The role of the L421P mutation in Penicillin-Binding Protein 1 (PBP1) in the evolution of chromosomally mediated penicillin resistance in Neisseria gonorrhoeae

The role of the L421P mutation in Penicillin-Binding Protein 1 (PBP1) in the evolution of chromosomally mediated penicillin resistance in Neisseria gonorrhoeae | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The role of the L421P mutation in Penicillin-Binding Protein 1 (PBP1) in the evolution of chromosomally mediated penicillin resistance in Neisseria gonorrhoeae View ORCID Profile Gabriella Gentile , View ORCID Profile Bryan Guzman , View ORCID Profile Adriana Le Van , View ORCID Profile Ann E. Jerse , View ORCID Profile Yonatan H. Grad , Daniel Dominguez , View ORCID Profile Tatum D. Mortimer , View ORCID Profile Robert A. Nicholas doi: https://doi.org/10.1101/2025.06.27.662027 Gabriella Gentile a Department of Pharmacology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gabriella Gentile Bryan Guzman a Department of Pharmacology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bryan Guzman Adriana Le Van b Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences , Bethesda, MD 20814, USA c Henry M. Jackson Foundation for the Advancement of Military Medicine , Inc, Bethesda, MD 20817, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Adriana Le Van Ann E. Jerse b Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences , Bethesda, MD 20814, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ann E. Jerse Yonatan H. Grad d Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health , Boston, MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yonatan H. Grad Daniel Dominguez a Department of Pharmacology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA e Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA f Department of Bioinformatics and Computational Biology Curriculum, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA g RNA Discovery Center, University of North Carolina , Chapel Hill, NC27599, USA h Lineberger Comprehensive Cancer Center, University of North Carolina , Chapel Hill, NC27599, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tatum D. Mortimer i Department of Population Health, College of Veterinary Medicine,University of Georgia , Athens, GA 30602, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tatum D. Mortimer Robert A. Nicholas a Department of Pharmacology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA j Department of Microbiology and Immunology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robert A. Nicholas For correspondence: robert_nicholas{at}med.unc.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract ponA L421P encodes a mutated variant of penicillin-binding protein 1 (PBP1) and is a key resistance determinant that increases the penicillin MIC (MIC PEN ) above the clinical breakpoint in Neisseria gonorrhoeae . Despite the removal of penicillin from treatment guidelines for gonococcal infections in the 1980s, ponA L421P is present in nearly 50% of current N. gonorrhoeae isolates in the PubMLST database. Bioinformatic analysis indicates that ponA L421P is exclusive to N. gonorrhoeae isolates, whereas Leu-421 is 100% conserved in other Neisseria species. To understand the involvement of ponA L421P in antibiotic resistance, we introduced ponA variants encoding 16 different amino acids at position-421 into FA6140, a penicillin-resistant gonococcal isolate that naturally harbors ponA L421P . Proline-421 was the only mutation that increased the MIC PEN to the same level as FA6140. We also assessed the fitness of strains with the 16 mutant ponA alleles over multiple serial passages, both with and without sub-MIC levels of penicillin. There was no fitness defect attributed to ponA L421P under these experimental conditions; instead, our analyses suggest that the widespread occurrence of ponA L421P is driven by its capacity to increase the MIC pen above the clinical breakpoint. In FA6140 transformed with the mosaic penA allele from strain H041, a ceftriaxone-resistant isolate, ponA L421P increased the MIC of ceftriaxone, suggesting that ceftriaxone targets PBP1 in this strain. We conclude that the ponA L421P allele emerged in gonococcal isolates, increasing the MIC PEN above the clinical breakpoint, and has remained in the population even after the removal of penicillin from treatment guidelines. Importance The emergence of antibiotic-resistant Neisseria gonorrhoeae threatens effective treatment of gonorrhea, one of the most common sexually transmitted infections worldwide. Understanding the genetic changes that drive and maintain resistance is crucial for anticipating future resistance trends. Here, we investigated the impact of a key resistance mutation in PBP1 (encoded by ponA L421P ). Although penicillin has not been used to treat gonorrhea for decades, this mutation remains widespread even in recent N. gonorrhoeae isolates. ponA L421P confers clinically relevant penicillin resistance without imposing an in vitro fitness cost. ponA L421P also increases resistance to ceftriaxone in strains with penA alleles that are associated with ceftriaxone resistance. This work highlights the role of the ponA L421P allele in shaping the current antibiotic resistance landscape and supports the need for ongoing surveillance and evolutionary studies of such mutations in the gonococcal population. Introduction Neisseria gonorrhoeae is the etiologic agent of the sexually transmitted infection gonorrhea, which causes ∼80 million infections annually ( 1 , 2 ). The β-lactam antibiotic penicillin was first used to treat gonorrhea in the early 1940s. For the following 40 years, penicillin remained effective, but the minimum inhibitory concentrations of penicillin (MIC PEN ) steadily increased, requiring higher doses to cure infections ( 3 ). By 1985, over 5% of circulating N. gonorrhoeae strains had MIC PEN values above the breakpoint (≥2 μg/ml), prompting a switch in treatment guidelines to other antibiotics. The rise in penicillin resistance was driven by two mechanisms: plasmid-mediated production of penicillinases (TEM-1 and TEM-135 β-lactamases) and expression of resistance determinants that are variants of chromosomal genes ( 4 – 11 ). In the US, the percentage of penicillinase-producing N. gonorrhoeae isolates (PPNG) decreased by the 1990s, while the percentage of chromosomally mediated penicillin-resistant N. gonorrhoeae isolates (CMRNG) increased ( 2 , 8 ). β-lactam antibiotics target penicillin-binding proteins (PBPs), which are involved in the cross-linking of the peptide chains of peptidoglycan. N. gonorrhoeae has four PBPs, two of which (PBP1 and PBP2) are essential. PBP1 is a bifunctional PBP that catalyzes both glycan polymerization (transglycosylation) and peptide cross-linking (transpeptidation), and PBP2 is a monofunctional PBP, catalyzing transpeptidation reactions. Because PBP2 has a much higher rate of acylation than PBP1 for penicillin and other β-lactam antibiotics used to treat N. gonorrhoeae in susceptible strains, PBP2 is considered the lethal target for these antibiotics. CMRNG is facilitated by the step-wise transfer ( 12 – 14 ) of at least four resistance determinants from a penicillin-resistant strain to an antibiotic-susceptible strain: 1) mutant penA alleles, which encode variants of PBP2 that reduce the acylation rate of penicillin by 16-fold ( 15 ); 2) mtr mutations, which increase mtrCDE operon expression and result in increased penicillin efflux through the MtrCDE efflux pump ( 9 , 16 ); 3) mutations in porB , which encodes the major outer membrane porin, that reduce the influx of penicillin into the periplasm ( 7 , 17 ); and 4) ponA L421P , encoding a PBP1 variant with an L421P missense mutation. Mutations in penC were thought to contribute to high-level penicillin resistance ( 8 ), but penC was later shown to encode the pore-forming secretion protein, PilQ ( 18 ). These pilQ mutations compromise its secretin function and increase the MIC of multiple antibiotics ( 18 , 19 ), but to date, pilQ mutations have not been observed in clinical isolates, as they disrupt the type IV pili that play a key role in gonococcal pathogenesis ( 20 , 21 ). While the first three resistance determinants are well characterized in the literature, the contribution of ponA L421P to penicillin resistance remains less well understood. Our laboratory has shown that the L421P mutation in PBP1 lowers the acylation rate by 2– to 3-fold for penicillin G and other β-lactam antibiotics ( 8 ), but it is not clear whether this lower acylation rate explains the role of ponA L421P in facilitating penicillin resistance. Transformation of the penA, mtr, and porB determinants from strain FA6140, a high-level-penicillin-resistant clinical isolate (MIC PEN = 4 µg/ml), into the antibiotic-susceptible strain FA19 (MIC PEN = 0.01 μg/ml) results in MIC PEN between 0.5 to 1.0 µg/ml ( 12 , 13 ), but further transformation with FA6140 DNA or ponA L421P does not increase the MIC PEN ( 22 ). All attempts to reach donor levels of resistance in gonococcal isolates by natural transformation have been unsuccessful ( 8 , 10 , 12 ). By contrast, we showed that reversion of ponA L421P in two penicillin-resistant clinical isolates, including FA6140, back to the wild-type ponA allele decreases the MIC PEN 2-fold ( 22 ). These data imply that ponA L421P requires a yet to be determined genetic background that cannot be transferred between strains by transformation to exert its phenotypic effect on resistance. We hypothesized that ponA L421P arose during the years of penicillin use and has continued to remain in the population because ponA L421P continues to confer a fitness benefit, even with the current β-lactam antibiotics recommended for use. In the present study, we sought to test this prediction by investigating the origins of ponA L421P to elucidate the relationship between ponA L421P and the evolution of β-lactam resistance in N. gonorrhoeae . Results ponA L421P is exclusive to N. gonorrhoeae among Neisseria species N. gonorrhoeae isolates can acquire resistance-conferring mutations from commensal Neisseria species and from N. meningitidis ( 23 – 25 ). We examined whether ponA L421P arose via transfer of DNA from other Neisseria species, or if this mutation is specific to gonococcal isolates. We accessed the sequences of all unique ponA alleles across Neisseria species from the PubMLST Database, and divided them into three groups: ( 1 ) N. gonorrhoeae (n = 20,513 isolates, 172 ponA alleles), ( 2 ) N. meningitidis (n = 75,612 isolates, 897 ponA alleles), and ( 3 ) all other commensal Neisseria species (n = 1,604 isolates, 369 ponA alleles). The PBP1 protein sequences across all three groups of Neisseria isolates are highly conserved (Fig. S1), particularly the region surrounding residue 421 ( Fig. 1 ). Leu-421 is 100% conserved across all N. meningitidis and commensal Neisseria ponA alleles ( Fig. 1B-C ), but for N. gonorrhoeae isolates, residue 421 is either a leucine or a proline ( Fig. 1A ). Moreover, ponA L421P was observed in 49.5% of all gonococcal isolates in the PubMLST database and was not found in other Neisseria species (Table S5). These data strongly suggest that ponA L421P arose in N. gonorrhoeae and not through horizontal gene transfer from other Neisseria species. Download figure Open in new tab FIG 1 Sequence logo diagrams comparing the conservation of positions 415-430 in PBP1 from various Neisseria isolates. Diagram generated on WebLogo with MUSCLE-aligned ponA sequences. Sequences were downloaded from PubMLST and are a representative sub-set of all of the ponA alleles documented for each Neisseria species group: N. gonorrhoeae (n=172), N. meningitidis (n=897), and Neisseria spp. (n=369). The X-axis indicates the position of the amino acid. The height of the logo element (letter) represents its log-transformed frequency displayed in bits of information, and the overall height of the stack indicates the sequence conservation at that position. ponA L421P is prevalent amongst gonococcal isolates with chromosomally-mediated penicillin resistance To investigate the emergence and distribution of ponA L421P in the gonococcal population, we assembled a dataset of sequenced N. gonorrhoeae isolates and the associated MIC PEN values (n=16,891). The first appearance of ponA L421P in a sequenced isolate was in 1961, and it rapidly expanded in the population in the 1980s ( Fig. 2 ). Interestingly, we first observed the insertion of aspartate after position 345 in penA (Asp345a), which is a hallmark of early penicillin-resistant isolates ( 26 , 27 ), in our dataset of sequenced isolates in 1956, only a few years prior to the L421P mutation in ponA ( Fig. 2 ). We examined the phylogenetic distribution of ponA alleles in a representative sample of N. gonorrhoeae isolates without plasmid-mediated resistance (n=5,192) and found ponA L421P emerged across multiple lineages in the gonococcal population ( Fig. 3 ). Download figure Open in new tab FIG 2 Historical analysis of N. gonorrhoeae isolates (n=16,891). Isolates are represented by the year the isolate was collected. Asp345a refers to the insertion of asparagine at position 345 in penA (encodes PBP2), a known penicillin-resistance-conferring mutation. Count represents the number of isolates that harbor ponA (blue) or ponA L421P (red). Download figure Open in new tab FIG 3 Phylogenetic distribution of ponA alleles in a representative sample of N. gonorrhoeae isolates without plasmid-mediated