests that these oligonucleotides can work with the right selection system, but perhaps not with the rpsL P91W system. DISCUSSION In this paper, we have demonstrated that several different mutations can give rise to an Sm D phenotype in Mycobacteria . As in many other organisms, several mutations of the rpsL P91 residue give rise to an Sm D phenotype. Many of these had substantial growth defects; however, Sm D rpsL P91W mutants were obtained in both M. smegmatis and Mtb , and grew similarly to wild-type and rpsL K43R strains. In both organisms, we found that one to two logs fewer Sm D colonies were obtained via oligonucleotide mediated recombineering rpsL P91 mutations than the number of Sm R colonies obtained via rpsL K43R. In M. smegmatis , despite the reduced efficiency, enough colonies were generated to reliably able to use this as a selection system to find clones that carried a mutation conferred by a second co-electroporated recombineering oligonucleotide. Reversion of Sm D strains to Sm S via rpsL W91P recombineering and plating onto drug free agar was also used as a selection system to find colonies that carried a mutation conferred by another recombineering oligonucleotide. By toggling between streptomycin-sensitivity and streptomycin-dependence, multiple sequential edits were made to the bacterial genome, without requiring any additional steps to remove or reset selection markers. Here, we have flipped the streptomycin phenotype in M. smegmatis four times, constituting two full round trips from streptomycin-sensitivity to -dependence and back again. In the process, we have introduced three sets of additional mutations, with a very low rate of additional spontaneous mutations arising during the process. It seems likely that one could continue to make an arbitrary number of mutations by continuing in this fashion. This powerfully expands the potential uses of oligonucleotide-mediated recombineering, by allowing facile sequential recombineering steps to generate strains with multiple mutations. This allows significantly easier exploration of combinatorial interactions between multiple mutations in mycobacteria. In Mtb , generating Sm D mutants via rpsL P91W recombineering and plating onto streptomycin agar produced bona fide streptomycin-dependent recombinants. As in M. smegmatis , this method yielded one to two logs fewer colonies than other methods. The total number of colonies obtained varied between experimental repetitions, but was generally only slightly above the background rate of spontaneous streptomycin-resistance or -dependence. In our experience using more established selection systems for oligonucleotide-mediated recombineering, it is typical for 5–10% of colonies to carry the desired mutation, similar to the success rate reported by others 2 , 4 . The recombineering oligonucleotides we attempted to use with the streptomycin-dependence system in Mtb have previously worked well with the more well-established hygromycin-based selection system, suggesting that the fault likely lies with the selection system rather than the payload oligos. It thus seems plausible that our difficulty in using streptomycin-dependence as a selection system in Mtb may reflect poor efficiency and a consequent inadequate number of transformants to find the relatively rare colonies which might carry the second mutation of interest. If this were the case, it would suggest that streptomycin-dependence could be a viable selection system in Mtb if other modifications could be made to boost the efficiency of the recombineering process. However, we cannot rule out the possibility that there may be other barriers to the streptomycin-dependence selection system working in Mtb . The reason for the decreased number of colonies obtained via recombineering an Sm D phenotype as compared to streptomycin- or hygromycin-resistance is not clear. One potential explanation could be that during the outgrowth period after transformation, Sm R cells can continue to reproduce, but Sm D cells do not. However, the Sm D strain of Mtb , 18b, can replicate for several days in the absence of streptomycin before entering a state of non-replicating persistence 16 . This argues against this explanation playing a major role in the discrepancy in efficiency of selection methods. Additionally, the magnitude of the difference seems higher than this explanation would predict. For example, rpsL P91W gives about two logs fewer Mtb colonies on streptomycin plates than does rpsL K43R after 4 days of outgrowth, although assuming a doubling time of approximately 24 hours, the cells would be able to reproduce at most 16-fold during this time. Thus, it seems likely that there are some other biological reasons for poorer transformation efficiency when introducing Sm D phenotypes, although this remains uncertain. In this work, we also attempted experimental testing of the impact of mutations in the 16S rRNA and infC which have previously been implicated in streptomycin-dependence in Mtb 18b. Spontaneous Sm D mutants obtained under other transformation conditions all carried 16S rRNA 515 SNPs, confirming that variations in this region of the 16S rRNA can give rise to a Sm D phenotype. When Mtb is transformed with an oligonucleotide encoding 16S rRNA 513_514insC mutation alone, very slow growing colonies became apparent only after about 8 weeks of incubation. Unfortunately, due to their very slow growth rate, we were unable to subculture these into liquid media or patch them onto agar plates. It is tempting to speculate that these may be Sm D bona fide recombinants with a very slow growth rate, however we have not been able to adequately test this. Attempting to introduce these mutations into M. smegmatis did not yield colonies. However, we note that M. smegmatis mc 2 155 genome encodes for two copies of its 16S rRNA , and that presumably both would need to be edited in order to result in a Sm D phenotype. Thus, it is reasonable to think that recombineering this mutation into M. smegmatis is statistically much less likely to occur. We consider that these results supply tantalizing hints supporting the proposal that Mtb 18b’s streptomycin dependence may be explained by a 16S rRNA cytosine insertion as well as another compensatory mutation, but no definitive proof. MATERIAL AND METHODS Strains and culture conditions Mycobacterium tuberculosis H37Rv 17 and Mycobacterium smegmatis mc 2 155 18 carrying plasmids pKM461 and pKM427 4 were used for all experiments. 7H9 (BD Difco) supplemented with 0.05% tween-80 (Sigma), 0.2% glycerol and 10% OAD (.05% oleic acid (Sigma), 5% fetal bovine serum albumin (Sigma) 2% dextrose (Sigma)) was used as liquid media, and 7H11 (BD Difco) supplemented with 0.5% glycerol and 10% OAD was used for solid media. Streptomycin sulfate (Sigma) was used at 1mg/mL. Kanamycin sulfate (Sigma) was used at 25 µg/mL. Anhydrotetracycline (aTC, Sigma) was used at 500 ng/mL for Mtb and 100 ng/mL for M. smegmatis . Cultures were incubated at 37°C. Recombineering Recombineering was carried out as previously reported 4 . Briefly, M. smegmatis or Mtb containing recombineering plasmids was grown in 7H9 with kanamycin to an OD of approximately 0.8 for Mtb or 0.5 for M. smegmatis . For strains which had already been made streptomycin-dependent in a previous step, this 7H9 was supplemented with 1mg/mL streptomycin. For Mtb , aTC and 1.5% glycine were then added and cells were incubated approximately 16 more hours; for M. smegmatis , aTC was added and the cells were incubated approximately 3 more hours. Cells were then collected by centrifugation and washed three times in sterile 10% glycerol (for M. smegmatis , 10% glycerol and centrifugation was done at 4°C, whereas for Mtb , 10% glycerol was pre-warmed to 37°C and centrifugation was done at room temperature), before being suspended in 1/10th the original volume. 200uL of these fresh competent cells were mixed with recombineering oligonucleotides and electroporated in a 0.2cm cuvette (BioRad) at 2.5 kV, 1000 Ω, 25 µF. Oligonucleotides were ordered from Eurofins Genomics, suspended in 10 mM Tris-HCl buffer (pH 8.0) at 5 µg/uL, and diluted to 0.5 µg/uL in water prior to use. For experiments evaluating only candidate Sm D oligonucleotides, 2 µg total of oligonucleotides were used. For experiments attempting to introduce another mutation, 2 µg of the oligonucleotide conferring that mutation and 200 ng of the selection oligonucleotide were used. Cells were then recovered in 5 mL of drug-free 7H9 and incubated shaking. M. smegmatis was allowed to incubate for 16 hours before plating. Multiple recovery periods for Mtb ranging from 2–8 days were trialed; data shown in Table 3 was obtained after 4 days of recovery. This transformation outgrowth was diluted or concentrated as appropriate, and plated onto 7H11 agar with or without streptomycin. Streptomycin phenotype evaluation For evaluation of streptomycin phenotype via patch plates, a sterile disposable inoculating loop or else a sterile pipette tip was touched to the colony, and then touched to first a drug-free 7H11 plate and then to a 7H11 streptomycin plate, and these were reincubated for at least 2 weeks (for M. smegmatis ) or 8 weeks (for Mtb ) with periodic examination. If only a few colonies were present, all were patched; if there were a large number of colonies an attempt would be made to take a representative sample of 20 of them. Clones which grew on both drug-free and streptomycin-containing plates were categorized as streptomycin-resistant (Sm R ). Clones which grew on streptomycin-containing but not drug-free plates were categorized as streptomycin-dependent (Sm D ). Clones which grew on both streptomycin-containing as well as drug-free plates, but for which the growth on streptomycin-containing plates was noticeably faster were categorized as streptomycin-enhanced (Sm E ). Recombineering screening and evaluation When attempting to recombineer a desired mutation, up to 24 colonies would be picked with a sterile inoculating loop or sterile pipette tip. This was touched to the surface of 1mL of 7H9 with kanamycin and, where appropriate, streptomycin, in a 24-well plate, which was subsequently incubated standing at 37°C. The remainder of the colony was then suspended in approximately 50uL of Chelex buffer (1 µM TRIS-HCl pH 8.0, 1 mM EDTA, 1 mg/mL NaN 3 , 0.2 g/mL Chelex (BioRad)) in a 96-well PCR plate. The PCR plate was then heated in a thermocycler (BioRad) at 98°C for 20 minutes, after which heat-killed MTB was removed from the BSL-3, vortexed, and centrifuged. 