Phages use contingency loci as a bet-hedging strategy

Phages use contingency loci as a bet-hedging strategy | 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 Phages use contingency loci as a bet-hedging strategy Jasper B. Gomez , Jeffrey E. Barrick , Christopher M. Waters doi: https://doi.org/10.1101/2025.10.21.683753 Jasper B. Gomez 1 Department of Microbiology, Genetics, & Immunology, Michigan State University , East Lansing, Michigan, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeffrey E. Barrick 2 Department of Molecular Biosciences, The University of Texas at Austin , Austin, Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christopher M. Waters 1 Department of Microbiology, Genetics, & Immunology, Michigan State University , East Lansing, Michigan, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: watersc3{at}msu.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Bacteriophages are estimated to outnumber bacteria by ∼10-fold 1 , 2 . Here, we show that phage genomes contain contingency loci (CL), hypermutable DNA regions that promote reversible frameshift mutations through DNA polymerase slippage 3 – 6 . CL have been described in bacteria, archaea, and eukaryotes but have not previously been reported in phages. We demonstrate that CL in coliphage T2 and T4 generates genomic and phenotypic diversity in resulting progeny to evade host defense, a process known as bet-hedging. Whole genome sequencing of T2 and T4 show similar levels of CL-driven sequence variation in dozens of other putative CL. Additional sequencing of T6, T7, Secφ27, ICP1 and ICP2, alongside bioinformatics of the BASEL phage collection reveals that putative CL are widespread in phages and are encoded in every functional class of genes. Collectively, our study describes a new paradigm for understanding phage replication in which CL drive genetic diversification and population heterogeneity to rapidly evolve. Introduction Bacteriophages (phages) encounter diverse bacterial hosts, and hundreds of bacterial phage defense systems, that enable hosts to survive infection 7 – 11 . To infect and replicate, phages must overcome such host defenses 12 . We discovered that phage genomes rapidly adapt to such selective pressures through contingency loci (CL). A key feature of CL is the presence of DNA repeats that drive high mutation rates due to slipped strand mispairing by DNA polymerase during replication 4 – 6 . These hypermutable regions of DNA mediate high frequency, reversible, heritable, stochastic, genotypic switching 3 , 13 . CL have been widely studied in eukaryotes, bacteria, and archaea 13 – 15 ; however, they have not been described in phages. Here, we demonstrate that phage CL drive diversity of progeny in a bet-hedging strategy to overcome bacterial defense. Furthermore, we find that CL are widespread in phage genomes, occurring in all functional gene classes, suggesting they mediate phage evolution to overcome other selective pressures in addition to host defenses. T2 mutants resistant to TgvAB rapidly evolve TgvAB, a Type IV restriction system, is encoded in Vibrio cholerae 16 , 17 . Heterologous expression of TgvAB in Escherichia coli protects it from T2, T4, and T6 phage infection by recognizing the glucosylated 5-hydroxymethylcytosine (5-hmC) on the phage DNA 16 , 17 . Addition of glucose to 5-hmC on phage DNA is encoded by the α-glucosyltransferase gene ( agt ) encoded in T-even phages 18 . T2 can escape TgvAB detection by acquiring null mutations in the agt gene 16 . However, loss of glucosylation on 5-hmC results in an evolutionary trade-off as unglucosylated 5-hmC is recognized and restricted by the McrA/BC Type IV restriction enzymes 16 , 19 , 20 ( Fig. 1A ). Download figure Open in new tab Fig. 1 CL in T2 agt facilitates exploiting hosts with varying restriction systems. A. Schematic of WT agt or agt null mutant infection on either host with McrA/BC or TgvAB. B. E. coli with p tgvAB and pEV (empty vector) infected with WT T2 at varying multiplicities of infection (MOI’s) at time 0. The mean and standard error of 3 biological replicates each with 3 technical replicates are presented. C. T2 phage isolated from the cultures in Fig 1B was used to reinfect E.coli DH10B with p tgvAB and pEV at varying MOI’s at time 0 to determine if the phage had gained resistance. D. 10-fold serial dilution plaque assay of T2 pre-and post-infection on pEV and T2 pre-and post-infection on p tgvAB on host pEV and p tgvAB. The mean and standard error of 3 biological replicates each with 3 technical replicates are presented. E. Schematic of WT T2 agt gene. Nucleotide sequence of mutants and the surrounding region in agt compared to WT T2 sequence. Regions highlighted in red indicate a nucleotide deletion while yellow indicates a nucleotide insertion and green indicates a substitution in corresponding position. Mutants chosen for the reversion experiment are boxed in red. F. Mutant frequency of T2 Mut 1 (single strand repeat) and Mut 8 (point mutation). Dots represent one biological replicate, and dashed line represents the average of 5 biological replicates per T2 mutant. Wilcoxon test performed, p-value = 0.01 and is indicated by * G. MG1655 infected with T2 Mut 1 or T2 Mut 8 at an MOI of 0.001 at time 0. The mean and standard error of 5 biological replicates each with 3 technical replicates are presented. During liquid culture infections, E. coli expressing TgvAB (p tgvAB ) exhibited only transient protection against T2 during the initial stages of growth for all multiplicities of infection (MOI) ( Fig. 1B ). This dynamic differs from other restriction systems in which phage infection of hosts encoding these defenses does not significantly impact growth dynamics 10 , 11 , 21 , 22 . We hypothesized that the inhibition of growth by T2 in these cultures was due to rapid selection of TgvAB-resistant T2 mutants. To test this, we collected T2 phages after infecting E. coli (p tgvAB ) and reinfected the same strain. These phages now exhibit complete killing of E. coli (p tgvAB ), demonstrating rapid evolution of T2 resistance to TgvAB ( Fig. 1C, D ). T2 agt functions as a contingency locus To identify which T2 mutations provide TgvAB resistance, we isolated spontaneous TgvAB-resistant plaques from 15 independently propagated T2 populations by plating WT T2 phages on E. coli (p tgvAB ). Whole genome sequencing (WGS) revealed that phages