resistance (n=5,192). The outer annotation ring indicates the presence of ponA (L421, grey) and ponA L421P (L421P, black). The inner ring indicates the level of penicillin resistance. S is susceptible (≤0.06 μg/mL), I is intermediate (0.25 – 1 μg/mL), and R is resistant (≥2 μg/mL) breakpoints of PenG as defined by the Clinical and Laboratory Standards Institute (CLSI) ( 83 ). We next examined the relationship between ponA L421P and penicillin-resistant CMRNG strains ( Fig. 4 , n=8,999). Less than 1% of isolates harboring the wild-type ponA allele included in Fig. 4 have resistant MIC PEN values (≥2 μg/mL), while the majority (90%) fall within the intermediate MIC PEN range (0.12–1 μg/mL). In contrast, 25% of isolates harboring ponA L421P have resistant MIC PEN (≥2 μg/mL), and 75% of isolates have intermediate MIC PEN . Few isolates, regardless of the ponA or ponA L421P allele, had susceptible MIC PEN. Additionally, 91.61% of isolates that harbor mosaic penA alleles, which encode highly remodeled PBP2 variants that confer increased resistance to cephalosporins, also harbor ponA L421P ( Fig. 4B ). Download figure Open in new tab FIG 4 Distribution of MIC values for penicillin G (PenG) among N. gonorrhoeae isolates (n = 8,999). Strains are separated by harboring either (A) a non-mosaic penA allele, or (B) a mosaic penA allele. The X-axis shows the PenG MIC (MIC PEN ), and dotted lines indicate the susceptible (≤0.06 μg/mL), intermediate (0.25 – 1 μg/mL), and resistant (≥2 μg/mL) breakpoints of PenG as defined by CLSI ( 83 ). The Y-axis shows the number of isolated N. gonorrhoeae strains for each MIC PEN , value. Red bars show distribution of strains with the ponA L421P allele and blue bars shows the number of strains with the wild-type ponA (L421) allele. The mosaic penA alleles in isolates were defined and typed according to the NG-STAR database ( 56 ). Identification of other polymorphisms in gonococcal ponA Amongst N. gonorrhoeae isolates in PubMLST, there were only two other mutations observed in gonococcal PBP1 at frequencies above 1%: A375T (7.07%) and Y537C (2.35%) ( Fig. 5 ). In keeping with these frequencies, a phylogeny of 6,082 representative gonococcal genomes reveals these mutations are present in fewer lineages, compared to the phylogenetic distribution of the L421P mutation (Fig. S2). We next examined the relationship between each mutation and penicillin-resistant CMRNG strains (n=8,999). The majority (95%) of isolates harboring the A375T mutation in ponA have intermediate (0.12–1 μg/mL) to resistant (≥ 2 μg/mL) MIC PEN. This proportion does not change in isolates with alanine at residue 375 of ponA , as 95% of these isolates also have intermediate to resistant MIC PEN . The same trend is observed with the Y537C mutation. We do not observe A375T and Y537C in sequenced isolates collected during the penicillin treatment era, as they first appear in 1998 and 2001, respectively, well after penicillin was removed as a treatment for gonorrhea infections in the US in 1985. It remains unclear whether A375T and Y537C are directly involved in facilitating resistance to penicillin, but, given the fact that isolates with those mutations are not enriched for penicillin resistance and they are present in fewer gonococcal lineages than L421P, we did not interrogate them further. Download figure Open in new tab FIG 5 Polymorphisms of N. gonorrhoeae PBP1 mapped onto the predicted protein structure. Polymorphisms are colored according to their prevalence in the gonococcal population. Only polymorphisms detected at frequencies greater than 1% are shown: L421P (47.15%), A375T (7.07%), and Y537C (2.35%). The Transpeptidase (TPase), Transglycosylase (TGase), and the Oligosaccharide-Binding (OB) domains of PBP1 are labeled, with the TPase active site residues marked in red. MUSCLE-aligned sequences of gonococcal PBP1 were taken from PubMLST isolate database in order to identify polymorphisms and calculate population frequencies, see Materials and Methods. Predicted structure of gonococcal PBP1 is from the AlphaFold Protein Structure Database (AFDB). Only proline and serine at PBP1 position 421 increase the MIC PEN To understand why only a proline mutation at residue 421 is observed in PBP1, we created an isogenic library of 16 mutant strains in FA6140, each with a different amino acid at position 421 ( Table 1 ). FA6140 (MIC PEN = 4 μg/mL) naturally harbors ponA L421P , and reversion to ponA 421L reduces the MIC PEN to 2 μg/mL ( 8 , 22 ) ( Table 1 ). In the strain library, FA6140 ponA L421P was the only strain with an MIC PEN of 4 μg/mL. FA6140 ponA L421S , which had a median MIC PEN of 2.5 μg/mL, was the only other mutation that increased the MIC PEN > 2 μg/mL, with all the other strains having the same MIC PEN as FA6140 ponA ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1 FA6140 ponA L421X Isogenic Mutant Library and penicillin MICs. In vitro serial passaging of the FA6140 ponA L421X mutant library in the absence or presence of sub-MIC concentrations of penicillin The 2-fold higher MIC PEN conferred by ponA L421P and the timing of its first appearance in sequenced isolates suggest that ponA L421P emerged in response to selection by penicillin. To test this, we performed in vitro serial passaging on our set of FA6140 ponA L421X mutants in increasing sub-MIC PEN concentrations (0.25, 0.5, and 1.0 μg/ml). Illumina sequencing revealed that equal amounts of each FA6140 ponA L421X mutant were used to inoculate the cultures at hour 0 across all 3 trials ( Fig. 6A , Fig. S3). In the absence of penicillin, the amino acids proline, cystine, asparagine, and glycine at position 421 appear to be more fit in this growth environment, as they have the largest fold increase in read counts at hour 36 relative to the inoculum ( Fig. 6C ). The ponA L421P reads consistently doubled by hour 36 in all three trials, while the ponA 421L reads remained similar to, or decreased from, the number of reads in the inoculum. The proline and serine mutants were the only strains with an MIC PEN above 2 μg/mL ( Table 1 ), but the read counts for the serine mutant at hour 36 did not surpass its read counts at hour 0 ( Fig. 6C ). None of the strains differed significantly in growth rate from the parental strain (Table S4). Download figure Open in new tab FIG 6 Optical densities (OD 600 ) and Illumina read counts from in vitro serial passaging of the FA6140 ponA L421X mutant library in the absence or presence of sub-MIC concentrations of penicillin. (A) Percentage of reads, relative to the total number of reads, for each mutant at hour 0 (inoculum). The inoculum was used to inoculate each culture at 0.08 OD 600 . (B) The serial passaging scheme included a total of 4 cultures, 3 of which were supplemented