1 µL of this supernatant would be used as the template for screening MAMA PCR 19 . MAMA PCR was performed with indicated primers and Taq polymerase. Each reaction contained 1.0 µL of template, 0.15 µL of MAMA specific forward primer, 0.05 µL of the control forward primer, 0.2 µL of reverse primer, 3.6 µL of H 2 O, and 5 µL of Taq 2X Master Mix (NEB). PCR products were visualized on an agarose gel. For those colonies which had a band at the expected size indicating presence of the desired mutation, PCR amplification of the relevant region of genomic DNA was performed from the Chelex extracted DNA template using Q5 polymerase (NEB) and the control forward primer and reverse primers. These were sent for confirmatory Oxford Nanopore Sequencing with Plasmidsaurus. Recombineering ssDNA oligonucleotides used in this study are provided in Table 4 . Table 4: Recombineering ssDNA oligonucleotides used in this study. Oligonucleotide name Sequence Mutation oPMF167 CGAGCGAACCGCGGATGATCTTGTAACGGACACC TT GCAGGTCCTTCACACGACCGCCACGCACCAGCAC M. smegmatis rpsL P91Q oPMF168 CGAGCGAACCGCGGATGATCTTGTAACGGACACC CCT CAGGTCCTTCACACGACCGCCACGCACCAGCAC M. smegmatis rpsL P91R oPMF169 CGAGCGAACCGCGGATGATCTTGTAACGGACACC TTT CAGGTCCTTCACACGACCGCCACGCACCAGCAC M. smegmatis rpsL P91K oPMF170 CGAGCGAACCGCGGATGATCTTGTAACGGACACC GGG CAGGTCCTTCACACGACCGCCACGCACCAGCAC M. smegmatis rpsL P91P oPMF176 AGTGCGTCTTCGATACGCACAATGCCTACCTGCTC CTC CCCCCCTGGGCCGATCAATCGGACTTCAGGTACGC Mtb infC 23_24insE oPMF177 CGGACAACGCTCGCACCCTACGTATTACCGCGGCT G GCTGGCACGTAGTTGGCCGGTGCTTCTTCTCCACC Mtb 16S 513_514insC oPMF178 AGAGCGTCTTCGATGCGCACGATGCCTACCTGCTC CTC CCCACCGGGGCCGATCAAACGGACTTCAGGTACTC M. smegmatis infC 23_24insE oPMF179 CGGACAACGCTCGGACCCTACGTATTACCGCGGCT G GCTGGCACGTAGTTGGCCGGTCCTTCTTCTGCACA M. smegmatis 16S 512_513insC oPMF180 CGAGCGAACCGCGGATGATCTTGTAACGGACACC CCA CAGGTCCTTCACACGACCGCCACGCACCAGCAC M. smegmatis rpsL P91W oPMF181 CGAGCGAACCGCGGATGATCTTGTAACGGACACCG T GCAGGTCCTTCACACGACCGCCACGCACCAGCAC M. smegmatis rpsL P91H oPMF182 CGAGCGAACCGCGGATGATCTTGTAACGGACACCG A GCAGGTCCTTCACACGACCGCCACGCACCAGCAC M. smegmatis rpsL P91L oPMF183 CGAGCGAACCGCGGATGATCTTGTAACGGACACC TTC CAGGTCCTTCACACGACCGCCACGCACCAGCAC M. smegmatis rpsL P91E oPMF184 CGCGCGCGACCTTCCGGAGCGCCGAGTTCGGCTTC C TCGGAGTGGTGGTGTAAACGCGCGTGCACACGCC M. smegmatis rpsL K43R oPMF252 ATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC CT GCAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT Mtb rpsL P91Q oPMF253 ATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC AGG CAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT Mtb rpsL P91P oPMF254 ATCCAGCGAACCGCGGATGATCTTGTAGCGCACACCA T GCAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT Mtb rpsL P91W oPMF255 ATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC CCA CAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT Mtb rpsL P91L oPMF256 ATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC CA GCAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT Mtb rpsL P91L oPMF257 ATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC CTC CAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT Mtb rpsL P91E oPMF258 ATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC CTT CAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT Mtb rpsL P91K oPMF259 GCGGGCAACCTTCCGAAGCGCCGAGTTCGGCTTC C TCGGAGTGGTGGTGTACACGCGGGTGCATACACC Mtb rpsL K43R oPMF348 GTGTCGAGCGAACCGCGGATGATCTTGTAACGGAC C CC CCA A AGGTCCTTCACACGACCGCCACGCACCAGCACCAT Reversible M. smegmatis rpsL P91W oPMF349 GTGTCGAGCGAACCGCGGATGATCTTGTAACGGAC A CC GGG C AGGTCCTTCACACGACCGCCACGCACCAGCACCAT Reversible M. smegmatis rpsL W91P oNP01 GCATCCTTGGTGGCGGCCTGGTACTCGCCGGTCGC CT C T TC C ACGAGGGTGGTCAGGTAGTACAGACCGGTCA M. smegmatis rpoC G494E oNP07 CAGGTAGGCCCCGGACTCGTGCACTTCGAACGGTT ATTA CGGATTCGCCACTACAGTTCCCCCTGTTCCGTGTGT M. smegmatis arr P5* K6* oNP13 CGCGCTCACGGGACAGACCGCCGGGGCCCAGCGCC AG C AGACGACGCTTGTGGGTCAGACCCGACAGCGGG M. smegmatis rpoB S450L oNPM01 TCTACGTTCCTCCAGAAAGCGTTGCAGGTTGTAGC A TCTGCCGCGAAAGCGTATCGCATTAACCATAGCGA Mtb whiB6 E8D oNPM07 CACGCTCACGTGACAGACCGCCGGGCCCCAGCGCC A ACAGTCGGCGCTTGTGGGTCAACCCCGACAGCGGG Mtb rpoB S450L oNPM13 GTGATCCCCGCTGGCCGGCTGGTATTCGCCGGT A TC TT C C GGGACCTCGGTGGTCAGGTAGTACAGCCCGGTCAC Mtb rpoC G594E Bases which introduce a change are indicated in bolded green text. Flanking silent mutations introduced to evade the mismatch repair system are underlined. Whole Genome Sequencing For M. smegmatis sequencing, a cell pellet was suspended in 1x DNA/RNA Shield (Zymo) and sent for extraction and whole genome Oxford Nanopore Sequencing with Plasmidsaurus. For Mtb sequencing, a cell pellet was heat killed, DNA was extracted using phenol/chloroform/isoamyl alcohol 20 , and DNA was sent for whole genome Oxford Nanopore Sequencing with Plasmidsaurus. We then performed variant calling with Burrows-Wheeler Aligner (BWA) aligner 21 and SAMtools 22 . In summary, the reference genome, Mycobacterium smegmatis mc 2 155 (GenBank accession CP000480) was indexed using BWA. The preprocessed reads were aligned to the reference using the BWA-MEM 23 to generate ‘*’.sam files. These files were then converted to ‘*’.bam format, sorted by coordinates to generate ‘*’_sorted.bam’ files, and subsequently indexed using SAMtools. SAMtools was then used to call raw variants, generating VCF files. The variants were then refined and filtered using BCFtools to produce a high-quality set of polymorphisms (SNPs) and insertion-deletion mutations (indels). Variants were visualized in IGV for manual inspection and quality assessment. Declarations DATA AVAILABILITY Whole genome sequences have been submitted to the NCBI SRA database under BioProject ID PRJNA1191427, with BioSample Accession Numbers SAMN45079471 through SAMN45079485. CONFLICT OF INTEREST The authors have no conflicts of interest to declare. FUNDING This research was funded (in part) by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases. Nicholaus Mnyambwa was supported by the African Postdoctoral Training Initiative (APTI) Fellowship funded by the US NIH and the Bill & Melinda Gates Foundation in partnership with the African Academy of Sciences, under the auspices of the Coalition of African Research and Innovation. AUTHOR CONTRIBUTIONS P. F. did conceptual work, and performed recombineering and data analysis. N. M. did conceptual work, and performed recombineering, MAMA-screening and data analysis. H. B. did conceptual work. C. B. did conceptual work. ACKNOWLEDGEMENTS We would like to thank Kenan Murphy for the kind donation of plasmids pKM461 and pKM427. References Ellis, H.M., Yu, D., DiTizio, T., Court, D.L.: High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proceedings of the National Academy of Sciences 98, 6742–6746, doi: (2001). 10.1073/pnas.121164898 van Kessel, J.C., Hatfull, G.F.: Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: characterization of antimycobacterial drug targets. Mol. Microbiol. 67 , 1094–1107 (2008). 10.1111/j.1365-2958.2008.06109.x Murphy, K.C., Papavinasasundaram, K., Sassetti, C.M.: in Mycobacteria Protocols (eds Tanya Parish & David M. Roberts) 177–199Springer New York, (2015) Murphy, K.C.: in Mycobacteria Protocols (eds Tanya Parish & Anuradha Kumar) 301–321Springer US, (2021) Wang, H.H., et al.: Programming cells by multiplex genome engineering and accelerated evolution. Nature. 460 , 894–898 (2009). 10.1038/nature08187 Miller, C.P., Bohnhoff, M.: Development of streptomycin-resistant variants of meningococcus. Science. 105 , 620–621 (1947) Yegian, D., Budd, V.: A variant of Mycobacterium ranae requiring streptomycin for growth. J. Bacteriol. 55 , 459–461 (1948) Yegian, D., Budd, V., Vanderlinde, R.J., STREPTOMYCIN-DEPENDENT TUBERCLE BACILLI:: A SIMPLE METHOD FOR ISOLATION. J. Bacteriol. 58 , 257–259 (1949). 10.1128/jb.58.2.257-259.1949 Hashimoto, T.: Experimental studies on the mechanism of infection and immunity in tuberculosis from the analytical standpoint of streptomycin-dependent tubercle bacilli. 1. Isolation and biological characteristics of a streptomycin-dependent mutant, and effect of streptomycin administration on its pathogenicity in guinea-pigs. Kekkaku:[Tuberculosis]. 30 , 4–46 (1955) Tsukamura, M.: Studies on the streptomycin-dependent mutants of a Mycobacterium (jucho). (1) Morphological changes and the relation between streptomycin dependence and kanamycin resistance. Iryo. 15 , 616–621 (1961) Lee, C., Ye, Q., Shin, E., Ting, T., Lee, S.-J.: Acquisition of Streptomycin Resistance by Oxidative Stress Induced by Hydrogen Peroxide in Radiation-Resistant Bacterium Deinococcus geothermalis. Int. J. Mol. Sci. 23 , 9764 (2022) van Acken, U.: Proteinchemical studies on ribosomal proteins S4 and S12 from ram (ribosomal ambiguity) mutants of Escherichia coli. Mol. Gen. Genet. MGG. 140 , 61–68 (1975). 10.1007/BF00268989 Gregory, S.T., Cate, J.H., Dahlberg, A.E.: Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus. J. Mol. Biol. 309 , 333–338 (2001). 10.1006/jmbi.2001.4676 Liu, X.Q., Gillham, N.W., Boynton, J.E.: Chloroplast Ribosomal Protein Gene rps12 of Chlamydomonas reinhardtii. J. Biol. Chem. 264 , 16100–16108 (1989). 10.1016/s0021-9258(18)71592-5 Benjak, A., et al.: Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b. BMC Genom. 17 , 190 (2016). 10.1186/s12864-016-2528-2 Sala, C., et al.: Simple Model for Testing Drugs against Nonreplicating Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 54 , 4150–4158 (2010). 10.1128/AAC.00821-10 Steenken, W. Jr., Oatway, W.H. Jr., Petroff, S.A., BIOLOGICAL STUDIES OF, THE TUBERCLE BACILLUS : III. DISSOCIATION AND PATHOGENICITY OF THE R AND S VARIANTS OF THE HUMAN TUBERCLE BACILLUS: (H37). J. Exp. Med. 60 , 515–540 (1934). 10.1084/jem.60.4.515 Snapper, S.B., Melton, R.E., Mustafa, S., Kieser, T., Jr, W.R.J.: Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4 , 1911–1919 (1990). https://doi.org/10.1111/j.1365-2958.1990.tb02040.x Cha, R.S., Zarbl, H., Keohavong, P., Thilly, W.G.: Mismatch amplification mutation assay (MAMA): application to the cH-ras gene. Genome Res. 2 , 14–20 (1992) Warren, R., et al.: Safe Mycobacterium tuberculosis DNA Extraction Method That Does Not Compromise Integrity. J. Clin. Microbiol. 44 , 254–256 (2006). 10.1128/jcm.44.1.254-256.2006 Li, H., Durbin, R.: Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 25 , 1754–1760 (2009). 10.1093/bioinformatics/btp324 Li, H., et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 25 , 2078–2079 (2009). 