from all 15 TgvAB-resistant plaques had mutations in agt. This result is consistent with our previous findings that null mutations in agt provide T2 resistance to TgvAB 16 . Five mutants had unique single nucleotide polymorphisms (SNPs) in the agt gene while 10 mutants had frameshift mutations of +/-1 in two monomeric adenine (A) single sequence repeats (SSRs) ( Fig. 1E ). Due to the frequency of mutations in these agt SSRs and the rapid evolution of agt resistant T2 mutants, we hypothesized that these SSRs were CL in agt . CL are repeat sequences in DNA that have an elevated rate of reversible mutations relative to the basal rate of DNA polymerase due to DNA polymerase slippage during replication 3 , 6 . To explore whether the agt SSRs exhibit an elevated reversible mutation rate, we quantified the agt reversion rates of T2 Mut 1 (deletion of an adenine in the agt SSR encoded from 288-296 bases into the agt gene) and T2 Mut 8 (an adenine to a guanine single nucleotide polymorphism (SNP)) ( Fig. 1E ). Equivalent numbers of phages from five independently harvested populations of each agt mutant were plated on E. coli strain MG1655 as it encodes the McrA/BC Type IV restriction system that recognizes and restricts agt- T2 phages ( Fig. 1A ), thus selecting for restoration of agt + phages. Our results from these five independent populations showed that T2 Mut 1 reacquired agt+ at a frequency ∼1000-fold that of the T2 Mut 8 ( Fig. 1F ). Sequencing of phages from McrA/BC resistant plaques showed that both the SSR and SNP mutants produced true agt revertants. Moreover, T2 Mut 8 (SNP) was significantly more restricted by E. coli MG1655 in a liquid culture infection whereas T2 Mut 1 (SSR) more rapidly evolved resistance at all MOIs tested ( Fig. 1G , Extended Fig. 1 ), illustrating the elevated reversion rate of the SSR allows rapid development of resistance to McrA/BC. These results indicate that the T2 SSR in agt at bases 288-296 is a CL. Putative CL are widespread in the T2 genome Because CL have an elevated mutation rate compared to the basal DNA polymerase error rate 3 , 6 , we hypothesized that agt CL mutations could be detected in individual sequence reads generated from Illumina short read sequencing of phage genomes. This approach could quantify the generation of sequence variation independent of phenotypic selection. To test this hypothesis, five independent WT T2 populations were propagated on a non-selective host and sequenced to a depth of ∼2,000 fold. We identified every SSR (of any base) with a length ≥6 bases and analyzed individual sequencing reads to quantify the number of single-base insertions or deletions in SSRs. We found that four out of the five WT T2 populations had detectable mutations in the agt CL encoded at bases 288-296. The average mutation frequency for this agt CL across all five populations was 7 × 10 - 4 (total mutant sequences/total WT sequences, Fig. 2A ), a mutation frequency that cannot be accounted for by the basal mutation rate of T2 DNA polymerase or errors in Illumina sequencing 23 – 25 . Download figure Open in new tab Fig. 2 Variation in putative CL detected by Illumina Sequencing. 5 independent populations of WT T2 were sequenced. Sequencing reads with genomic regions with single sequence repeats (SSRs) ≥6 were analyzed and the number of reads that differed from WT sequence was calculated as Mutant Frequency. A. agt is shown with each dot representing an independent sequencing result. ipIV is shown with one enlarged dot representing all 5 independent sequencing results resulted in the same value of 0. Dot sizes correspond to number of independent sequencing with similar values. B. Percent mutants with all 5 independent sequencing values above 0. Slashes between gene names indicate the SSR is in an intergenic region between two ORFs. Color of dots represent position of repeat region in T2 genome. Start of genome is represented as dark blue while light blue indicates the end of genome. C. All SSRs in WT T2 plotted from the start of the genome (right) to end of the genome (left). The red box highlights the SSRs in agt . Including the agt CL at bases 288-296, we identified 168 SSRs ≥6 bases spread across the genome of T2 ( Fig. 2B and Supplemental Table 5) and found that 77.9% of SSRs had at least one population with detectable frameshift mutations from our whole genome sequencing analysis ( Fig. 3A ). Ten SSRs (5.95%) had mutations in all five populations that were sequenced ( Fig. 2C , Supplemental Table 5). 22% of SSRs had no detectable mutations in any of the five mutant populations that were sequenced ( Fig. 3A ), and one example is shown in Fig. 2A . Mutations in individual sequencing reads could be generated during the Illumina library preparation or sequencing process. However, three pieces of evidence suggest this is not the explanation. First, the calculated average mutation rate determined from all 168 SSRs is 2.94 × 10 -4 , which is higher than the basal Illumina error rate 26 . Second, as mentioned 22% of SSRs had no mutations detected, suggesting that a repeat of ≥6 bases is not in itself sufficient to generate Illumina sequencing errors at this rate. And third, the mutation rates calculated from direct analysis of sequence reads were less than that determined by quantifying agt + revertants (2.89 x 10 -1 , Fig. 1F ) and a CL in rIIA described below ( Fig. 4C ), showing that biological quantification of CL mutation rates is on par with our sequencing read analysis. We therefore show that the 77.9% of SSRs with reliably detectable mutations are putative CL. The lack of observed mutations in 22% of SSRs could be due to inherent differences in mutation rates caused by cis acting sequences or by strong counter selection against mutations in these specific SSRs. Collectively, our results indicate that T2 encodes 131 SSRs that exhibit elevated mutation rates and are putative CL. Download figure Open in new tab Fig. 3 CL are widespread in other phages. A. Percentage of populations with mutations detected from 5 independent populations of T2, T4, T6, T7, Secφ27, ICP-1 and ICP-2 phages. B. Total number of unique genomic regions with putative CL in an open reading frame (ORF) or intergenic region for T2, T4, T6, T7, Secφ27, ICP-1 and ICP-2. C. Number of genomic regions (ORF/Intergenic region) with different numbers of putative CL for T2, T4, T6, T7, Secφ27, ICP-1 and