with different sub-MIC concentrations of penicillin (0.25, 0.5, and 1.0 µg/mL), and one with no penicillin added. Cultures were passaged every 6 hours, for a total of 36 hours, always with a starting OD 600 of 0.08. (C) OD 600 and (D) Illumina read counts of the cultures with no penicillin, compared to cultures supplemented with 0.5 µg/mL penicillin. (D) Reads for each mutant in the inoculum were normalized to 1, as indicated by the dotted line at y=1. Bars represent read counts for each mutant at hour 36, relative to their inoculum value. This experiment was performed 3 separate times (trials) and each trial represents a separate Illumina sequencing run; (C-D) Trial 1 is shown in red, trial 2 in yellow, and trial 3 in blue. The pie chart in (A) is a representative figure from trial 1, but all 3 trials had similar results. See supplemental for read counts from the cultures supplemented with 0.25 µg/mL of penicillin (Fig. S4) and for the inoculum used in trials 2 and 3 (Fig. S3). We grew the collection of strains in growth medium with increasing concentrations of penicillin to examine which strains would grow in the presence of selective pressure. Optical Density at 600 nm (OD 600 ) values during growth in medium containing 0.25 mg/ml penicillin were very similar to no antibiotic (Fig. S4), while there was more variability in the OD 600 values across trials for the cultures supplemented with 0.5 μg/mL of penicillin ( Fig. 6B ). Of the 16 mutants, the two with the highest MIC PEN , FA6140 ponA L421P and FA6140 ponA L421S , increased in read counts when grown in 0.5 μg/mL penicillin, while all the others decreased in read counts. FA6140 ponA L421P consistently increased in read counts across all three trials, while read counts for FA6140 ponA L421S increased in trials 1 and 2, but not in trial 3. In trial 1, FA6140 ponA L421P had more read counts at 36 h than FA6140 ponA L421S , and both strains had similar reads counts at 36 h in trial 2 ( Fig. 6C ). FA6140 ponA L421P is the only mutant that consistently increased in read counts over 36 h in cultures either with or without sub-MIC PEN concentrations. At the highest sub-MIC PEN concentration tested, we observed more variable results. After 6 h of growth and passaging into fresh medium with 1 μg/ml penicillin, the OD 600 increased very little over the next 12 h ( Fig. 7A ). However, in two of the three trials (1 & 3), there were still live cells present, as later passages showed measurable growth during the 6 h period. These data suggest that gonococcal cells remained viable but were not dividing during the prior 6 h growth periods and eventually acquired either phenotypic or genetic changes that allowed for measurable growth at the later growth periods. In trial 1, we observed increasing growth during consecutive passages, beginning at 18 h. Illumina sequencing of the ponA region revealed that serine was the predominant mutation at position 421 ( Fig. 7B ). Download figure Open in new tab FIG 7 A pilQ mutation arose in the culture supplemented with 1.0 ug/ml of penicillin. Optical densities (A) and Illumina read counts (B) for the FA6140 ponA L421X strains grown in cultures supplemented with 1.0 µg/mL of penicillin. (B) Read counts for each mutant at hour 36, relative to their inoculum (Hr 0) value. Inoculum reads for each mutant were normalized to 1, as indicated by the dotted line at y=1. (C) Read counts over time for FA6140 ponA L421S (orange) FA6140 ponA L421P (black), relative to their inoculum value (y=1), during trials 1 and 3. Each trial represents a separate Illumina sequencing run (A-B, trial 1 = red, trial 2 = yellow, trial 3 = blue). Cells growing in the culture from trial 2 appeared to die at hour 18 (A; yellow line), resulting in poor sequencing reads. See supplemental Fig. S5 for trial 2 data. Whole genome sequencing of cells from in vitro serial passaging FA6140 ponA L421X mutant library reveals emergence of a mutation in pilQ To determine if the FA6140 ponA L421X mutants acquired additional mutations during the 36 hours of in vitro growth, we sequenced the genomes of cells collected at 0 h and 36 h. We looked for mutations that were distinct from the inoculum and present in greater than 20% of the reads, as anything detected below this would likely not be responsible for any changes in the overall codon counts from the Illumina reads ( Figs. 6D , 7B ). Across all 3 trials, most, but not all, mutations above the 20% threshold were either errors in the reference genome sequence used for alignment (not true genetic changes), also present in the inoculum, or in genes that would most likely be inconsequential to resistance. In trial 1, we detected a mutation in pilQ at 36 h at 85% frequency in the culture supplemented with 1.0 μg/mL of penicillin. The mutation was an insertion of two bases (+CG) at position 459, which would result in a frameshift and early termination. Previous work from our lab showed that missense and frameshift mutations in pilQ increase MIC PEN by inactivating PilQ and decreasing permeation of drug into the gonococcal cell ( 8 , 18 , 19 , 28 ), which likely explains the marked increase in codon counts for the serine mutation at 24 h seen in trial 1 ( Fig. 7C ). Our data suggest that during trial 1, FA6140 ponA L421S acquired a mutation in pilQ between hours 18 and 24, which allowed it to expand in the culture compared to FA6140 ponA L421P . ponA L421P increases the ceftriaxone MICs of gonococcal isolates that also harbor a mosaic penA allele Our data indicate that ponA L421P is involved in penicillin resistance, but penicillin was removed as a recommended treatment in 1985, and ceftriaxone has been the only recommended β-lactam antibiotic since 2012 for treating gonococcal infections. Given that ponA L421P appears to confer a modest fitness deficit in vivo ( 29 ), it might be expected that ponA L421P would decrease in prevalence without selection by penicillin use. However, ponA L421P is observed in a high percentage of isolates with a mosaic penA allele (91.61%, Fig. 5B ), which is the predominant determinant for decreased susceptibility to ceftriaxone ( 30 , 31 ). One possibility to explain the prevalence of ponA L421P is that ponA L421P influences the MIC of ceftriaxone in the presence of a mosaic penA allele. To test this hypothesis, we replaced the penA4 allele in FA6140 and FA6140 ponA strains with penA41 , the mosaic allele from the multi-drug resistant gonococcal isolate, H041 ( 32 ). This allele encodes a PBP2 with a markedly reduced acylation rate for penicillin, cefixime, and ceftriaxone ( 31 , 33 , 34 ). Reversion of ponA L421P to wild-type ponA reduced the MIC PEN in both strain backgrounds, regardless of the penA allele ( Fig. 8A ), suggesting that PBP1 is the lethal target