10.1093/bioinformatics/btp352 Li, H.: Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv preprint arXiv:1303.3997 (2013) Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5639512","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":403584508,"identity":"989d3107-2ea3-47df-8058-9a41dcd844fb","order_by":0,"name":"Clifton Barry, III","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACCTBpw8DGwNgAZEskEKsljXQth+Fswlr4Zzc/+1zZdj6Pj/1w4w3GHRZ5DPyLj0ngteTOMeOZZ9tuF7PxJDZbMJ6RKGaQeJaGV4uBRIIxY2Pb7cQ2hsQ2CcY2icQGiTPGBvi1pH8GajmX2Mb/kGgtOSBbDgCtgNnC32P4AL9fzhQzNpxLBqp/2GwB0ciWiFcL/+z2zYwNZXaJ8/vTH9742FaX2M9/+MABfFrAgJENykgAYjZiYpOB4Q+KxYTtGAWjYBSMgpEFAHf1SEvGuZtfAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2927-270X","institution":"National Institutes of Health","correspondingAuthor":true,"prefix":"","firstName":"III","middleName":"Clifton","lastName":"Barry","suffix":""},{"id":403584509,"identity":"e0c3d3d2-cd23-4135-8720-fc6eb2121e9d","order_by":1,"name":"Peter Finin","email":"","orcid":"","institution":"National Institute of Allergy and Infectious Disease","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Finin","suffix":""},{"id":403584510,"identity":"7368806b-3eae-4fa3-9f4c-dae86f168154","order_by":2,"name":"Nicholaus Mnyambwa","email":"","orcid":"https://orcid.org/0000-0002-8584-4477","institution":"National Institute for Medical Research, Muhimbili Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Nicholaus","middleName":"","lastName":"Mnyambwa","suffix":""},{"id":403584511,"identity":"965684b9-e77f-4a40-b91e-58158174ed61","order_by":3,"name":"Helena Boshoff","email":"","orcid":"https://orcid.org/0000-0002-4333-206X","institution":"National Institute for Allergy and Infectious Disease","correspondingAuthor":false,"prefix":"","firstName":"Helena","middleName":"","lastName":"Boshoff","suffix":""}],"badges":[],"createdAt":"2024-12-13 16:21:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5639512/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5639512/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78805380,"identity":"92d57075-1f4f-4b36-8a41-4ff11f080a99","added_by":"auto","created_at":"2025-03-19 07:48:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1317144,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5639512/v1/7dc3740d-21fc-4dbe-bff2-e1b7a19d7df1.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Use of Streptomycin-Dependence as Selection/Counterselection for Sequential Oligonucleotide Mediated Recombineering in Mycobacteria","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cspan fontcategory=\"NonProportional\" class=\"\" name=\"Emphasis\"\u003ePlease have a look at courier new font provided for text in article.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eOligonucleotide mediated recombineering was first developed for \u003cem\u003eE. coli\u003c/em\u003e using the bacteriophage λ Red system \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and subsequently adapted for mycobacteria using the mycobacteriophage Che9c ssDNA annealing protein gp61 \u003csup\u003e2\u003c/sup\u003e. A plasmid encoding Che9c gp61 under an inducible promoter is first transformed into mycobacterial cells, expression is induced, and mycobacteria are then electroporated with ssDNA oligonucleotides generally 50\u0026ndash;100 bp in length. Che9c gp61 anneals the ssDNA oligonucleotides to the lagging strand at the replication fork of the target bacterial DNA. If the recombineering oligonucleotide is mostly identical to the host cell DNA, but contains a small number of mutations, daughter cells can inherit these mutations, resulting in a targeted genetic edit. If this edit confers a selectable phenotype, it is simple to isolate clones which carries the desired mutation. If the edit does not confer a known selectable phenotype, one can electroporate cells with both the oligonucleotide conferring the desired edit, as well as additional DNA which does confer a selectable phenotype. This can be a plasmid encoding a hygromycin resistance cassette \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, an ssDNA oligonucleotide resulting in an \u003cem\u003erpsL\u003c/em\u003e K43R mutation conferring streptomycin resistance (Sm\u003csup\u003eR\u003c/sup\u003e) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, or a ssDNA oligonucleotide resulting in a \u003cem\u003eaph(4)-1A\u003c/em\u003e *190W mutation repairing a defective hygromycin resistance cassette\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. After selecting onto appropriate antibiotic-containing media, up to 10% of colonies may carry the desired edit. By screening a manageable number of colonies, successful edits can often be found.\u003c/p\u003e \u003cp\u003eAlthough this system works well for single edits, it is much more cumbersome to make multiple edits. The system is not efficient enough to practically introduce multiple edits simultaneously in mycobacteria, as can be done in \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Multiple edits can be made sequentially, however once resistance to a given antibiotic is used as a selection marker, it cannot be reused without first reversing it somehow.\u003c/p\u003e \u003cp\u003eStreptomycin-dependence (Sm\u003csup\u003eD\u003c/sup\u003e) is a phenotype first described in \u003cem\u003eNeisseria meningitidis\u003c/em\u003e in 1947 \u003csup\u003e6\u003c/sup\u003e, whereby cells are only able to grow in the presence of streptomycin, and are unable to grow without it. It has since been described in many other bacteria, including the mycobacterial species \u003cem\u003eMycobacterium fortuitum\u003c/em\u003e \u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (\u003cem\u003eMtb\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We considered whether streptomycin-dependence might not make a better selectable marker for sequential genetic editing. Whereas antibiotic resistance can be easily selected for (by plating onto antibiotic-containing media), it cannot generally be easily selected against. Antibiotic dependence, on the other hand, is easy to select either for (by plating onto antibiotic containing media) or against (by plating onto drug-free media). We thus hypothesized that by using ssDNA oligonucleotides that either introduce or reverse mutations conferring a Sm\u003csup\u003eD\u003c/sup\u003e phenotype as a selection system, an unlimited number of sequential edits could be made without requiring additional intermediate steps to remove resistance markers.\u003c/p\u003e \u003cp\u003eA genetic basis for streptomycin-dependence has been well characterized in a variety of organisms. Mutations in the highly conserved proline 91 (mycobacterial numbering) residue of \u003cem\u003erpsL\u003c/em\u003e have been described to result in streptomycin-dependence in a wide variety of organisms across the tree of life, including the gram positive prokaryote \u003cem\u003eDeinococcus geothermalis\u003c/em\u003e \u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, gram negative prokaryotes \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eThermus thermophilus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and the chloroplast of the eukaryote \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The only streptomycin-dependent mycobacterial strain which has undergone genetic sequencing is \u003cem\u003eMtb\u003c/em\u003e strain 18b. This is a derivative of the lab strain \u003cem\u003eMtb\u003c/em\u003e H2 \u003csup\u003e9\u003c/sup\u003e, which unfortunately seems to have been lost, and for which no sequence is available for comparison. The mutations responsible for 18b\u0026rsquo;s streptomycin-dependent phenotype have been proposed to be insertions in both the \u003cem\u003e16S rRNA\u003c/em\u003e and in \u003cem\u003einfC\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, although this has not been experimentally verified. When deprived of streptomycin, \u003cem\u003eMtb\u003c/em\u003e 18b can grow for several generations before entering a state of non-replicating persistence, in which it neither grows nor experiences substantial cell death \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this work, we attempt to create a streptomycin-dependent phenotype in both \u003cem\u003eM. smegmatis\u003c/em\u003e and \u003cem\u003eMtb\u003c/em\u003e. Multiple \u003cem\u003erpsL\u003c/em\u003e P91 mutations cause a streptomycin-dependent phenotype in both \u003cem\u003eMtb\u003c/em\u003e and \u003cem\u003eM. smegmatis\u003c/em\u003e, although \u003cem\u003erpsL\u003c/em\u003e P91W results in the best growth characteristics in both organisms. The number of colonies obtained on streptomycin containing agar after these recombineering steps is substantially less than those obtained using \u003cem\u003erpsL\u003c/em\u003e K43R or \u003cem\u003eaph(4)-1a\u003c/em\u003e *190W selection systems. In \u003cem\u003eM. smegmatis\u003c/em\u003e, introduction of an \u003cem\u003erpsL\u003c/em\u003e P91W mutation can be used to select for a desired mutation using streptomycin selection, and introduction of an \u003cem\u003erpsL\u003c/em\u003e W91P mutation can be used to select for a second desired mutation using growth on drug-free media as selection. In \u003cem\u003eMtb\u003c/em\u003e, the total number of streptomycin dependent colonies was very low, and despite multiple efforts we were unfortunately not able to identify our desired mutations among these.