ICP-2 genome. D. Number of SSRs per Kb identified in original BASEL phage collection. Download figure Open in new tab Fig. 4 Evidence for CL reversion rate in T4 rIIA . A. Schematic of WT rIIA or rIIA null mutant infection on either E.coli B host with or without superinfection or host with RexAB (λ) B. Nucleotide sequence of mutants and the surrounding region in rIIA compared to WT T4 sequence. The region highlighted in yellow indicate a nucleotide insertion, and in blue indicate a substitution in corresponding position. C. Mutant frequency of T4 Mut 123 (CL) and Mut 127 (point mutation). Dots represent one biological replicate, and dashed line represents the average of 5 biological replicates per T2 mutant. Wilcoxon test performed, p-value = 0.007 and is indicated by ** D. MG1655(λ) infected with T4 Mut 123 or T4 Mut 127 at an MOI of 0.001 at time 0. The mean and standard error of 5 biological replicates each with 3 technical replicates are presented. E. Model for CL driven evolution in phages. When an individual phage replicates in its host, nucleotide repeat mutants arise through polymerase slippage at different CLs, resulting in a phage population with many distinct genomes. Diverse phages encode contingency loci The T-even phages T2, T4, and T6 are morphologically, antigenically, and genetically similar 28 – 30 , and we hypothesized that the T4 and T6 phage genomes would also encode multiple putative CL like T2. To test this hypothesis, we propagated five independent populations each of T4 and T6, then sequenced their genomes and analyzed mutations in their SSRs as described above. We identified 191 SSRs in phage T4 and 188 SSRs in phage T6 (Supplemental Table 6, 7). Of these SSRs identified in T4 and T6, we observed 140 and 120 SSRs with detectable mutations, respectively, and we consider these putative CL. This result closely matched with our observations for phage T2, demonstrating that each of these phages encode over a hundred putative CL in their genome ( Fig. 3A ). To explore whether genetically distinct phages encode putative CL, we sequenced five independent isolates of two additional coliphages, including the well-studied T7 31 phage and the poorly studied SecΦ27 32 phage, and the two vibriophages, ICP-1 and ICP-2 33 – 35 . We assessed the presence of SSRs by analyzing individual sequencing reads as described above. Our results identified SSRs for each phage; however, the number varied across phages with ICP-1 and SecΦ27 having 57 and 75 SSRs, respectively, while ICP-2 and T7 only had 2 and 1 SSRs, respectively (Supplemental Tables 8, 9, 10 and 11). Analogous to the T-even phages, the majority of SSRs had detectable sequencing reads with base insertions or deletions in at least one of the populations ranging from 77.2% in ICP1, 97.3% in SecΦ27, and 100% in ICP2 and T7 ( Fig. 3A ). Most putative CL for all phages analyzed are present in open reading frames (ORFs) while a few are encoded in intergenic (IG) regions ( Fig. 3B ). Although this result is not surprising given that most of the phage genome is dedicated to encoding ORFs, it does suggest that many phage genes have the potential to be inactivated by frameshift mutations in CL. Additionally, we found that 100% of genomic regions in ICP-2 and T7, 90% in ICP-1, 89.4-81.2% in the T-even phages, and 72.5% in SecΦ27 had only one putative CL, but other genomic regions encoded 2-4 putative CLs, suggesting that such regions are mutational hot spots ( Fig. 3C ). We analyzed the predicted function of genomic regions with putative CL in each E. coli phage and found they occurred in all functional categories designated by PhageScope 36 (Extended Fig. 2A , Supplemental Table 12). SSRs are widespread in phage genomes To further ascertain the range of SSRs in phages, we analyzed the genome sequences of 106 dsDNA E. coli phages from the BASEL phage collection, which contains representatives of the major E. coli phage ICTV Genus 37 . Every phage ICTV Genus from the original BASEL library ( Fig. 3D ) and newly described expanded library (Extended Fig. 2B , Supplemental Table 13) encoded SSRs ≥6 bases and within each ICTV Genus, the density of SSRs per kilobase (kb) in the genome varied. Three phage ICTV Genus ( Teseptimavirus , Kayfunavirus , and Berlinvirus ) had the lowest average SSR/kb ranging from 0.02-0.07 while the phage ICTV Genus (Warwickvirus) had the highest SSR/kb at an average of 1.52. Consistent with our sequencing results, the Tequatrovirus genus, which includes the T-even phages, encoded the 8th highest average SSR/kb out of 32 genera, suggesting abundant SSRs, while the T7 family Teseptimavirus encoded the 31st lowest average SSR/kb, suggesting the loss of SSRs. We compared the average SSR/kb of each phage genus to the %GC in their genome but found no correlation, suggesting differences in SSR abundance are not due to base content (Extended Fig. 3 , Supplemental Table 14). To more broadly understand the role of SSRs in phage evolution, we examined what types of protein-coding genes contained SSRs ≥6 base pairs in a diverse set of 41 coliphage genomes from the BASEL collection (Extended Fig. 2B ). The 41 phages selected exhibit <90% sequence identity to remove closely related phages (see Methods). We found that these SSRs were significantly underrepresented in infection proteins, such as tail fibers, and replication proteins, such as polymerases, with only about 0.77× as many SSRs in these genes as expected from randomizations tests (see Methods) (Extended Fig. 4 , Bonferroni adjusted p < 0.001). This result is not surprising as these proteins are likely to be essential, and there may be selection against the evolution of hypermutable sequences that can cause frameshift mutations in their genes. On the other hand, we observed 2.03× as many SSRs as expected in the regulatory protein category, which includes phage repressors, and this overrepresentation was highly significant (Extended Fig. 4 , adj p < 0.001). There were also excess SSRs in the hypothetical and unclassified protein category (Extended Fig. 4 , adj p = 0.014), which includes the agt gene. These results suggest that SSRs have evolved in diverse phage genes to enable evolutionary bet-hedging to survive a variety of selective pressures. T4 rIIA encodes a contingency locus To investigate the function of another putative CL, we examined the repeat of 6 A’s encoded in the rIIA gene in