of penicillin in these strains. While reversion of ponA L421P to ponA in strain FA6140 had no effect on the MIC of ceftriaxone ( Fig. 8B ), reversion of ponA L421P to ponA in strain FA6140 penA41 caused a 3-fold decrease in the MIC of ceftriaxone, suggesting again that PBP1 is the lethal target of ceftriaxone in this strain. Download figure Open in new tab FIG 8 MICs of penicillin G and ceftriaxone for FA6140 transformants. The MICs reported above each bar are the median values (± the standard deviation) determined from three independent experiments. Data points within each bar represent the MIC values measured during each experiment. Discussion In this study, we sought to understand the origins and role of the PBP1-L421P mutation (encoded by the ponA L421P allele) in β-lactam antibiotic resistance in N. gonorrhoeae. Our data revealed that ponA L421P is found exclusively in N. gonorrhoeae and its emergence in gonococcal isolates is likely due to it increasing the MIC PEN above the clinical breakpoint of 2 µg/ml in CMRNG. While ponA L421P has a modest fitness cost in vivo ( 29 ), there is no apparent fitness cost during growth in vitro . Our data suggest that this cost turns into a fitness benefit during growth in sub-MIC PEN concentrations. Moreover, while penicillin G is no longer used to treat gonococcal infections, ponA L421P also increases the MIC of ceftriaxone in strains harboring mosaic penA41 alleles that confer ceftriaxone resistance. Thus, the increases in MIC PEN (in FA6140) and MIC CEF (in FA6140 penA41 ) that result from harboring ponA L421P appear to have provided enough of an evolutionary advantage to remain in the population despite its modest fitness cost. Mosaic penA alleles are defined by a sub-set of mutations that surround the active site of PBP2 and decrease the conformational dynamics of the PBP, which reduces acylation rates and increases MICs for various β-lactam antibiotics ( 15 , 30 , 31 ). These mutations emerged in N. gonorrhoeae through interspecies recombination events with Neisseria commensals. In contrast, ponA L421P appears to have arisen spontaneously in N. gonorrhoeae, and, unlike resistance-conferring mutations in PBP2, L421P is located on a hinge that links the oligosaccharide-binding domain to the start of the transpeptidase domain in PBP1 and is over 20 Å from the active-site serine residue ( Fig. 5 ). How the L421P mutation decreases acylation is unknown, and compared to mosaic PBP2 variants, the effects on k 2 /K S are modest ( 8 ). The small decrease in acylation, however, could explain the 2-fold increase in the MIC PEN . While ponA L421P was the only mutation in FA6140 to increase the MIC PEN to 4 μg/ml, another mutation, ponA L421S , increased the MIC PEN to 2.5 μg/mL. However, to date ponA L421S has not been documented in clinical isolates. This could be due to three reasons: 1) the L421P mutation requires only a single base change in the Leu-421 codon in ponA (C T G ➜ C C G), whereas the L421S mutation would require at least two base changes (CTG ➜ TCT / TCC / TCA / TC G/ AGT / AGC ); 2) strain FA6140 ponA L421S is less fit in vitro compared to other strains; and 3) the increase in resistance is insufficient to overcome its fitness cost. Following the six serial passages of the 16 mutants, we sequenced the genomes of the 36-hour cultures from all three biological replicates to be sure that the results were not skewed by the acquisition of random mutations, particularly under antibiotic selective pressure. No consistent or biologically relevant mutations were detected, with the exception of a single frameshift mutation in pilQ observed in the culture supplemented with 1.0 μg/mL of penicillin during trial 1. PilQ increases the MICs of a range of antimicrobials in N. gonorrhoeae strains that also have the mtr and porB resistance determinants ( 18 , 19 ), which decrease the concentration of antibiotics in the periplasm by increasing efflux and decreasing influx of antibiotics, respectively. This implies that permeation through the PilQ secretin is low and does not impact the MIC unless both mtr and porB determinants are present, as they are in FA6140 ( 3 , 8 , 18 , 22 , 35 ). Based on the increase in read counts for ponA L421S at 24 hours, we surmise that the PilQ mutation arose in FA6140 ponA L421S sometime between 18-24 hours of growth, which increased its MIC PEN above that of FA6140 ponA L421P and allowed it to survive in higher penicillin concentrations. However, this is an outcome that would likely occur only during in vitro growth, as expression of type IV pili (of which PilQ is an essential component) is important for N. gonorrhoeae survival during pathogenesis ( 36 ). This is supported by the absence of inactive PilQ mutants identified in clinical isolates ( 37 , 38 ). We recently reported that N. gonorrhoeae lineages harboring ponA L421P between 1993 and 2013 were associated with a modest fitness disadvantage, compared to the baseline type (lineages that do not carry any resistance determinants), suggesting that ponA L421P imposes a fitness cost ( 29 ). These data were consistent with results from competitive co-infections between ponA – and ponA L421P -containing strains of FA19 and FA6140 ( 29 ). Despite this observed fitness cost, we showed here that ponA L421P remains prevalent in the gonococcal population and is present in over 90% of isolates that also harbor a mosaic penA allele (as classified by the NG-STAR database). Why is ponA L421P found so frequently in strains with a mosaic penA allele? There are several possible explanations for this: 1) mosaic penA alleles emerged by transformation almost exclusively into penicillin-resistant strains that already had ponA L421P , as these strains also contain other resistance determinants and presumably compensatory mutations that lessen their fitness cost; 2) with penicillin resistance at or near 100% in Asia ( 39 ), where ceftriaxone resistance has been emerging, the link between ponA L421P and ceftriaxone resistance may reflect the gonococcal diversity/population structure in the places experiencing the most selection for ceftriaxone resistance; and 3) ponA L421P confers additional resistance to ceftriaxone and thus provides a benefit to these strains, given the widespread use of ceftriaxone to treat current infections. We tested the latter possibility by determining the MIC CEF values of FA6140 and FA6140 ponA also harboring the mosaic penA41 allele from H041, which confers ceftriaxone resistance to these strains. Importantly, the MIC CEF drops ∼3-fold when ponA L421P is replaced with ponA , indicating that ponA L421P confers additional resistance to ceftriaxone and potentially explaining, at least in part, why ponA L421P is so prevalent in strains also harboring