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMultiple\u003c/b\u003e \u003cb\u003erpsL\u003c/b\u003e \u003cb\u003eP91 mutations result in streptomycin-dependence in\u003c/b\u003e \u003cb\u003eM. smegmatis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe first tested whether a streptomycin-dependent phenotype could be introduced to \u003cem\u003eM. smegmatis\u003c/em\u003e by oligonucleotide mediated recombineering. \u003cem\u003eM. smegmatis\u003c/em\u003e carrying the recombineering plasmids pKM461 and pKM427 was transformed with oligonucleotides designed to introduce multiple candidate mutations that we expected might result in a streptomycin-dependent phenotype. These included multiple \u003cem\u003erpsL\u003c/em\u003e P91 mutations, which have been associated with streptomycin dependence in other organisms. We also tested oligonucleotides designed to introduce \u003cem\u003e16S rRNA\u003c/em\u003e 512_513insC and \u003cem\u003einfC\u003c/em\u003e 23_24insE mutations, which have been proposed to explain \u003cem\u003eMtb\u003c/em\u003e 18b\u0026rsquo;s streptomycin-dependent phenotype. Transformants were plated onto streptomycin containing agar. The negative control \u003cem\u003erpsL\u003c/em\u003e P91P oligonucleotide gave rise to no colonies on agar plates with streptomycin, and the positive control \u003cem\u003erpsL\u003c/em\u003e K43R oligonucleotide (a mutation known to confer high-level streptomycin-resistance) resulted in numerous colonies. All tested \u003cem\u003erpsL\u003c/em\u003e P91 mutating oligonucleotides resulted in colonies on streptomycin plates, although these were present at 1-2.5 logs reduced frequency as compared to \u003cem\u003erpsL\u003c/em\u003e K43R colonies, and grew at markedly different rates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003erpsL\u003c/em\u003e P91W colonies grew the fastest, being visible after 4 days of incubation, and being similar in size to the \u003cem\u003erpsL\u003c/em\u003e K43R colonies at that time. \u003cem\u003erpsL\u003c/em\u003e P91K colonies grew the slowest, becoming visible only after 11 days of incubation, at which time they were still very small; after additional time they slowly grew and were noted to additionally be quite dysmorphic. The \u003cem\u003e16S rRNA\u003c/em\u003e and \u003cem\u003einfC\u003c/em\u003e mutating oligonucleotides did not result in any colonies on streptomycin plates, either alone or in combination.\u003c/p\u003e \u003cp\u003eColonies were patched onto both fresh streptomycin-containing and drug-free agar plates to assess the streptomycin phenotype. As expected, \u003cem\u003erpsL\u003c/em\u003e K43R colonies grew well with or without streptomycin, indicating streptomycin-resistance (Sm\u003csup\u003eR\u003c/sup\u003e). \u003cem\u003erpsL\u003c/em\u003e P91H colonies grew on both streptomycin-containing and drug-free agar plates, however, grew faster on streptomycin-containing plates; this phenotype is sometimes described as streptomycin-enhanced (Sm\u003csup\u003eE\u003c/sup\u003e). All other tested colonies grew only on streptomycin-containing plates, indicating an Sm\u003csup\u003eD\u003c/sup\u003e phenotype.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRecombineering oligonucleotides thought likely to result in a streptomycin dependent phenotype in \u003cem\u003eM. smegmatis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAttempted Mutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003essDNA recombineering oligo(s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e plate CFU\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e plate CFU\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDay visible\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNote\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePatching results\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e K43R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTMTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20/20 Sm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSimilar in size to rpsL 43R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17/17 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTMTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmaller than rpsL 43R or 91W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20/20 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20/20 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5/5 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17/17 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatched colonies grew faster on streptomycin plates than drug free plates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12/12 Sm\u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVery small, dysmorphic colonies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11/11 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003einfC\u003c/em\u003e 23_24insE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e16S rRNA\u003c/em\u003e 512_513insC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003einfC\u003c/em\u003e 23_24insE and \u003cem\u003e16S rRNA\u003c/em\u003e 512_513insC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF178 and oPMF179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eResults of attempt at recombineering streptomycin-dependence in \u003cem\u003eM. smegmatis\u003c/em\u003e. TMTC: Too many to count. NA: Not applicable.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter plating appropriate dilutions of outgrowth onto streptomycin-containing agar plates, colonies were patched onto streptomycin-containing and drug-free plates to assess the streptomycin phenotype. Multiple rpsL P91 mutations produced Sm\u003csup\u003eD\u003c/sup\u003e colonies. Most of these had a noticeably slower growth rate than positive control Sm\u003csup\u003eR\u003c/sup\u003e \u003cem\u003erpsL\u003c/em\u003e K43R colonies. \u003cem\u003erpsL\u003c/em\u003e P91W had the closest growth rate to \u003cem\u003erpsL\u003c/em\u003e K43R.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStreptomycin-dependence can be used as a selection marker to introduce a desired mutation into\u003c/b\u003e \u003cb\u003eM. smegmatis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven the good growth rate of \u003cem\u003erpsL\u003c/em\u003e P91W mutants, we decided to attempt to use this as a selection method to introduce a desired mutation. We transformed \u003cem\u003eM. smegmatis\u003c/em\u003e (carrying the recombineering plasmids pKM461 and pKM427) with a reversible \u003cem\u003erpsL\u003c/em\u003e P91W oligonucleotide (oPMF348), as well as a nucleotide designed to introduce an \u003cem\u003earr\u003c/em\u003e P5* K6* mutation (oNP07), and plated onto streptomycin-containing agar. We screened 24 colonies using MAMA PCR, identified two candidate successes, and confirmed them both via sequencing. This demonstrates that the Sm\u003csup\u003eD\u003c/sup\u003e phenotype can be used as a selection marker for introducing another desired mutation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReversion of streptomycin-dependence can be used as a selection marker to introduce another desired mutation into\u003c/b\u003e \u003cb\u003eM. smegmatis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next tested whether we could use reversion from Sm\u003csup\u003eD\u003c/sup\u003e \u003cem\u003erpsL\u003c/em\u003e 91W to Sm\u003csup\u003eS\u003c/sup\u003e \u003cem\u003erpsL\u003c/em\u003e 91P as a selection method to introduce a second desired mutation. We started with the \u003cem\u003eM. smegmatis rpsL\u003c/em\u003e P91W \u003cem\u003earr\u003c/em\u003e P5* K6* strain generated in the previous step, and transformed with an \u003cem\u003erpsL\u003c/em\u003e W91P oligonucleotide (oPMF349) alone or in combination with oligonucleotides designed to introduce either an \u003cem\u003erpoC\u003c/em\u003e G494E mutation (oNP01) or an \u003cem\u003erpoB\u003c/em\u003e S450L mutation (oNP13). These were plated onto drug-free agar plates. After MAMA screening and confirmatory sequencing, we found successfully recombineered colonies for all three. We had thus created Sm\u003csup\u003eS\u003c/sup\u003e strains of \u003cem\u003eM. smegmatis\u003c/em\u003e with the \u003cem\u003earr\u003c/em\u003e mutation alone, as well as two double mutants carrying \u003cem\u003earr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003erpoB\u003c/em\u003e and \u003cem\u003earr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003erpoC\u003c/em\u003e mutations. This demonstrated that reverting the \u003cem\u003erpsL\u003c/em\u003e 91 residue from tryptophan to proline can restore an Sm\u003csup\u003eS\u003c/sup\u003e phenotype, and that this can be used select for additional edits.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSequentially toggling between streptomycin-dependence and -sensitivity can be used to introduce further mutations\u003c/h2\u003e \u003cp\u003eWe next tested whether we could make further edits by continuing to switch between Sm\u003csup\u003eD\u003c/sup\u003e and Sm\u003csup\u003eS\u003c/sup\u003e phenotypes. Starting with the Sm\u003csup\u003eS\u003c/sup\u003e \u003cem\u003earr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003erpoC\u003c/em\u003e double mutant, we transformed it with the \u003cem\u003erpsL\u003c/em\u003e P91W (oPMF348) and the \u003cem\u003erpoB\u003c/em\u003e S450L oligonucleotide (oNP13) and then plated onto streptomycin plates. After MAMA PCR screening and sequencing, we found successfully recombineered streptomycin-dependent colonies carrying \u003cem\u003earr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003erpoC\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003erpoB\u003c/em\u003e mutations. We had thus obtained, in an \u003cem\u003earr\u003c/em\u003e P5* K6* background, Sm\u003csup\u003eD\u003c/sup\u003e strains with either no additional edits, \u003cem\u003erpoB\u003c/em\u003e S450L, \u003cem\u003erpoC\u003c/em\u003e G494E, or both. We wanted to have a Sm\u003csup\u003eS\u003c/sup\u003e strain with the \u003cem\u003earr\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, and \u003cem\u003erpoC\u003c/em\u003e mutations, so we transformed this with the reversion oligo oPMF349, resulting in a Sm\u003csup\u003eS\u003c/sup\u003e strain carrying the set of three desired mutations but with otherwise unmarked chromosomal DNA. This demonstrated that multiple rounds of switching between Sm\u003csup\u003eS\u003c/sup\u003e and Sm\u003csup\u003eD\u003c/sup\u003e could be used to generate unmarked mutants carrying multiple other mutations, suitable for exploring combinatorial effects of mutations of interest.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRate of additional spontaneous mutations during serial recombination is low\u003c/h3\u003e\n\u003cp\u003eWe wanted to assess how many other mutations spontaneously arose in other locations in the genome over the course of multiple rounds of recombineering. We obtained whole genome sequences for \u003cem\u003eM. smegmatis\u003c/em\u003e before recombineering and after multiple rounds of recombineering via toggling Sm\u003csup\u003eD\u003c/sup\u003e. Genome sequencing confirmed the presence of all the mutations intended to be introduced during recombineering and identified 1\u0026ndash;2 additional new SNPs from the generated mutants (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The initial recombineering strain had 42 SNPs compared to the reference genome, of which only one SNP was not detected in all generated mutants. One more SNP was not detected in the third-generation mutant with oNP07\u0026thinsp;+\u0026thinsp;01\u0026thinsp;+\u0026thinsp;13.