T2, T4, and T6 sequencing reads, which had detectable mutations in all three phages as determined by sequence analysis (Supplemental Tables 5, 6, and 7). The rIIA gene was key in elucidating fundamental aspects of molecular biology including determination of a triplet genetic code and demonstration of homologous recombination 38 – 42 , but a CL has never been described in this gene. To examine whether the putative CL in rIIA exhibits a higher reversion rate relative to other mutations, we engineered an addition of an adenine in the putative CL (T4 Mut 123) and a substitution of an adenine to a guanine 53 bp upstream (T4 Mut 127), regenerating previously identified rIIA null mutations 40 , 43 ( Fig. 4A ). During superinfection, when phages outnumber hosts, WT T4 activates lysis inhibition (LIN) to delay infection. T4 rIIA mutants undergo rapid lysis, overcoming the delayed infection in E.coli B. However, the loss of rIIA results in an evolutionary tradeoff as these phages are now susceptible to RexAB, a phage defense system encoded by a lambda lysogen 40 , 44 – 46 ( Fig. 4B ). Thus, we quantified restoration of rIIA+ in T4 Mut 123 and T4 Mut 127 from five independent populations by plating on E. coli K-12 (λ). T4 Mut123, which had a mutation in the CL, reverted at ∼10,000× higher frequency than the SNP mutation T4 Mut 127, confirming this SSR repeat in rIIA is a CL ( Fig. 4C ). Analogous to our results with agt, the rIIA CL provided increased fitness upon infection of K12(λ) as the RexAB defense system only exhibited transient protection against the rIIA CL null mutant, T4 Mut123, but complete protection against the rIIA SNP mutant, T4 Mut127, ( Fig. 4D , Extended Fig. 5). Discussion Our results indicate that during genome replication of dsDNA phages, DNA polymerase slippage at CL produces frameshift mutations at rates thousands of times higher than basal mutation rates ( Fig. 4E ). This process generates genetic diversity and provides a bet-hedging strategy that enables phages to survive diverse selective pressures, such as bacterial phage defense systems. Results from analyzing whole-genome sequencing of various phages for ≥6 SSRs revealed putative CL in ORFs with a wide range of functions that include assembly, lysis, packaging, regulation, and replication. The prevalence of CLs in genes of many functions suggests this evolutionary strategy likely extends beyond evolving to overcome phage defense to other selective pressures such as divergent bacterial hosts or environments. CL have never been ascribed to phages, however, Fletchervirus phages encode 7-11 guanine (polyG) tracts in receptor binding proteins, generating diversity that allows a sub-population to infect Campylobacter when its phase-variable receptor is not expressed 47 . These repeats were not designated CL nor was their mutation rate quantified. PolyG tracts were identified in a thymine methylase gene of bacteriophages BBP-1 48 and in PB1-like phages in a baseplate gene 49 , but they were also not designated CL nor studied further. The mutational variation that arises from phage CL may reflect a history of “Red Queen” coevolution between bacteria and phages, where each side must constantly evolve to counter evolution by the antagonist. By evolving reversible, elevated frameshift mutations in CL, phages can harness their short life cycle and high numbers of progeny to rapidly and reversibly sample genetic variation to enhance evolution. For example, using a simple probabilistic model F mut =1-(1-m) r , where m is the average mutation rate and r is the total number of repeats, we estimate using T2 as an example, that ∼1% of progeny produced during an infection will have a mutation in a CL. Thus, CL can generate vast genetic variation in phages that has not been appreciated nor explored. Methods Strains and growth conditions Bacterial and phage strains used in this study are listed in Supplementary Table 1. Unless otherwise stated, cultures were grown in Luria broth (LB) at 37 ° C with shaking at 210 rpm or LB plates at 37 ° C and supplemented with the following antibiotics for plasmid maintenance as necessary: kanamycin (100 μg mL -1 ) and/or carbenicillin (100 μg mL -1 ). Coliphages were propagated by streaking on MMB agar (LB + 0.1mM MnCl 2 + 5 mM CaCl 2 + 5 mM MgCl 2 + 0.5% agar) with E. coli DH10B at a 1:1,000 dilution of an overnight culture and incubated overnight. Plates were then flooded with 10 mL of phage buffer (0.1 mM Tris HCl 7.5 pH + 10mM MgSO 4 + 0.4% NaCl 2 + dH 2 O) and incubated statically at 4 ° C for 24 hours. Phage buffer was then filtered through a 0.22 μM filter. Vibriophages were propagated exactly like coliphages phages except the host is V. cholerae E7946. Plasmid construction Plasmids are listed in Supplementary Table 2 and primers in Supplementary Table 3. All PCR products were amplified using Q5-High Fidelity DNA polymerase according to the manufacturer’s instructions (New England Biolabs). All plasmid constructs were generated using fast cloning as described in Gomez et al. 2024 16 . All plasmids and PCR products were confirmed by sequencing (Plasmidsaurus). Plasmid sequencing results were mapped to T4 reference genome (NC_000866.4) using Geneious Prime 2023.0.2. To construct pJBG122, rIIA was amplified from wild-type T4 Carolina with overlapping ends to pUC19 using oJBG206 and oJBG207. pUC19 was linearized using HF_pUC19-MCS_F and HF_pUC19-MCS_R. To construct pJBG123 and pJBG127, one region of pJBG122 was amplified with site-directed mutagenesis primer oJBG208 (pJBG123) or oJBG216 (pJBG217) with oMJF006 and another region was amplified with site-directed mutagenesis primer oJBG209 (pJBG123) or oJBG217 (pJBG127) with oMJF005. Both PCR products were used for fast cloning to create pJBG123 or pJBG127, respectively. Inserts were verified by PCR using primers oJBG191 and oJBG192. T4 rIIA site directed mutagenesis To generate T4 Mut 123 and Mut 127, E. coli strain DH10B containing pJBG123 and pJBG127 respectively, were grown overnight in LB at 37 ° C. Overnight cultures were mixed 1:1,000 with 20 mL of MMB agar + carbenicillin and poured into 150 x 15mm Petri dish. Plates were left to solidify for 30 minutes at room temperature. After 30 minutes, 150 μl of wild-type T4 was spread across the plates. Plates were dried and placed at 37 ° C overnight. Plates were covered with 10 mL of phage buffer and placed at 4 ° C