mosaic penA alleles, particularly with mosaic penA alleles that confer the largest increases in MIC CEF . The MIC data we present in this study can be best interpreted by knowing which PBP is being targeted by penicillin and ceftriaxone ( Fig. 9 ). The acylation constant k 2 /K S for each antibiotic determines which PBP first becomes acylated as the antibiotic concentration increases (the higher the k 2 /K S , the lower the concentration of antibiotic needed to inhibit the PBP). Thus, k 2 /K S and the MIC have an inverse relationship, and the essential PBP that first becomes acylated as the antibiotic concentration increases is the killing target for that antibiotic and determines the MIC. For penicillin, the killing target for FA6140 is likely a combination of PBP2 FA6140 and PBP1 L421P , but when PBP1 L421P is replaced by PBP1, the MIC decreases because PBP1 becomes the exclusive killing target of penicillin. We showed previously that in 35/02, a ceftriaxone-intermediate resistant (and penicillin-resistant) gonococcal isolate, reversion of ponA L421P to ponA did not alter MIC CEF values ( 40 ). In contrast, expression of PBP2 H041 in FA6140, which has a much lower k 2 /K s for ceftriaxone than PBP2 35/02 , shifts the killing target to PBP1 L421P . Thus, exchanging PBP1 L421P for PBP1 in the FA6140 genetic background decreases MIC CEF by 3-fold, suggesting that when strains acquire a “strong” mosaic PBP2 variant, e.g. PBP2 H041 , that has a sufficiently low acylation rate constant for ceftriaxone, the target shifts from PBP2 to PBP1 and ponA L421P confers a selective advantage over ponA . Download figure Open in new tab FIG 9. Diagram outlining the drug targets for penicillin and ceftriaxone in gonococcal strains with different penA and ponA alleles. PBP1 variants are marked by green rectangles and PBP2 variants are marked as purple rectangles. The left axis indicates the acylation rate, and the right axis indicates the MIC of the indicated strain. Arrows point to the killing target for each of the indicated strains. A). Penicillin has a low k 2 /K S for all PBP2 variants, so it kills gonococcal cells by inhibiting PBP1. Replacing PBP1 L421P with PBP1 WT decreases the MIC. B). Ceftriaxone normally kills by inhibiting PBP2, but PBP2 H041 has a much lower k 2 / K S and this shifts the killing target to PBP1 (and maybe some PBP2 H041 ). Replacing PBP1 L421P with PBP1 WT decreases the MIC. In conclusion, this is the first study to investigate the origins of ponA L421P in the context of penicillin resistance and address why it is maintained in the N. gonorrhoeae population. During the 40+ years of penicillin use, N. gonorrhoeae strains acquired β-lactamases, as well as mutations in PBP1, PBP2, mtr , and porB , which eventually increased the MIC PEN enough to result in its removal as a treatment option. When expanded-spectrum cephalosporins (ESCs) were introduced to treat gonococcal infections, mosaic PBP2 variants emerged (because PBP2 was the exclusive killing target of ESCs), in existing penicillin-resistant strains, resulting in the proliferation of mosaic penA alleles that increased resistance to ESCs. Over time, the MICs of ESCs for gonococcal isolates increased as the mosaic penA alleles picked up additional mutations, and the killing target shifted to PBP1, allowing the L421P mutation to further increase the MIC. These findings reinforce the need to identify antibiotics that are PBP1-selective and thus can avoid the highly mutated mosaic PBP2 variants that are compromising the use of ESCs. β-lactam antibiotics that target both PBPs or a combination of two β-lactam antibiotics that target PBP2 and PBP1 may provide a new strategy for treating strains with mosaic penA alleles. Methods Strains and plasmids Strains, plasmids, and primers used in this study are listed in Tables S1 and S2. To aid in subsequent screening for generating random mutations at codon-421, we transformed FA6140 with the ponA allele from FA19, which naturally has a PstI site encompassing codons 421 and 422. A portion of the ponA gene (bp 956 – 2400) from FA19 genomic DNA was amplified via PCR and transformed into FA6140 via spot transformation, performed as described ( 41 ). Colonies were screened for ponA by PCR amplification of ponA and digestion with PstI. To generate FA6140 and FA6140 ponA harboring the mosaic penA allele ( penA41 ) from the ceftriaxone-resistant strain H041, piliated cells were transformed with pUC18us- penA41 plasmid containing penA41 starting at bp 133 and ending 200 bp downstream penA41 to facilitate recombination. Transformants that acquired penA41 were selected on GCB agar plates containing 1.0 !g/mL cefixime. All transformants were verified by PCR and Sanger sequencing. Descriptions of the cloning plasmids used to create the library of isogenic FA6140 ponA L421X mutants can be found in Table S3. pUC18us- ponA 421X -Ω contains a portion of the ponA gene (from bp 831 to 54 bp downstream of the stop codon) harboring randomized codons to introduce amino acid substitutions at codon-421, the aad1 resistance cassette (Ω) conferring spectinomycin/streptomycin resistance ( 42 ), and 531 bp of sequence downstream of ponA to facilitate recombination. We employed overlap-extension ( 43 ) PCR mutagenesis to introduce amino acid substitutions at codon-421 in ponA fragments, using the primers listed in Table S2. pUC18us- ponA 421X -Ω plasmids were created using the NEBuilder HiFi DNA Assembly Master Mix and transformed into Escherichia coli XL-10 Gold cells, according to manufacturer’s protocol. Clones were screened by PCR amplification of the ponA gene followed by Sanger sequencing. Plasmids containing unique mutations were isolated using a QIAPrep miniprep kit (Qiagen). Plasmids (Table S3) were transformed into FA6140 ponA by allelic exchange, and transformants were selected on GCB agar plates containing 25 !g/mL spectinomycin. Individual colonies from each transformation were screened by PCR by amplifying the ponA gene and digesting with PstI (loss of PstI indicated the presence of the mutation). All clones were verified by Sanger sequencing. PCR reaction conditions were as follows; 94°C for 30s (denaturation), 60°C for 30s (annealing), and 72°C for 60s (elongation) for 35 cycles, and fragments were purified using the QIAquick PCR Purification kit (Qiagen) as per manufacturers guidelines. All gonococcal strains were propagated on solid GCB agar containing Kellogg’s supplement I ( 38 ) and 12 mM Fe(NO 3 ) 3 for 18 to 20 h at 37°C in a 5% CO 2 -enriched humidified atmosphere. Serial passaging was minimized to reduce the risk of acquiring secondary mutations. Bacterial growth FA6140 ponA L421X mutants were grown in liquid culture to verify individual growth phenotypes (Table