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSNPs and indels identified from each recombineered \u003cem\u003eM. smegmatis\u003c/em\u003e mutant\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo of edits introduced\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNew SNPs identified\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo of indels detected\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecombineering strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarrying plasmid pKM461 and pKM427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutant with oNP07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSTR dependent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP91W: 5 edits\u003c/p\u003e \u003cp\u003eoNP07: 4 edits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u0026thinsp;\u0026minus;\u0026thinsp;9\u0026thinsp;=\u0026thinsp;41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutant with oNP07\u0026thinsp;+\u0026thinsp;oNP01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSTR susceptible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eoNP01: 4 edits\u003c/p\u003e \u003cp\u003eoNP07: 4 edits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51\u0026thinsp;\u0026minus;\u0026thinsp;8\u0026thinsp;=\u0026thinsp;42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e114\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutant with oNP07\u0026thinsp;+\u0026thinsp;01\u0026thinsp;+\u0026thinsp;13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSTR dependent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP91W: 5 edits\u003c/p\u003e \u003cp\u003eoNP01: 4 edits\u003c/p\u003e \u003cp\u003eoNP07: 4 edits\u003c/p\u003e \u003cp\u003eoNP13: 3 edits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57\u0026thinsp;\u0026minus;\u0026thinsp;16\u0026thinsp;=\u0026thinsp;41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e111\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eNumber of mutations introduced by each oligonucleotide: P91W\u0026thinsp;=\u0026thinsp;5, oNP01\u0026thinsp;=\u0026thinsp;4, oNP07\u0026thinsp;=\u0026thinsp;4, and oNP13\u0026thinsp;=\u0026thinsp;3 mutations. Number of SNPs and small indels between each isolate and the \u003cem\u003eM. smegmatis\u003c/em\u003e mc\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e155 reference genome are listed. SNPs due to deliberately introduced edits are subtracted, giving the net number of unexplained differences to the reference genome.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMultiple\u003c/b\u003e \u003cb\u003erpsL\u003c/b\u003e \u003cb\u003eP91 mutations result in streptomycin dependence in\u003c/b\u003e \u003cb\u003eMtb\u003c/b\u003e, \u003cb\u003ealthough with low efficiency\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next sought to repeat our creation of an Sm\u003csup\u003eD\u003c/sup\u003e phenotype in \u003cem\u003eMtb\u003c/em\u003e. \u003cem\u003eMtb\u003c/em\u003e carrying the recombineering plasmids pKM461 and pKM427 was transformed with oligonucleotides designed to introduce several \u003cem\u003erpsL\u003c/em\u003e P91 mutations, a \u003cem\u003e16S rRNA\u003c/em\u003e insertion, an \u003cem\u003einfC\u003c/em\u003e insertion, and the \u003cem\u003e16S rRNA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003einfC\u003c/em\u003e combination, and then plated onto streptomycin plates. As with \u003cem\u003eM. smegmatis\u003c/em\u003e, the total number of colonies obtained with Sm\u003csup\u003eD\u003c/sup\u003e conferring oligonucleotides was several orders of magnitude lower than with traditional Sm\u003csup\u003eR\u003c/sup\u003e ones. The number of colonies appeared to be slightly above the background rate of the negative control for several \u003cem\u003erpsL\u003c/em\u003e P91 mutating oligonucleotides. Intriguingly, we also obtained numerous very slow-growing colonies with the 16S rRNA oligonucleotide \u0026ndash; these were barely visible pinpoint colonies after more than 8 weeks of incubation, however technical difficulties relating to their growth rate prevented us from further analyzing these. The \u003cem\u003einfC\u003c/em\u003e insertion oligonucleotide yielded around the background number of colonies. The combination of the \u003cem\u003e16S rRNA\u003c/em\u003e and \u003cem\u003einfC\u003c/em\u003e oligonucleotides yielded a small number of colonies (we plated an additional plate with 10X concentrated outgrowth, to increase the chances of catching potentially rare double recombinants) with roughly wild-type growth rate. We additionally saw a larger number of small pinpoint colonies on these plates after 8 weeks of incubation, which we though likely represented 16S rRNA single mutations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eR\u003c/em\u003eecombineering oligonucleotides thought likely to result in an Sm\u003csup\u003eD\u003c/sup\u003e phenotype in \u003cem\u003eMtb\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003essDNA recombineering oligo(s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003csup\u003e1\u003c/sup\u003e plate CFU\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e0\u003c/sup\u003e plate CFU\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e plate CFU\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWeek visible\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNote\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePatching results\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/4 Sm\u003csup\u003eD\u003c/sup\u003e, 3/4 Sm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e K43R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLawn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTMTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20/20 Sm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20/20 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3/3 Sm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11/11 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2/4 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSeveral patched colonies grew faster on streptomycin plates than drug free plates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/5 Sm\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3/5 Sm\u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e1/5 Sm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erpsL\u003c/em\u003e P91K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/1 Sm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e16S rRNA\u003c/em\u003e insertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003efew\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eVery slow growth rate prevented further analysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003einfC\u003c/em\u003e insertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2/4 Sm\u003csup\u003eD\u003c/sup\u003e, 2/4 Sm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e16S rRNA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003einfC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eoPMF 176\u0026thinsp;+\u0026thinsp;oPMF177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAdditional very slow colonies became apparent after approximately 8 weeks.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10/14 Sm\u003csup\u003eD\u003c/sup\u003e, 4/14 Sm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003eResults of attempt at recombineering streptomycin-dependence in \u003cem\u003eMtb\u003c/em\u003e. ND: Not done. TMTC: Too many to count.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter plating appropriate dilutions of outgrowth onto streptomycin-containing agar plates, colonies were patched onto streptomycin-containing and drug-free plates to assess the streptomycin phenotype. \u003cem\u003erpsL\u003c/em\u003e P91W and P91Q oligonucleotides gave rise to consistently Sm\u003csup\u003eD\u003c/sup\u003e colonies. Negative control (\u003cem\u003erpsL\u003c/em\u003e P91P) and other oligonucleotides gave rise to a mix of Sm\u003csup\u003eR\u003c/sup\u003e and Sm\u003csup\u003eD\u003c/sup\u003e colonies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSequencing\u003c/b\u003e \u003cb\u003eMtb\u003c/b\u003e \u003cb\u003estreptomycin-dependent strains confirms successful\u003c/b\u003e \u003cb\u003erpsL\u003c/b\u003e \u003cb\u003eP91W recombineering\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe number of colonies obtained by attempts at recombineering streptomycin-dependence into \u003cem\u003eMtb\u003c/em\u003e was generally not much more than the negative control, raising the question of whether these represented successful recombineering at a very low efficiency or spontaneous mutants unrelated to the recombineering process. We noted that some oligos seemed to result in a higher rate of streptomycin-dependence than the negative control, suggesting that at least some of them might represent successful recombineering. To clarify this question, we performed whole-genome sequencing on several Sm\u003csup\u003eD\u003c/sup\u003e strains of MTB isolated after recombineering. Both sequenced \u003cem\u003erpsL\u003c/em\u003e P91W strains carried the precise edit we had attempted to make, suggesting that these were \u003cem\u003ebona fide\u003c/em\u003e successful recombinants. Both \u003cem\u003erpsL\u003c/em\u003e P91Q strains did carry the desired edit, however both also carried an additional mutation in the \u003cem\u003erpsL\u003c/em\u003e gene; one carried an \u003cem\u003erpsL\u003c/em\u003e G85A, and another an \u003cem\u003erpsL\u003c/em\u003e R94G mutation. This suggests that we had successfully recombineered an \u003cem\u003erpsL\u003c/em\u003e P91Q mutation, but that additional spontaneous compensatory mutations may have been additionally selected for, perhaps by rescuing a slower growth rate. These results suggest that recombineering \u003cem\u003erpsL\u003c/em\u003e P91 mutations into \u003cem\u003eMtb\u003c/em\u003e can be accomplished, that it can result in an Sm\u003csup\u003eD\u003c/sup\u003e phenotype, and that \u003cem\u003erpsL\u003c/em\u003e P91W is the most promising mutation, as we previously found to be the case in \u003cem\u003eM. smegmatis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eFor the Sm\u003csup\u003eD\u003c/sup\u003e strains obtained after attempting \u003cem\u003erpsL\u003c/em\u003e P91L or \u003cem\u003einfC\u003c/em\u003e mutation, or \u003cem\u003e16S\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003einfC\u003c/em\u003e double mutation, we found no evidence of the desired mutation. In 5 cases, we found a \u003cem\u003e16S\u003c/em\u003e 515G\u0026thinsp;\u0026gt;\u0026thinsp;C mutation, and in one case we found an \u003cem\u003e16S\u003c/em\u003e 515 G\u0026thinsp;\u0026gt;\u0026thinsp;T mutation. This location is intriguingly close to the \u003cem\u003e16S\u003c/em\u003e 513_514insC mutation we had attempted to create, and is within the footprint of expected annealing of our recombineering ssDNA oligonucleotide to the genome, raising the possibility that it might have been a consequence of misannealing of the recombineering oligonucleotide to the genome. However, the presence of these same mutations in the colonies isolated after attempting \u003cem\u003erpsL\u003c/em\u003e P91L or \u003cem\u003einfC\u003c/em\u003e mutations suggests that these more likely represented spontaneous Sm\u003csup\u003eD\u003c/sup\u003e mutations not derived from recombineering. The consistency with which mutations were found at this location suggests that this may be the location in the genome at which spontaneous mutations resulting in a Sm\u003csup\u003eD\u003c/sup\u003e phenotype are most likely to arise.