overnight. Phage buffer was collected and filtered using a 0.22 μM filter. Lysates were then taken and spread on plates made with an overnight culture of E.coli B at 1:1,000 and mixed with 20 mL of MMB agar and poured into a 150 x 15mm Petri dish and incubated at 37 ° C overnight. Plates were covered with 10 mL of phage buffer and placed at 4 ° C overnight. Phage buffer was collected and filtered using a 0.22 μM filter. Lysate was then streaked on plates made with an overnight of E. coli B at 1:1,000 and mixed with 10 mL of MMB agar to obtain isolated plaques. Plaques were then picked based on the large plaque phenotype on E. coli B. T4 Mut 123 and Mut 127 were verified by plating on MG1655(λ) and with PCR using primers oJBG191 and oJBG192. Phage infection and reinfection in liquid culture Overnight cultures of DH10B p tgvAB and pEV (pJBG078 and pMJF103) grown in LB + kanamycin were diluted to an OD 600 of 0.01 into 10 mL flasks containing LB + kanamycin. Cultures were grown for ∼2 hours to an OD 600 0.1. The cultures were distributed into a 96 well plate and wild-type T2 was added at MOIs of 0.1, 0.01, and 0.001. Infection was monitored by measuring OD 600 every 2.5 minutes for 10 hours at 37 ° C using a BioTek 800 TS (Agilent) plate reader with continuous, linear shaking. After 10 hours plates were placed in 4 ° C. Cultures from the wells were then collected and transferred to a AcroPrep Advance 96-well filter plate for aqueous filtration with a 0.2 μM filter. The 96-well filter plate was placed on top of a another 96 well plate to collect phage lysate. Plates were centrifuged at 4,000rpm at 4 ° C. Phage concentration was calculated from infection on DH10B. Overnight cultures of DH10B p tgvAB and pEV grown in LB + kanamycin were diluted to an OD 600 of 0.01 into 10 mL flasks containing LB + kanamycin. Cultures were grown for ∼2 hours to an OD 600 0.1. Once OD 600 = 0.1, cultures were distributed into a 96 well plate. Using phage lysate from the initial E. coli p tgvAB infection, MOIs initially at 0.1, 0.01 and 0.001 were added again at the exact MOI into 96 well plate and measured with the same parameters as initial infection. Experiments were replicated 3 times independently and representative images are shown. Phage infection in liquid culture Overnight cultures of MG1655 or K-12 (λ) were diluted to an OD 600 of 0.01 into 10 mL flasks containing LB. Cultures were grown for ∼1.5 hours to OD 600 0.1. Once OD 600 = 0.1, cultures were distributed into a 96 well plate and T2 Mut 1 and Mut 8 were added to MG1655 or T4 Mut 123 and Mut 127 were added to K-12 (λ) at MOIs of 0.1, 0.01, and 0.001. Infection was monitored as described above. Experiments were replicated 3 times independently and representative images are shown. Efficiency of plaquing (EOP) assays Assays were performed as described in Gomez et al 16 . Briefly, overnight cultures of DH10B p tgvAB and pEV were mixed 1:1,000 with 20 mL MMB + kanamycin and poured into 150 x 15mm Petri dishes. Wild-type, post, and pre T2 stocks were diluted 1:10 up to a 10 - 12 dilution. 5 μl of each dilution was spotted on DH10B p tgvAB and pEV plates. Plates were dried before placing at 37 ° C overnight. Experiments were replicated 3 times independently and representative images are shown. Mutant frequency assay T2 Mut 1 and T2 Mut 8 were struck out on overnight cultures of p tgvAB mixed 1:1,000 with 10 mL of MMB agar + kanamycin and poured into 100 x 15mm Petri dish. 5 isolated plaques were picked and resuspended in 150 μl of distilled H 2 O (dH 2 O). Overnight cultures of DH10B were mixed 1:1,000 with 20 mL of MMB agar and poured into 150 x 15mm Petri dish. Plates were left to solidify for 30 minutes at room temperature. After 30 minutes, 150 μl of each isolated plaque was spread, dried and placed at 37 ° C. Plates were covered with 10 mL of phage buffer and placed at 4 ° C overnight. Phage buffer was collected and filtered using a 0.22 μM filter. T4 Mut 123 and T4 Mut 127 were struck out on overnight cultures of E. coli B mixed 1:1,000 with 10 mL of MMB agar + kanamycin and poured into 100 x 15mm Petri dish. 5 isolated plaques were picked and resuspended in 150 μl of distilled H 2 O (dH 2 O). Overnight cultures of MG1655 were mixed 1:1,000 with 20 mL of MMB agar and poured into 150 x 15mm Petri dish. Plates were left to solidify for 30 minutes at room temperature. After 30 minutes, 150 μl of each isolated plaque was spread, dried and placed at 37 ° C. Plates were covered with 10 mL of phage buffer and placed at 4 ° C overnight. Phage buffer was collected and filtered using a 0.22 μM filter. EOP of all isolated phage populations for T2 Mut 1 and Mut 8 were determined on MG1655 and DH10B. EOP of T4 Mut 123 and Mut 127 were determined on MG1655 and K-12 (λ). Using EOPs we performed plaque-forming unit quantification assays as described in Gomez et al 16 for all independent phage lysates. The following equation was used to calculate mutant frequency, m = ((D1/C1)*(C2/D2)) where D1 is the dilution factor from non-selective host lysate, C1 are PFU counts on non-selective host, D2 is the dilution factor from selective host lysate and C2 are PFU counts on selective host and m is the mutation rate. Experiments for each individual population were replicated 3 times independently and representative images are shown. Illumina sequencing E. coli phage DNA extraction was done as described in Gomez et al. 2024 16 . Vibriophage extractions were done as described in Minmin et al. 2017 50 . High-titer phage lysates (>10 6 PFU μl -1 ) were used for extraction. DNA extracted samples were sent to Seqcoast for short read whole genome Illumina sequencing. Short read whole genome Illumina sequencing was performed using their small 200 Mbp/1.3 million reads service with 150 bp paired end reads. Sequencing was performed using either a standard SBS flow cell or a XLEAP flow cell on a NextSeq 2000.T2 agt null mutations information is provided in Supplemental Table 4. Breseq analysis Illumina sequencing results were analyzed using breseq 51 with runs automated using brefito 52 . Sequences were aligned with their corresponding reference genome T2 (NC_054931.1), T4 (NC_000866.4), T6 (NC_05407.1), T7 (NC_001604.1), Secφ27 (NC_047938.1), ICP-1 (MH310933), and ICP-2 (NC_015158). Sequence information is provided in Supplemental Tables 5, 6, 7, 8, 9, 10 and 11. Genomic regions were pulled based on SSRs ≥6 using