S4). Nonpiliated bacterial colonies were swabbed from GCB agar grown for 16 h and inoculated into GCB (per liter: 15 g protease peptone 3, 4 g K 2 HPO 4 , dibasic, 1 g KH 2 PO 4 , 5 g NaCl) supplemented with Kellogg’s supplement I ( 38 ) and 12 mM Fe(NO 3 ) 3 at a starting optical density at 600 nm (OD 600 ) of 0.08. Cultures were shaken at 180 rpm at 37°C in a 5% CO 2 -enriched atmosphere, and bacterial growth was assessed by measuring the OD 600 at hourly intervals for a total of 8 h. Experiments were repeated in biological triplicate, and statistical significance between individual strains was determined by using a repeated-measures 2-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Differences in growth rate were measured by comparing the average time to reach an OD 600 of 0.8 for each strain as previously described ( 44 ). Results were compared using a one-way analysis of variance to determine overall significance followed by Dunnett’s multiple comparison test to determine significance between individual strains. Neisseria sequence diversity analysis The datasets used to identify polymorphisms and assess overall sequence diversity of the ponA gene across all Neisseria species were all exported from the Neisseria page of the PubMLST data base (last accessed September 30, 2024) ( 45 ). Allele mining analysis of the ponA gene was performed as described ( 46 ). An individual search of the profiles was made via the locus/sequence definitions tool to identify the locus assigned to ponA (NEIS0414). Data was exported from the two-field breakdown section of the Neisseria isolate database as a Microsoft Excel file, which provided a list of unique ponA allelic profiles and a count for how many isolates possess that unique allele within each Neisseria spp. Microsoft Excel was used to calculate the number of isolates that possess each allele. Mutations identified in the ponA alleles from N. gonorrhoeae isolates with frequency greater than 5% were mapped onto the predicted PBP1 structure, which was generated using Alphafold ( 47 ). The PDB file for PBP1 from N. gonorrhoeae (UniProt: O05131) was downloaded from the AlphaFold Protein Structure Database (AFDB) ( 48 , 49 ) and mutations detected frequencies greater than 5% were mapped to the structure using Pymol (2.0.7) ( 50 ). ponA sequences from PubMLST were exported using the script available in the allele/sequence definition database. Molecular Evolutionary Genetics Analysis software, version 7 (MEGA7) ( 51 ) was used to translate the sequences and perform a multiple sequence comparison by log-expectation (MUSCLE) ( 52 ). Sequence logo images were created using WebLogo ( 53 ). MUSCLE alignments of unique ponA alleles from PubMLST were uploaded to WebLogo, background composition of proteome was set to equiprobable, and y-axis units were set to bits. Genomic Analyses Genome assembly and typing. Publicly available N. gonorrhoeae genomic data with available MICs of penicillin were downloaded from the European Nucleotide Archive ( 54 – 72 ). Resistance-associated SNPs were determined from variant calls produced by Pilon v 1.23 ( 73 ) after mapping to the NCCP11945 reference genome (NC_011035.1) using BWA-MEM v 0.7.17 ( 74 ). Mosaic alleles and the presence or absence of resistance-associated accessory genes (e.g. bla TEM ) were identified using blastn v 2.14.0 ( 75 ) searches of de novo assemblies generated with SPAdes v 3.12.0 ( 76 ). Mosaic penA alleles were typed according to the NG-STAR database ( 77 ). Genomic data was included if the total de novo assembly length was within the range of expected lengths for N. gonorrhoeae (1.89 Mb – 2.31 Mb) and that the number of contigs was less than 250. We also required more than 40X coverage of the reference genome, that at least 80% of reads mapped to the reference genome, and less than 12% of positions in the reference genome had ambiguous calls defined as at least 90% of reads supporting either the reference or alternate nucleotide. Lastly, we removed isolates with ambiguous calls in genes that contribute to penicillin resistance, including ponA, penA , and the mtr operon. Phylogenetic analysis. An initial alignment and core genome phylogeny was estimated using ParSNP v 2.0.5 ( 78 ) from 10,282 gonococcal de novo assemblies. To reduce the computational resources needed for a recombination-corrected phylogenetic analysis, we used Treemmer v 0.3 ( 79 ) to prune the ParSNP tree to 99.9% of the original tree length, removing the most closely related pairs of isolates from our dataset. For this representative subset of 6,082 isolates, pseudogenomes were generated from variant calls and concatenated to produce an alignment for recombination detection and phylogenetic analysis with Gubbins v 3.3.3 ( 80 ). Phylogenetic trees were visualized using iTOL ( 81 ). We additionally pruned the dataset to remove isolates encoding blaTEM , leaving a representative dataset of 5,192 isolates to investigate the distribution of ponA among CMRNG. Antimicrobial susceptibility testing All MIC values were determined by the agar dilution method exactly as described previously ( 34 ). Briefly, FA6140 transformants were grown overnight on GCB plates, resuspended in GCB broth at a final concentration of ∼1 × 10 7 colony forming units (CFU)/ml. Aliquots (5 μl) of each strain were spotted onto GCB plates containing antibiotics at <2-fold dilutions. The MIC was defined as the lowest concentration of antibiotic on which fewer than 5 colonies appeared after 24 hrs of incubation. In vitro serial passaging Non-piliated colonies from each individual FA6140 ponA L421X strain ( Table 1 ) were harvested and used to prepare a starting inoculum, which consisted of an equal ratio (verified through Illumina sequencing) of all 16 strains. An equal volume of the starting inoculum was used to inoculate liquid cultures at 0 h at an OD 600 of 0.08. Three cultures were supplemented with a different sub-MIC concentration of penicillin (0.25, 0.5, and 1.0 μg/mL), and one culture remained penicillin-free. Penicillin was added at hour 0 at the specified concentration and was maintained throughout each of the six 6-hr growth periods. After inoculation, cultures grew for 6 hours, at which time the OD 600 of each culture was measured to calculate the volume of cells needed to inoculate the next culture at an OD 600 of 0.08, and an aliquot was removed for analysis. This passaging procedure was performed every 6 h for a total of 36 h. At every 6-hour timepoint, the cultures were checked for contamination by dipping a sterile swab in the media and streaking it out on a GCB agar plate. This experiment was repeated 3 separate times. All liquid cultures consisted of 40 mL of GCB + supplements and were shaken at 180 rpm at 37°C in a 5% CO 2 -enriched