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStreptomycin-dependence may not be an effective selection marker for introducing another desired mutation into\u003c/b\u003e \u003cb\u003eMtb\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, we attempted to replicate our previous successes using streptomycin-dependence as a selection method for recombineering in \u003cem\u003eMtb\u003c/em\u003e. We transformed \u003cem\u003eMtb\u003c/em\u003e with a reversible \u003cem\u003erpsL\u003c/em\u003e P91W oligonucleotide (oPMF389), as well as a nucleotide designed to introduce \u003cem\u003eMtb whiB6\u003c/em\u003e E8D (oNPM01), \u003cem\u003eMtb rpoB\u003c/em\u003e S450L (oNPM07) or \u003cem\u003eMtb rpoC\u003c/em\u003e G594E (oNPM13) mutations, and plated the outgrowth onto streptomycin plates. Despite multiple attempts, we consistently obtained only a small number of colonies, typically on the order of 10, and we were not able to identify our desired mutations in any of these. We have had recombineering success in \u003cem\u003eMtb\u003c/em\u003e using oNPM01, oNPM07 and oNPM13 oligonucleotides with the more established \u003cem\u003eaph(4)-1A\u003c/em\u003e *190W hygromycin-based selection system, which typically yields many more colonies. This suggests that these oligonucleotides can work with the right selection system, but perhaps not with the \u003cem\u003erpsL\u003c/em\u003e P91W system.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this paper, we have demonstrated that several different mutations can give rise to an Sm\u003csup\u003eD\u003c/sup\u003e phenotype in \u003cem\u003eMycobacteria\u003c/em\u003e. As in many other organisms, several mutations of the \u003cem\u003erpsL\u003c/em\u003e P91 residue give rise to an Sm\u003csup\u003eD\u003c/sup\u003e phenotype. Many of these had substantial growth defects; however, Sm\u003csup\u003eD\u003c/sup\u003e \u003cem\u003erpsL\u003c/em\u003e P91W mutants were obtained in both \u003cem\u003eM. smegmatis\u003c/em\u003e and \u003cem\u003eMtb\u003c/em\u003e, and grew similarly to wild-type and rpsL K43R strains. In both organisms, we found that one to two logs fewer Sm\u003csup\u003eD\u003c/sup\u003e colonies were obtained via oligonucleotide mediated recombineering rpsL P91 mutations than the number of Sm\u003csup\u003eR\u003c/sup\u003e colonies obtained via \u003cem\u003erpsL\u003c/em\u003e K43R.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eM. smegmatis\u003c/em\u003e, despite the reduced efficiency, enough colonies were generated to reliably able to use this as a selection system to find clones that carried a mutation conferred by a second co-electroporated recombineering oligonucleotide. Reversion of Sm\u003csup\u003eD\u003c/sup\u003e strains to Sm\u003csup\u003eS\u003c/sup\u003e via \u003cem\u003erpsL\u003c/em\u003e W91P recombineering and plating onto drug free agar was also used as a selection system to find colonies that carried a mutation conferred by another recombineering oligonucleotide. By toggling between streptomycin-sensitivity and streptomycin-dependence, multiple sequential edits were made to the bacterial genome, without requiring any additional steps to remove or reset selection markers. Here, we have flipped the streptomycin phenotype in \u003cem\u003eM. smegmatis\u003c/em\u003e four times, constituting two full round trips from streptomycin-sensitivity to -dependence and back again. In the process, we have introduced three sets of additional mutations, with a very low rate of additional spontaneous mutations arising during the process. It seems likely that one could continue to make an arbitrary number of mutations by continuing in this fashion. This powerfully expands the potential uses of oligonucleotide-mediated recombineering, by allowing facile sequential recombineering steps to generate strains with multiple mutations. This allows significantly easier exploration of combinatorial interactions between multiple mutations in mycobacteria.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eMtb\u003c/em\u003e, generating Sm\u003csup\u003eD\u003c/sup\u003e mutants via \u003cem\u003erpsL\u003c/em\u003e P91W recombineering and plating onto streptomycin agar produced \u003cem\u003ebona fide\u003c/em\u003e streptomycin-dependent recombinants. As in \u003cem\u003eM. smegmatis\u003c/em\u003e, this method yielded one to two logs fewer colonies than other methods. The total number of colonies obtained varied between experimental repetitions, but was generally only slightly above the background rate of spontaneous streptomycin-resistance or -dependence. In our experience using more established selection systems for oligonucleotide-mediated recombineering, it is typical for 5\u0026ndash;10% of colonies to carry the desired mutation, similar to the success rate reported by others \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The recombineering oligonucleotides we attempted to use with the streptomycin-dependence system in \u003cem\u003eMtb\u003c/em\u003e have previously worked well with the more well-established hygromycin-based selection system, suggesting that the fault likely lies with the selection system rather than the payload oligos. It thus seems plausible that our difficulty in using streptomycin-dependence as a selection system in \u003cem\u003eMtb\u003c/em\u003e may reflect poor efficiency and a consequent inadequate number of transformants to find the relatively rare colonies which might carry the second mutation of interest. If this were the case, it would suggest that streptomycin-dependence could be a viable selection system in \u003cem\u003eMtb\u003c/em\u003e if other modifications could be made to boost the efficiency of the recombineering process. However, we cannot rule out the possibility that there may be other barriers to the streptomycin-dependence selection system working in \u003cem\u003eMtb\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe reason for the decreased number of colonies obtained via recombineering an Sm\u003csup\u003eD\u003c/sup\u003e phenotype as compared to streptomycin- or hygromycin-resistance is not clear. One potential explanation could be that during the outgrowth period after transformation, Sm\u003csup\u003eR\u003c/sup\u003e cells can continue to reproduce, but Sm\u003csup\u003eD\u003c/sup\u003e cells do not. However, the Sm\u003csup\u003eD\u003c/sup\u003e strain of \u003cem\u003eMtb\u003c/em\u003e, 18b, can replicate for several days in the absence of streptomycin before entering a state of non-replicating persistence\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This argues against this explanation playing a major role in the discrepancy in efficiency of selection methods. Additionally, the magnitude of the difference seems higher than this explanation would predict. For example, \u003cem\u003erpsL\u003c/em\u003e P91W gives about two logs fewer \u003cem\u003eMtb\u003c/em\u003e colonies on streptomycin plates than does \u003cem\u003erpsL\u003c/em\u003e K43R after 4 days of outgrowth, although assuming a doubling time of approximately 24 hours, the cells would be able to reproduce at most 16-fold during this time. Thus, it seems likely that there are some other biological reasons for poorer transformation efficiency when introducing Sm\u003csup\u003eD\u003c/sup\u003e phenotypes, although this remains uncertain.\u003c/p\u003e \u003cp\u003eIn this work, we also attempted experimental testing of the impact of mutations in the \u003cem\u003e16S rRNA\u003c/em\u003e and \u003cem\u003einfC\u003c/em\u003e which have previously been implicated in streptomycin-dependence in \u003cem\u003eMtb\u003c/em\u003e 18b. Spontaneous Sm\u003csup\u003eD\u003c/sup\u003e mutants obtained under other transformation conditions all carried \u003cem\u003e16S rRNA\u003c/em\u003e 515 SNPs, confirming that variations in this region of the 16S rRNA can give rise to a Sm\u003csup\u003eD\u003c/sup\u003e phenotype. When \u003cem\u003eMtb\u003c/em\u003e is transformed with an oligonucleotide encoding \u003cem\u003e16S rRNA\u003c/em\u003e 513_514insC mutation alone, very slow growing colonies became apparent only after about 8 weeks of incubation. Unfortunately, due to their very slow growth rate, we were unable to subculture these into liquid media or patch them onto agar plates. It is tempting to speculate that these may be Sm\u003csup\u003eD\u003c/sup\u003e \u003cem\u003ebona fide\u003c/em\u003e recombinants with a very slow growth rate, however we have not been able to adequately test this. Attempting to introduce these mutations into \u003cem\u003eM. smegmatis\u003c/em\u003e did not yield colonies. However, we note that \u003cem\u003eM. smegmatis\u003c/em\u003e mc\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e155 genome encodes for two copies of its \u003cem\u003e16S rRNA\u003c/em\u003e, and that presumably both would need to be edited in order to result in a Sm\u003csup\u003eD\u003c/sup\u003e phenotype. Thus, it is reasonable to think that recombineering this mutation into \u003cem\u003eM. smegmatis\u003c/em\u003e is statistically much less likely to occur. We consider that these results supply tantalizing hints supporting the proposal that \u003cem\u003eMtb\u003c/em\u003e 18b\u0026rsquo;s streptomycin dependence may be explained by a \u003cem\u003e16S rRNA\u003c/em\u003e cytosine insertion as well as another compensatory mutation, but no definitive proof.