the tabulate-CL utility command of breseq in strict mode, which requires an exact match in a read to the five bases on either side of the SSR to count a read. Mutant frequency detected in the population was calculated by N/T where N = total number of mutant reads and T = the total number of reads. All analyses were done for 5 independent populations of each phage strain. BASEL phage CL analysis Phage genomes from the BASEL collection were downloaded using GenBank accessions provided in Humolli et al 2025 37 . Putative CL in each genome were identified by using a Python script to scan for ≥6 base pair homopolymer SSRs. Gene contexts (ORF/intergenic region) of SSRs were classified based on annotations loaded from GenBank files using BioPython 53 . GenBank accession numbers and SSR information are provided in Supplemental Table 13. Association of putative CL with phage gene categories Coliphage genomes from the BASEL collection were analyzed 37 . First, we de-duplicated this set to 41 genomes that all had a pairwise average nucleotide identity (ANI) <90% as calculated using FastANI (v1.34) 54 . GenBank accession numbers and information about these sequences are provided in Supplemental Table 15. We transferred annotations of phage protein categories from the GenBank portion of PhageScope 36 to proteins in these genomes via BLASTP (v2.15.0 +) searches 55 , taking top matches and requiring 70% query covered and an E-value 1E-6 to assign a category to a protein. Because there were few proteins in the “immune” and “tRNA_related” categories, we combined those with “hypothetical” and “unsorted” proteins into one “Other or unknown” category. Finally, we assessed the statistical significance of depletion or enrichment of SSRs (homopolymer SSRs ≥6 base pairs or longer) in the sequence of proteins in specific functional categories using randomization tests. For each randomization, we selected a new number of SSRs from a Poisson distribution with the actual number observed in the phage genomes as the mean, then shuffled SSRs among proteins in these phages, weighting by gene length. We repeated this procedure 20,000 times for each of eight categories. Two-tailed p-values for rejecting the hypothesis that the observed number of SSRs in a protein category would be observed by chance were calculated as twice the number of trails with the same or a more extreme number of SSRs compared to the actual genomes and Bonferroni adjusted by multiplying by the number of categories tested before assessing statistical significance. Author contributions J.B.G. performed the experiments, J.B.G. and J.E.B. analyzed the data and performed computations analysis, J.B.G. and C.M.W. wrote the manuscript and J.E.B. edited the manuscript. Supplemental Tables List Table S1 – Strains used in this study Table S2 –Plasmids used in this study Table S3 – Primers used in this study Table S4 – Illumina sequencing results of 15 independent T2 mutants passaged in presence of ptgvAB Table S5 – Wildtype T2 SSR ≥6 Illumina whole genome sequencing reads Table S6 – Wildtype T4 SSR ≥6 Illumina whole genome sequencing reads Table S7 – Wildtype T6 SSR ≥6 Illumina whole genome sequencing reads Table S8 – Wildtype ICP2 SSR ≥6 Illumina whole genome sequencing reads Table S9 – Wildtype secΦ27 SSR ≥6 Illumina whole genome sequencing reads Table S10 – Wildtype ICP1 SSR ≥6 Illumina whole genome sequencing reads Table S11 – Wildtype T7 SSR ≥6 Illumina whole genome sequencing reads Table S12 – Predicted gene functions of genomic regions with putative CL in E.coli phages Table S13 – Total number of SSRs and SSRs per kb in BASEL phage library Table S14 – BASEL phage ICTV Genus average SSRs and average GC content Table S15 – 41 BASEL phage with ANI <90% sequence identity Download figure Open in new tab Extended Fig. 1 Contingency loci in T2 agt protects against varying restriction systems. MG1655 infected with T2 Mut 1 or T2 Mut 8 at an MOI of 1 and 0.1 at time 0. The mean and standard error of 5 biological replicates each with 3 technical replicates are presented. Download figure Open in new tab Extended Fig. 2 Simple sequence repeats ≥6 bases identified in other phages A. Functional regions with putative contingency loci for T2, T4, T6, T7 and Secφ27 B. Number of SSRs per kb identified in extended BASEL phage collection. Download figure Open in new tab Extended Fig. 3 Simple sequence repeats ≥6 bases identified in other phages. Average putative CL per kb compared to average % GC of BASEL phage ICTV genus Download figure Open in new tab Extended Fig. 4 CL in BASEL phage collection. Homopolymer SSRs ≥6 base pairs identified in phage protein categories for 41 genetically distinct BASEL phage. Randomization test for each protein category was performed. Bonferroni adjusted p < 0.001 is indicated as ***, p = 0.014 is indicated as * Download figure Open in new tab Extended Fig. 5 Evidence for CL reversion rate in T4 rIIA . MG1655(λ) infected with T4 Mut 123 or T4 Mut 127 at an MOI of 1 and 0.1 at time 0. The mean and standard error of 5 biological replicates each with 3 technical replicates are presented. Acknowledgments We thank Elizabeth N. Ottosen and Micah Ferrell for sharing primers used to construct plasmids, Chris Adami for consultation regarding calculating the percentage of phage population containing a CL mutation, and Bonnie Bassler and Richard Lenski for advice and comments on this paper. This research was supported and funded by NIH grants GM139537 and AI158433 to C.M.W, GM088344 to J.E.B., and F31AI186463 to J.B.G., and NSF grants DEB-1813069 and DEB-1951307 to J.E.B. Funder Information Declared National Institutes of Health , GM139537 , AI158433 , GM088344 , F31AI186463 U.S. National Science Foundation , DEB-1813069 , DEB-1951307 Footnotes Declarations of interest: none References 1. ↵ Hendrix , R. W. , Smith , M. C. M. , Burns , R. N. , Ford , M. E. & Hatfull , G. F . Evolutionary relationships among diverse bacteriophages and prophages: All the world’s a phage . Proc. Natl. Acad. Sci . 96 , 2192 – 2197 ( 1999 ). OpenUrl Abstract / FREE Full Text 2. ↵ Wigington , C. H. , Sonderegger , D. , Brussaard , C. P. D. , Buchan , A. , Finke , J. F. , Fuhrman , J. A. , Lennon , J. T. , Middelboe , M. , Suttle , C. A. , Stock , C. , Wilson , W. H. , Wommack , K. E. , Wilhelm , S. W. & Weitz , J. S . Re-examination of the relationship between marine virus and microbial cell abundances . Nat. Microbiol . 