atmosphere. Genomic DNA extraction DNA was extracted from cells collected every 6 h during the in vitro serial passaging experiments using the Promega Wizard genomic DNAs purification kit (Promega, Madison, WI) according to the manufacturer’s protocol. Extracted gDNA is from a mixture of cells that represent the ratio of all sixteen FA6140 ponA L421X mutants growing in the culture at that time point. DNA concentrations were measured on the Invitrogen Qubit 2.0 Fluorometer. Samples were prepped using the Qubit™ dsDNA Quantification Assay Kits according to the manufacturer’ guidelines (Invitrogen). PCR and DNA sequencing of FA6140 ponA L421X mutants during in vitro serial passaging Library preparation. Fragments for the library were prepped in a series of two PCR reactions. The first PCR reaction (PCR 1; 20 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 15 s) used genomic DNA extracted from cells collected during the serial in vitro passaging experiments as a template. Primers were designed to target a region of the ponA gene (bp 1212-1309), which encompasses codon-421, and to attach the appropriate adaptor sequences to the 5’ and 3’ ends of each amplified fragment. The amplicons obtained were then purified and used as templates in the second PCR (PCR 2; 12 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 15 s). Primers used in PCR 2 target the 5’ and 3’ Illumina adaptor sequences from PCR 1. Barcoding was achieved by adding a unique, 5 bp sequence to the 5’ end of fragments to signify the time point it refers to. A unique index sequence was attached to the 3’ ends of the fragments to mark the penicillin concentration of the culture from which the fragment was obtained. All PCR fragments were extracted from 3% agarose gels and purified using the QIAquick Gel Extraction Kit (QIAGEN) according to manufacturers’ guidelines. Concentrations were measured on the Invitrogen Qubit 2.0 Fluorometer. Illumina sequencing. Purified fragments from PCR 2 were pooled and sequencing was performed on an Illumina Next-seq 1000. This was done three separate times, once per in vitro serial passaging trial. Data analysis. Read files generated from our libraries contained between 10 and 70 million reads. The reads in this library are essentially all genetically identical copies of the amplified region of ponA , except for codon-421, meaning each read correlates to a strain from the FA6140 ponA L421X library, based on the amino acid at codon-421. Data were parsed using a UNIX script to count codons and condition specific barcodes. This process yielded a table for each timepoint, listing all of the codons identified at position-421, and a count of how many reads have that codon. Read counts across time points were normalized to the total number of reads in the file. Only informative reads, containing the expected codons at the expected positions, were analyzed to limit normalization and counting based on uncertain read calls. After filtering, we retained ∼99% of reads that were informative. Bar graphs were generated by normalizing codon counts at 36 hr to the strain’s inoculum value (codon count at 0 h). Since this is a zero-sum competitive experiment, any gain in codon counts by one isolate directly corresponds to a loss in codon counts by the others. Whole genome sequencing and assembly Whole genome sequencing was performed by SeqCenter (Pittsburgh, PA) on genomic DNA from the inoculum and the 36 h timepoint. Illumina sequencing libraries were prepared using the tagmentation-based and PCR-based Illumina DNA Prep kit and custom IDT 10 bp unique dual indices (UDI) with a target insert size of 280 bp. No additional DNA fragmentation or size selection steps were performed. Illumina sequencing was performed on an Illumina NovaSeq X Plus sequencer in one or more multiplexed shared-flow-cell runs, producing 2 x 151 bp paired-end reads. Demultiplexing, quality control and adapter trimming was performed with bcl-convert1 (v4.2.4). Variant calling analysis on the reads was also performed by SeqCenter, using their standard methods. Illumina-generated 2 x 151 bp paired-end read data was used as the input for variant calling against the reference genome of gonococcal clinical isolate, FA6140 ( 82 ), the parental strain used to create the library of mutants (GCF_001047255.1). Variant calling was carried out by SeqCenter using BreSeq1 under default settings. Data availability This publication made use of the PubMLST website ( http://pubmlst.org/ ) developed by Keith Jolley ( 45 ) and sited at the University of Oxford. The development of that website was funded by the Welcome Trust. Data was retrieved and is available from https://pubmlst.org/organisms/neisseria-spp . Data supporting the findings are included in the manuscript. All metadata and quality metrics for isolates included in the datasets for our genomic and phylogenetic analysis are available at https://github.com/mortimer-lab/ponA421 . Experimental data, raw FASTQ files, and tables with codons and counts for each Illumina sequencing trial, as well as the code used to generate these tables are available upon request. Supplemental Material TABLE S1 TABLE S2 TABLE S3 TABLE S5 FIG S1 FIG S2 FIG S3 FIG S4 FIG S5 Acknowledgements The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the Uniformed Services University of the Health Sciences, the Department of Defense, the University of North Carolina, Harvard University, and the University of Georgia. This work was supported by grants AI153521 (YHG, AEJ, and RAN) and AI164794 (RAN), as well as the Pharmacological sciences T32 Training Program award, GM135095 (GG), and the National Institute of Allergy and Infectious Disease (NIAID) STI/HIV T32 Training Program award, T32AI007001 (GG). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. GG and RAN designed and conceptualized the research, GG performed all experiments, analyzed data, provided opinions, and wrote the manuscript. TDM performed genome assembly and typing and phylogenetic analysis, reviewed and edited the article, and provided opinions. BG and DD assisted with Illumina sequencing, providing insightful advice on experimental design and data analysis. ALV, AEJ, and YHG provided input and opinions during the course of this study, and critical review of the manuscript. Supervision, funding acquisition, and article revisions provided by RAN. Funder Information Declared National Institute of Health , AI153521 , AI164794 , GM135095 National Institute of Allergy and Infectious Disease , T32AI007001 Footnotes Methods (line 482) revised to cite Table 1. https://github.com/mortimer-lab/ponA421 https://pubmlst.org/organisms/neisseria-spp References 1. ↵ World Health Organization . 2024 . 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