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eStrains and culture conditions\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e H37Rv \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e mc\u003csup\u003e2\u003c/sup\u003e155 \u003csup\u003e18\u003c/sup\u003e carrying plasmids pKM461 and pKM427 \u003csup\u003e4\u003c/sup\u003e were used for all experiments. 7H9 (BD Difco) supplemented with 0.05% tween-80 (Sigma), 0.2% glycerol and 10% OAD (.05% oleic acid (Sigma), 5% fetal bovine serum albumin (Sigma) 2% dextrose (Sigma)) was used as liquid media, and 7H11 (BD Difco) supplemented with 0.5% glycerol and 10% OAD was used for solid media. Streptomycin sulfate (Sigma) was used at 1mg/mL. Kanamycin sulfate (Sigma) was used at 25 \u0026micro;g/mL. Anhydrotetracycline (aTC, Sigma) was used at 500 ng/mL for \u003cem\u003eMtb\u003c/em\u003e and 100 ng/mL for \u003cem\u003eM. smegmatis\u003c/em\u003e. Cultures were incubated at 37\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eRecombineering\u003c/h2\u003e\n \u003cp\u003eRecombineering was carried out as previously reported\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Briefly, \u003cem\u003eM. smegmatis\u003c/em\u003e or \u003cem\u003eMtb\u003c/em\u003e containing recombineering plasmids was grown in 7H9 with kanamycin to an OD of approximately 0.8 for \u003cem\u003eMtb\u003c/em\u003e or 0.5 for \u003cem\u003eM. smegmatis\u003c/em\u003e. For strains which had already been made streptomycin-dependent in a previous step, this 7H9 was supplemented with 1mg/mL streptomycin. For \u003cem\u003eMtb\u003c/em\u003e, aTC and 1.5% glycine were then added and cells were incubated approximately 16 more hours; for \u003cem\u003eM. smegmatis\u003c/em\u003e, aTC was added and the cells were incubated approximately 3 more hours. Cells were then collected by centrifugation and washed three times in sterile 10% glycerol (for \u003cem\u003eM. smegmatis\u003c/em\u003e, 10% glycerol and centrifugation was done at 4\u0026deg;C, whereas for \u003cem\u003eMtb\u003c/em\u003e, 10% glycerol was pre-warmed to 37\u0026deg;C and centrifugation was done at room temperature), before being suspended in 1/10th the original volume. 200uL of these fresh competent cells were mixed with recombineering oligonucleotides and electroporated in a 0.2cm cuvette (BioRad) at 2.5 kV, 1000 Ω, 25 \u0026micro;F. Oligonucleotides were ordered from Eurofins Genomics, suspended in 10 mM Tris-HCl buffer (pH 8.0) at 5 \u0026micro;g/uL, and diluted to 0.5 \u0026micro;g/uL in water prior to use. For experiments evaluating only candidate Sm\u003csup\u003eD\u003c/sup\u003e oligonucleotides, 2 \u0026micro;g total of oligonucleotides were used. For experiments attempting to introduce another mutation, 2 \u0026micro;g of the oligonucleotide conferring that mutation and 200 ng of the selection oligonucleotide were used. Cells were then recovered in 5 mL of drug-free 7H9 and incubated shaking. \u003cem\u003eM. smegmatis\u003c/em\u003e was allowed to incubate for 16 hours before plating. Multiple recovery periods for \u003cem\u003eMtb\u003c/em\u003e ranging from 2\u0026ndash;8 days were trialed; data shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e was obtained after 4 days of recovery. This transformation outgrowth was diluted or concentrated as appropriate, and plated onto 7H11 agar with or without streptomycin.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eStreptomycin phenotype evaluation\u003c/h3\u003e\n\u003cp\u003eFor evaluation of streptomycin phenotype via patch plates, a sterile disposable inoculating loop or else a sterile pipette tip was touched to the colony, and then touched to first a drug-free 7H11 plate and then to a 7H11 streptomycin plate, and these were reincubated for at least 2 weeks (for \u003cem\u003eM. smegmatis\u003c/em\u003e) or 8 weeks (for \u003cem\u003eMtb\u003c/em\u003e) with periodic examination. If only a few colonies were present, all were patched; if there were a large number of colonies an attempt would be made to take a representative sample of 20 of them. Clones which grew on both drug-free and streptomycin-containing plates were categorized as streptomycin-resistant (Sm\u003csup\u003eR\u003c/sup\u003e). Clones which grew on streptomycin-containing but not drug-free plates were categorized as streptomycin-dependent (Sm\u003csup\u003eD\u003c/sup\u003e). Clones which grew on both streptomycin-containing as well as drug-free plates, but for which the growth on streptomycin-containing plates was noticeably faster were categorized as streptomycin-enhanced (Sm\u003csup\u003eE\u003c/sup\u003e).\u003c/p\u003e\n\u003ch3\u003eRecombineering screening and evaluation\u003c/h3\u003e\n\u003cp\u003eWhen attempting to recombineer a desired mutation, up to 24 colonies would be picked with a sterile inoculating loop or sterile pipette tip. This was touched to the surface of 1mL of 7H9 with kanamycin and, where appropriate, streptomycin, in a 24-well plate, which was subsequently incubated standing at 37\u0026deg;C. The remainder of the colony was then suspended in approximately 50uL of Chelex buffer (1 \u0026micro;M TRIS-HCl pH 8.0, 1 mM EDTA, 1 mg/mL NaN\u003csub\u003e3\u003c/sub\u003e, 0.2 g/mL Chelex (BioRad)) in a 96-well PCR plate. The PCR plate was then heated in a thermocycler (BioRad) at 98\u0026deg;C for 20 minutes, after which heat-killed MTB was removed from the BSL-3, vortexed, and centrifuged. 1 \u0026micro;L of this supernatant would be used as the template for screening MAMA PCR \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMAMA PCR was performed with indicated primers and Taq polymerase. Each reaction contained 1.0 \u0026micro;L of template, 0.15 \u0026micro;L of MAMA specific forward primer, 0.05 \u0026micro;L of the control forward primer, 0.2 \u0026micro;L of reverse primer, 3.6 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO, and 5 \u0026micro;L of Taq 2X Master Mix (NEB). PCR products were visualized on an agarose gel. For those colonies which had a band at the expected size indicating presence of the desired mutation, PCR amplification of the relevant region of genomic DNA was performed from the Chelex extracted DNA template using Q5 polymerase (NEB) and the control forward primer and reverse primers. These were sent for confirmatory Oxford Nanopore Sequencing with Plasmidsaurus. Recombineering ssDNA oligonucleotides used in this study are provided in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Recombineering ssDNA oligonucleotides used in this study.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse;border: none;width: 616px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;line-height:150%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Segoe UI\",sans-serif;'\u003eOligonucleotide name\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;line-height:150%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Segoe UI\",sans-serif;'\u003eSequence\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;line-height:150%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Segoe UI\",sans-serif;'\u003eMutation\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;font-family:Calibri;color:black;\"\u003eoPMF167\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Courier New\";color:black;'\u003eCGAGCGAACCGCGGATGATCTTGTAACGGACACC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Courier New\";color:#00B050;'\u003eTT\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Courier New\";color:black;'\u003eGCAGGTCCTTCACACGACCGCCACGCACCAGCAC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;line-height:150%;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Calibri\",sans-serif;'\u003eM. smegmatis rpsL\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;P91Q\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF168\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCGAGCGAACCGCGGATGATCTTGTAACGGACACC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eCCT\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCAGGTCCTTCACACGACCGCCACGCACCAGCAC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eM. smegmatis rpsL\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;P91R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF169\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCGAGCGAACCGCGGATGATCTTGTAACGGACACC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eTTT\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCAGGTCCTTCACACGACCGCCACGCACCAGCAC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eM. smegmatis rpsL\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;P91K\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF170\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCGAGCGAACCGCGGATGATCTTGTAACGGACACC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eGGG\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCAGGTCCTTCACACGACCGCCACGCACCAGCAC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eM. smegmatis rpsL\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;P91P\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF176\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eAGTGCGTCTTCGATACGCACAATGCCTACCTGCTC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eCTC\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCCCCCCTGGGCCGATCAATCGGACTTCAGGTACGC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: 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style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eCCA\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003eMtb rpsL\u003c/em\u003e P91L\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF256\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eCA\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eGCAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003eMtb rpsL\u003c/em\u003e P91L\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF257\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eCTC\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003eMtb rpsL\u003c/em\u003e P91E\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF258\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eATCCAGCGAACCGCGGATGATCTTGTAGCGCACACC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eCTT\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCAGGTCCTTCACCCGGCCGCCGCGCACCAGCACCAT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003eMtb