1 , 15024 ( 2016 ). 3. ↵ Moxon , E. R. , Rainey , P. B. , Nowak , M. A. & Lenski , R. E . Adaptive evolution of highly mutable loci in pathogenic bacteria . Curr. Biol . 4 , 24 – 33 ( 1994 ). OpenUrl CrossRef PubMed Web of Science 4. ↵ Streisinger , G. , Okada , Y. , Emrich , J. , Newton , J. , Tsugita , A. , Terzaghi , E. & Inouye , M . Frameshift mutations and the genetic code . Cold Spring Harb. Symp. Quant. Biol . 31 , 77 – 84 ( 1966 ). OpenUrl Abstract / FREE Full Text 5. Levinson , G. & Gutman , G. A . Slipped-strand mispairing: a major mechanism for DNA sequence evolution . Mol. Biol. Evol . 4 , 203 – 221 ( 1987 ). OpenUrl CrossRef PubMed Web of Science 6. ↵ Kroutil , L. C. , Register , K. , Bebenek , K. & Kunkel , T. A . Exonucleolytic proofreading during replication of repetitive DNA . Biochemistry 35 , 1046 – 1053 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 7. ↵ Attrill , E. L. , Claydon , R. , Łapińska , U. , Recker , M. , Meaden , S. , Brown , A. T. , Westra , E. R. , Harding , S. V. & Pagliara , S . Individual bacteria in structured environments rely on phenotypic resistance to phage . PLoS Biol . 19 , e3001406 ( 2021 ). OpenUrl CrossRef PubMed 8. Kortright , K. E. , Chan , B. K. & Turner , P. E . High-throughput discovery of phage receptors using transposon insertion sequencing of bacteria . Proc. Natl. Acad. Sci . 117 , 18670 – 18679 ( 2020 ). OpenUrl Abstract / FREE Full Text 9. Mutalik , V. K. , Adler , B. A. , Rishi , H. S. , Piya , D. , Zhong , C. , Koskella , B. , Kutter , E. M. , Calendar , R. , Novichkov , P. S. , Price , M. N. , Deutschbauer , A. M. & Arkin , A. P . High-throughput mapping of the phage resistance landscape in E. coli . PLoS Biol . 18 , e3000877 ( 2020 ). OpenUrl CrossRef PubMed 10. ↵ Bernheim , A. & Sorek , R . The pan-immune system of bacteria: antiviral defence as a community resource . Nat Rev Microbiol 18 , 113 – 119 ( 2020 ). OpenUrl CrossRef PubMed 11. ↵ Millman , A. , Melamed , S. , Leavitt , A. , Doron , S. , Bernheim , A. , Hör , J. , Garb , J. , Bechon , N. , Brandis , A. , Lopatina , A. , Ofir , G. , Hochhauser , D. , Stokar-Avihail , A. , Tal , N. , Sharir , S. , Voichek , M. , Erez , Z. , Ferrer , J. L. M. , Dar , D. , Kacen , A. , Amitai , G. & Sorek , R . An expanded arsenal of immune systems that protect bacteria from phages . Cell Host Microbe 30 , 1556 – 1569 .e5 ( 2022 ). OpenUrl CrossRef PubMed 12. ↵ Samson , J. E. , Magadán , A. H. , Sabri , M. & Moineau , S . Revenge of the phages: defeating bacterial defences . Nat. Rev. Microbiol . 11 , 675 – 687 ( 2013 ). OpenUrl CrossRef PubMed 13. ↵ Moxon , R. , Bayliss , C. & Hood , D . Bacterial Contingency Loci: The role of simple sequence DNA repeats in bacterial adaptation . Genetics 40 , 307 – 333 ( 2006 ). OpenUrl 14. Snowbarger , J. , Koganti , P. & Spruck , C . Evolution of repetitive elements, their roles in homeostasis and human disease, and potential therapeutic applications . Biomolecules 14 , 1250 ( 2024 ). 15. ↵ Vashakidze , R. P. & Prangishvili , D. A . Simple repetitive sequences in the genomes of archaebacteria . FEBS Lett . 216 , 217 – 220 ( 1987 ). OpenUrl PubMed 16. ↵ Gomez , J. B. & Waters , C. M . A Vibrio cholerae Type IV restriction system targets glucosylated 5-hydroxymethylcytosine to protect against phage infection . J. Bacteriol . 206 , e00143 – 24 ( 2024 ). OpenUrl PubMed 17. ↵ Vizzarro , G. , Lemopoulos , A. , Adams , D. W. & Blokesch , M . Vibrio cholerae pathogenicity island 2 encodes two distinct types of restriction systems . J. Bacteriol . 206 , e00145 – 24 ( 2024 ). OpenUrl PubMed 18. ↵ Revel , H. R. & Luria , S. E . DNA-glucosylation in T-even phage: genetic determination and role in phage-host interaction 1 . Annual Review of Genetics ( 1970 ). 19. ↵ Krüger , T. , Wild , C. & Noyer-Weidner , M . McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues . EMBO J . 14 , 2661 – 2669 ( 1995 ). OpenUrl PubMed Web of Science 20. ↵ Sutherland , E. , Coe , L. & Raleigh , E. A . McrBC: a multisubunit GTP-dependent restriction endonuclease . J. Mol. Biol . 225 , 327 – 348 ( 1992 ). OpenUrl CrossRef PubMed Web of Science 21. ↵ Thomas , J. A. , Orwenyo , J. , Wang , L.-X. & Black , L. W . The odd “RB” phage— identification of arabinosylation as a new epigenetic modification of DNA in T4-like phage RB69 . Viruses 10 , 313 ( 2018 ). 22. ↵ Enikeeva , F. N. , Severinov , K. V. & Gelfand , M. S . Restriction–modification systems and bacteriophage invasion: Who wins? J. Theor. Biol . 266 , 550 – 559 ( 2010 ). OpenUrl CrossRef PubMed 23. ↵ Georjon , H. & Bernheim , A . The highly diverse antiphage defence systems of bacteria . Nat. Rev. Microbiol . 1 – 15 ( 2023 ). doi: 10.1038/s41579-023-00934-x OpenUrl CrossRef PubMed 24. Boezen , D. , Ali , G. , Wang , M. , Wang , X. , Werf , W. van der , Vlak , J. M. & Zwart , M. P. Empirical estimates of the mutation rate for an alphabaculovirus . PLoS Genet . 18 , e1009806 ( 2022 ). OpenUrl PubMed 25. ↵ Sanjuán , R. , Nebot , M. R. , Chirico , N. , Mansky , L. M. & Belshaw , R. Viral mutation rates . J. Virol . 84 , 9733 – 9748 ( 2010 ). OpenUrl Abstract / FREE Full Text 26. ↵ Stoler, N. & Nekrutenko, A. Sequencing error profiles of Illumina sequencing instruments. NAR Genom. Bioinform. 3, lqab019 ( 2021 ). 27. Schirmer , M. , D’Amore , R. , Ijaz , U. Z. , Hall , N. & Quince , C . Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data . BMC Bioinform . 17 , 125 ( 2016 ). 28. ↵ Cowie , D. B. , Avery , R. J. & Champe , S. P . DNA homology among the T-even bacteriophages . Virology 45 , 30 – 37 ( 1971 ). OpenUrl CrossRef PubMed 29. Luria , S. E. , Delbrück , M. & Anderson , T. F . Electron microscope studies of bacterial viruses . J. Bacteriol . 46 , 57 – 77 ( 1943 ). OpenUrl FREE Full Text 30. ↵ Kim , J.-S. & Davidson , N . Electron microscope heteroduplex study of sequence relations of T2, T4, and T6 bacteriophage DNAs . Virology 57 , 93 – 111 ( 1974 ). OpenUrl CrossRef PubMed Web of Science 31. ↵ Molineux , I. J . The T7 Group . The Bacteriophages: Second Edition 0 ( 2005 ). doi: 10.1093/oso/9780195148503.003.0020 OpenUrl CrossRef 32. ↵ Doron , S. , Melamed , S. , Ofir , G. , Leavitt , A. , Lopatina , A. , Keren , M. , Amitai , G. & Sorek , R . Systematic discovery of antiphage defense systems in the microbial pangenome . Science 359 , ( 2018 ). 33. ↵ Seed , K. D. , Bodi , K. L. , Kropinski , A. M. , Ackermann , H.