rpsL\u003c/em\u003e P91K\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF259\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eGCGGGCAACCTTCCGAAGCGCCGAGTTCGGCTTC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eC\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eTCGGAGTGGTGGTGTACACGCGGGTGCATACACC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003eMtb rpsL\u003c/em\u003e K43R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF348\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eGTGTCGAGCGAACCGCGGATGATCTTGTAACGGAC\u003c/span\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eC\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eCCA\u003cu\u003eA\u003c/u\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eAGGTCCTTCACACGACCGCCACGCACCAGCACCAT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eReversible \u003cem\u003eM. smegmatis rpsL\u003c/em\u003e P91W\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoPMF349\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eGTGTCGAGCGAACCGCGGATGATCTTGTAACGGAC\u003c/span\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eA\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eGGG\u003cu\u003eC\u003c/u\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eAGGTCCTTCACACGACCGCCACGCACCAGCACCAT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eReversible \u003cem\u003eM. smegmatis rpsL\u003c/em\u003e W91P\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoNP01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eGCATCCTTGGTGGCGGCCTGGTACTCGCCGGTCGC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eCT\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eC\u003c/span\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eT\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eTC\u003c/span\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eC\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eACGAGGGTGGTCAGGTAGTACAGACCGGTCA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eM. smegmatis\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003erpoC\u003c/em\u003e G494E\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoNP07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCAGGTAGGCCCCGGACTCGTGCACTTCGAACGGTT\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eATTA\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCGGATTCGCCACTACAGTTCCCCCTGTTCCGTGTGT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eM. smegmatis\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003earr\u003c/em\u003e P5* K6*\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoNP13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCGCGCTCACGGGACAGACCGCCGGGGCCCAGCGCC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eAG\u003cu\u003eC\u003c/u\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eAGACGACGCTTGTGGGTCAGACCCGACAGCGGG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eM. smegmatis rpoB\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;S450L\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoNPM01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eTCTACGTTCCTCCAGAAAGCGTTGCAGGTTGTAGC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eA\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eTCTGCCGCGAAAGCGTATCGCATTAACCATAGCGA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eMtb\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003ewhiB6\u003c/em\u003e E8D \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoNPM07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eCACGCTCACGTGACAGACCGCCGGGCCCCAGCGCC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eA\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eACAGTCGGCGCTTGTGGGTCAACCCCGACAGCGGG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eMtb\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003cem\u003erpoB\u003c/em\u003e S450L\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003eoNPM13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eGTGATCCCCGCTGGCCGGCTGGTATTCGCCGGT\u003c/span\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eA\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eTC\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eTT\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eC\u003c/span\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:#00B050;'\u003eC\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003eGGGACCTCGGTGGTCAGGTAGTACAGCCCGGTCAC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border: none;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eMtb rpoC G594E\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"font-size:13px;font-family:Calibri;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 315.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Courier New\";color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 462.1pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 0.2in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Calibri\",sans-serif;color:black;'\u003eBases which introduce a change are indicated in bolded green text. \u0026nbsp;Flanking silent mutations introduced to evade the mismatch repair system are underlined.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;line-height:150%;'\u003e\u003cspan style='font-family:\"Segoe UI\",sans-serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eWhole Genome Sequencing\u003c/h2\u003e\n \u003cp\u003eFor \u003cem\u003eM. smegmatis\u003c/em\u003e sequencing, a cell pellet was suspended in 1x DNA/RNA Shield (Zymo) and sent for extraction and whole genome Oxford Nanopore Sequencing with Plasmidsaurus. For \u003cem\u003eMtb\u003c/em\u003e sequencing, a cell pellet was heat killed, DNA was extracted using phenol/chloroform/isoamyl alcohol\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and DNA was sent for whole genome Oxford Nanopore Sequencing with Plasmidsaurus. We then performed variant calling with Burrows-Wheeler Aligner (BWA) aligner\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and SAMtools\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In summary, the reference genome, \u003cem\u003eMycobacterium smegmatis mc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e155\u003c/em\u003e (GenBank accession CP000480) was indexed using BWA. The preprocessed reads were aligned to the reference using the BWA-MEM\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e to generate \u0026lsquo;*\u0026rsquo;.sam files. These files were then converted to \u0026lsquo;*\u0026rsquo;.bam format, sorted by coordinates to generate \u0026lsquo;*\u0026rsquo;_sorted.bam\u0026rsquo; files, and subsequently indexed using SAMtools. SAMtools was then used to call raw variants, generating VCF files. The variants were then refined and filtered using BCFtools to produce a high-quality set of polymorphisms (SNPs) and insertion-deletion mutations (indels). Variants were visualized in IGV for manual inspection and quality assessment.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eWhole genome sequences have been submitted to the NCBI SRA database under BioProject ID PRJNA1191427, with BioSample Accession Numbers SAMN45079471 through SAMN45079485.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e\u003cp\u003eThis research was funded (in part) by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases. Nicholaus Mnyambwa was supported by the African Postdoctoral Training Initiative (APTI) Fellowship funded by the US NIH and the Bill \u0026amp; Melinda Gates Foundation in partnership with the African Academy of Sciences, under the auspices of the Coalition of African Research and Innovation.\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e\u003cp\u003eP. F. did conceptual work, and performed recombineering and data analysis. N. M. did conceptual work, and performed recombineering, MAMA-screening and data analysis. H. B. did conceptual work. C. B. did conceptual work.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eWe would like to thank Kenan Murphy for the kind donation of plasmids pKM461 and pKM427.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEllis, H.M., Yu, D., DiTizio, T., Court, D.L.: High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 98, 6742\u0026ndash;6746, doi: (2001). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.121164898\u003c/span\u003e\u003cspan address=\"10.1073/pnas.121164898\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Kessel, J.C., Hatfull, G.F.: Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: characterization of antimycobacterial drug targets. 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In mycobacteria, researchers usually transform bacteria with two oligonucleotides: one conferring the mutation of interest, and a second conferring selectable antibiotic resistance. Multiple genetic modifications may be performed sequentially, however, this requires either the introduction of multiple antibiotic resistances or a tedious process of reversing antibiotic resistance between steps. Rather than using antibiotic resistance for selection, we instead develop a system that uses streptomycin-dependence. Because streptomycin-dependence can easily be selected both for and against, this allows a theoretically unlimited number of recombineering edits to be sequentially selected for, by toggling between streptomycin-sensitivity and dependence. After an even number of editing cycles, strains are automatically unmarked. Strains which have undergone an odd number of edits can be unmarked with a single additional recombineering step. This allows significantly easier exploration of combinatorial interactions between multiple mutations in mycobacteria.\u003c/p\u003e","manuscriptTitle":"Use of Streptomycin-Dependence as Selection/Counterselection for Sequential Oligonucleotide Mediated Recombineering in Mycobacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-19 07:48:42","doi":"10.21203/rs.3.rs-5639512/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cfeb0b95-1062-4563-85a8-743fa7ddeaac","owner":[],"postedDate":"March 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":43031520,"name":"Biological sciences/Microbiology/Pathogens"},{"id":43031521,"name":"Biological sciences/Genetics/Microbial genetics"}],"tags":[],"updatedAt":"2025-03-19T07:48:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-19 07:48:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5639512","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5639512","identity":"rs-5639512","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}