-W. , Calderwood , S. B. , Qadri , F. & Camilli , A . Evidence of a dominant lineage of Vibrio cholerae -specific lytic bacteriophages shed by Cholera patients over a 10-year period in Dhaka, Bangladesh . Mbio 2 , e00334 – 10 ( 2011 ). OpenUrl CrossRef PubMed 34. Hays , S. G. & Seed , K. D . Dominant Vibrio cholerae phage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite . eLife 9 , e53200 ( 2020 ). OpenUrl PubMed 35. ↵ Madi , N. , Cato , E. T. , Sayeed , M. A. , Creasy-Marrazzo , A. , Cuénod , A. , Islam , K. , Khabir , M. I. U. , Bhuiyan , M. T. R. , Begum , Y. A. , Freeman , E. , Vustepalli , A. , Brinkley , L. , Kamat , M. , Bailey , L. S. , Basso , K. B. , Qadri , F. , Khan , A. I. , Shapiro , B. J. & Nelson , E. J . Phage predation, disease severity, and pathogen genetic diversity in cholera patients . Science 384 , ( 2024 ). 36. ↵ Wang , R. H. , Yang , S. , Liu , Z. , Zhang , Y. , Wang , X. , Xu , Z. , Wang , J. & Li , S. C . PhageScope: a well-annotated bacteriophage database with automatic analyses and visualizations . Nucleic Acids Res . 52 , D756 – D761 ( 2023 ). OpenUrl 37. ↵ Humolli , D. , Piel , D. , Maffei , E. , Heyer , Y. , Agustoni , E. , Shaidullina , A. , Willi , L. , Imwinkelried , P. , Estermann , F. , Cuénod , A. , Buser , D. P. , Alampi , C. , Chami , M. , Egli , A. , Hiller , S. , Dunne , M. & Harms , A . Completing the BASEL phage collection to unlock hidden diversity for systematic exploration of phage–host interactions . PLOS Biol . 23 , e3003063 ( 2025 ). OpenUrl PubMed 38. ↵ Crick , F. H. C. , Barnett , L. , Brenner , S. & Watts-Tobin , R. J . General nature of the genetic code for proteins . Nature 192 , 1227 – 1232 ( 1961 ). OpenUrl CrossRef PubMed Web of Science 39. Fisher , K. M. & Bernstein , H . The additivity of intervals in the rIIA cistron of phage T4D . Genetics 52 , 1127 – 1136 ( 1965 ). OpenUrl FREE Full Text 40. ↵ Benzer , S . Fine structure of a genetic region in bacteriophage . Proc. Natl. Acad. Sci . 41 , 344 – 354 ( 1955 ). OpenUrl FREE Full Text 41. Benzer , S . On the topography of the genetic fine structure . Proc. Natl. Acad. Sci . 47 , 403 – 415 ( 1961 ). OpenUrl FREE Full Text 42. ↵ Edgar , R. S. , Feynman , R. P. , Klein , S. , Lielausis , I. & Steinberg , C. M . Mapping experiments with r mutants of bacteriophage T4D . Genetics 47 , 179 – 186 ( 1962 ). OpenUrl FREE Full Text 43. ↵ Shinedling , S. , Singer , B. S. , Gayle , M. , Pribnow , D. , Jarvis , E. , Edgar , B. & Gold , L . Sequences and studies of bacteriophage T4 rII mutants . J. Mol. Biol . 195 , 471 – 480 ( 1987 ). OpenUrl CrossRef PubMed 44. ↵ Wong , S. , Alattas , H. & Slavcev , R. A . A snapshot of the λ T4rII exclusion (Rex) phenotype in Escherichia coli . Curr. Genet . 67 , 739 – 745 ( 2021 ). OpenUrl CrossRef PubMed 45. Shinedling , S. , Parma , D. & Gold , L . Wild-type bacteriophage T4 is restricted by the lambda rex genes . J. Virol . 61 , 3790 – 3794 ( 1987 ). OpenUrl Abstract / FREE Full Text 46. ↵ Paddison , P. , Abedon , S. T. , Dressman , H. K. , Gailbreath , K. , Tracy , J. , Mosser , E. , Neitzel , J. , Guttman , B. & Kutter , E . The roles of the bacteriophage T4 r genes in lysis inhibition and fine-structure genetics: A new perspective . Genetics 148 , 1539 – 1550 ( 1998 ). OpenUrl Abstract / FREE Full Text 47. ↵ Sørensen , M. C. H. , Vitt , A. , Neve , H. , Soverini , M. , Ahern , S. J. , Klumpp , J. & Brøndsted , L . Campylobacter phages use hypermutable polyG tracts to create phenotypic diversity and evade bacterial resistance . Cell Rep . 35 , 109214 ( 2021 ). 48. ↵ Liu , M. , Gingery , M. , Doulatov , S. R. , Liu , Y. , Hodes , A. , Baker , S. , Davis , P. , Simmonds , M. , Churcher , C. , Mungall , K. , Quail , M. A. , Preston , A. , Harvill , E. T. , Maskell , D. J. , Eiserling , F. A. , Parkhill , J. & Miller , J. F . Genomic and genetic analysis of Bordetella bacteriophages encoding reverse transcriptase-mediated tropism-switching cassettes . J. Bacteriol . 186 , 1503 – 1517 ( 2004 ). OpenUrl Abstract / FREE Full Text 49. ↵ Ceyssens , P. , Miroshnikov , K. , Mattheus , W. , Krylov , V. , Robben , J. , Noben , J. , Vanderschraeghe , S. , Sykilinda , N. , Kropinski , A. M. , Volckaert , G. , Mesyanzhinov , V. & Lavigne , R . Comparative analysis of the widespread and conserved PB1-like viruses infecting Pseudomonas aeruginosa . Environ. Microbiol . 11 , 2874 – 2883 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ Yen , M. , Cairns , L. S. & Camilli , A . A cocktail of three virulent bacteriophages prevents Vibrio cholerae infection in animal models . Nat. Commun . 8 , 14187 ( 2017 ). 51. ↵ Deatherage , D. E. & Barrick , J. E . Engineering and analyzing multicellular systems, methods and protocols . Methods Mol. Biol . 1151, 165 – 188 ( 2014 ). 52. ↵ Barrick , J. E. & Zubbu , I. Brefito: Snakemake pipelines for analyzing resequenced bacterial genomes . Brefito: Snakemake pipelines for analyzing resequenced bacterial genomes . ( 2025 ). at 53. ↵ Cock , P. J. A. , Antao , T. , Chang , J. T. , Chapman , B. A. , Cox , C. J. , Dalke , A. , Friedberg , I. , Hamelryck , T. , Kauff , F. , Wilczynski , B. & Hoon , M. J. L. de. Biopython: freely available Python tools for computational molecular biology and bioinformatics . Bioinformatics 25 , 1422 – 1423 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 54. ↵ Jain , C. , Rodriguez-R , L. M. , Phillippy , A. M. , Konstantinidis , K. T. & Aluru , S . High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries . Nat. Commun . 9 , 5114 ( 2018 ). 55. ↵ Camacho , C. , Coulouris , G. , Avagyan , V. , Ma , N. , Papadopoulos , J. , Bealer , K. & Madden , T. L. BLAST+: architecture and applications . BMC Bioinform . 10 , 421 ( 2009 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted October 21, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Phages use contingency loci as a bet-hedging strategy Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Phages use contingency loci as a bet-hedging strategy Jasper B. Gomez , Jeffrey E. Barrick , Christopher M. Waters bioRxiv 2025.10.21.683753; doi: https://doi.org/10.1101/2025.10.21.683753 Share This Article: Copy Citation Tools Phages use contingency loci as a bet-hedging strategy Jasper B. Gomez , Jeffrey E. Barrick , Christopher M. Waters bioRxiv 2025.10.21.683753; doi: https://doi.org/10.1101/2025.10.21.683753 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)