Epigenetic Control of Salmonella enterica O-Antigen Chain Length: A Tradeoff between Virulence and Bacteriophage Resistance

RESEARCH ARTICLE Epigenetic Control of Salmonella enterica O- Antigen Chain Length: A Tradeoff between Virulence and Bacteriophage Resistance Ignacio Cota 1 , María Antonia Sánchez-Romero 1 , Sara B. Hernández 1¤ ,M. Graciela Pucciarelli 2,3 , Francisco García-del Portillo 2 , Josep Casadesús 1 * 1Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain, 2Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain, 3Departamento de Biología Molecular, Universidad Autónoma de Madrid, Centro de Biología Molecular Severo Ochoa (CBMSO-CSIC), Madrid, Spain ¤Current address: UmeåCentre for Microbial Research, UmeåUniversity, Umeå, Sweden *[email protected] Abstract The Salmonella enterica opvAB operon is a horizontally-acquired locus that undergoes phase variation under Dam methylation control. The OpvA and OpvB proteins form intertwining ribbons in the inner membrane. Synthesis of OpvA and OpvB alters lipopolysaccharide O-antigen chain length and confers resistance to bacteriophages 9NA (Siphoviridae), Det7 (Myoviridae), and P22 (Podoviridae). These phages use the O-antigen as receptor. Because opvAB undergoes phase variation, S.enterica cultures contain subpopulations of opvAB OFF and opvAB ON cells. In the presence of a bacteriophage that uses the O-antigen as receptor, the opvAB OFF subpopulation is killed and the opvAB ON subpopulation is selected. Acquisition of phage resistance by phase variation of O-antigen chain length requires a payoff: opvAB expression reduces Salmonella virulence. However, phase variation permits resuscitation of the opvAB OFF subpopulation as soon as phage challenge ceases. Phenotypic heterogeneity generated by opvAB phase variation thus preadapts Salmonella to survive phage challenge with a fitness cost that is transient only. Author Summary A tradeoff can increase the adaptive capacity of an organism at the expense of lowering the fitness conferred by specific traits. This study describes a tradeoff that confers bacteriophage resistance in Salmonella enterica at the expense of reducing its pathogenic capacity. Phase variation of the opvAB operon creates two subpopulations of bacterial cells, each with a distinct lipopolysaccharide structure. One subpopulation is large and virulent but sensitive to phages that use the lipopolysaccharide O-antigen as receptor, while the other is small and avirulent but phage resistant. In the presence of a phage that targets the O- antigen, only the avirulent subpopulation survives. However, phase variation permits resuscitation of the virulent opvAB OFF subpopulation as soon as phage challenge ceases. PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 1/18 OPEN ACCESS Citation: Cota I, Sánchez-Romero MA, Hernández SB, Pucciarelli MG, García-del Portillo F, Casadesús J (2015) Epigenetic Control of Salmonella enterica O- Antigen Chain Length: A Tradeoff between Virulence and Bacteriophage Resistance. PLoS Genet 11(11): e1005667. doi:10.1371/journal.pgen.1005667 Editor: Sangryeol Ryu, Seoul National University, REPUBLIC OF KOREA Received: June 5, 2015 Accepted: October 25, 2015 Published: November 19, 2015 Copyright: © 2015 Cota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants BIO2013-44220-R (to JC) and BIO2013-46281-P (to FGdP) from the Ministerio de Economía e Innovación of Spain and the European Regional Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. This transient tradeoff may illustrate the adaptive value of epigenetic mechanisms that generate bacterial subpopulations in a reversible manner. Introduction The study of differentiation in bacterial species that undergo developmental programs has played a historic role in biology [1,2,3]. In addition, phenotypic differences between colonies [4] and within colonies [5,6] were described many years ago in bacterial species that do not undergo development. Despite their technical limitations, these early studies contributed to bring about the idea that phenotypic heterogeneity might be a common phenomenon in the bacterial world [7]. This view has been confirmed by single cell analysis technologies [8,9,10,11,12]. Furthermore, theoretical analysis has provided evidence that phenotypic heterogeneity can have adaptive value, especially in hostile or changing environments [13,14,15]. In certain cases, the adaptive value of subpopulation formation is illustrated by experimental evidence [16,17,18]. Formation of bacterial lineages is governed by diverse mechanisms, including programmed genetic rearrangement [19] and contraction or expansion of DNA repeats at genome regions [20,21]. In other cases, however, lineage formation is controlled by epigenetic mechanisms: certain cell-to-cell differences serve as physiological signals, and signal propagation by a feedback loop generates an inheritable phenotype [12,22]. Cell-to-cell differences can be a consequence of environmental inputs or result from the noise intrinsical to many cellular processes [10,12,15]. In turn, the feedback loops that propagate the initial state beyond can be relatively simple or involve complex mechanisms like the formation of inheritable DNA adenine methylation patterns in the genome [12,23,24]. Some feedback loops are stable enough to cause bistability, the bifurcation of a bacterial population into two distinct phenotypic states [22]. If a feedback loop is metastable, reversion of the epigenetic state will occur after a certain number of cell divisions. Reversible bistability is usually known as phase variation, and typically involves reversible switching of gene expression from OFF to ON or from low to high expression [25,26,27]. Examples of phase variation have been described mostly in bacterial pathogens, and subpopulation formation is frequently viewed as a strategy that may facilitate evasion of the immune system during infection of animals [25,26]. This view is supported by the observation that phase-variable loci often encode envelope components or proteins involved in modification of the bacterial envelope [25,26]. Some phase-variable envelope modifications controlled by DNA adenine methylation play roles in bacteriophage resistance. For instance, phase variation in the gtrABC1 cluster protects S.enterica against the T5-like phage SPC35, probably by an indirect mechanism [28]. In Haemophilus influenzae, DNA adenine methylation controls phase-variable resistance to bacteriophage HP1c1 but the underlying mechanism remains hypothetical [29]. Phase variation can also contribute to phage resistance without alteration of the bacterial surface. For instance, certain genes encoding restriction-modification systems show phase variation [30,31]. In this study, we describe a phase variation system that confers resistance to bacteriophages that use the lipopolysaccharide (LPS) O-antigen as receptor. The genome of Salmonella enterica contains a horizontally-acquired locus, known as opvAB or STM2209-STM2208 [32]. The opvA and opvB genes form a bicistronic operon [32] and encode inner membrane proteins [32]. OpvA is a small peptide of 34 amino acids, and OpvB is a larger protein of 221 amino acids with homology to the Wzz superfamily of regulators of LPS O-antigen chain length [32]. We show that expression of the S.enterica opvAB operon confers resistance to bacteriophages Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 2/18 P22 (Podoviridae), 9NA (Siphoviridae), and Det7 (Myoviridae) by modification of the phage receptor, the LPS O-antigen. Because expression of opvAB is phase-variable, bacteriophage resistance occurs in the subpopulation of opvAB ON cells only. This subpopulation, which is extremely small, preadapts Salmonella to survive phage challenge albeit at the cost of reduced virulence. However, because the opvAB ON state is reversible, the virulence payoff is temporary, and a virulent bacterial population resuscitates as soon as phage challenge ceases. Results Localization of the OpvA and OpvB proteins in the S.enterica envelope OpvA and OpvB were previously shown to be inner membrane proteins involved in LPS synthesis [32]. Because the LPS is known to have a helical distribution in the cell envelope [33], the OpvA and OpvB subcellular localization was investigated. For this purpose, a chromosomal opvB::mCherry fusion was constructed downstream of the opvB gene (so that the strain remains OpvAB + ). In a wild type background, expression of opvB::mCherrywas low in most cells (Fig 1A). However, rare cells with high levels of expression of opvB::mCherry were detected (Fig 1A), an observation consistent with the occurrence of phase variation skewed towards the OFF state [32]. Expression of opvB::mCherry was also monitored in an opvAB- constitutive (opvAB ON ) strain engineered by elimination of GATC sites upstream of the opvAB promoter [32]. In an opvAB ON background, all cells displayed high levels of fluorescence, similar to those of the rare fluorescent cells visualized in a wild type background (Fig 1B). In Fig 1. Analysis of OpvA and OpvB localization by fluorescence microscopy. A. Localization of OpvB- mCherry in a wild type background. B. Localization of OpvB-mCherry in an opvAB ON strain. In both panels, cells enclosed in boxes are shown with higher magnification on the right. C. Localization of plasmid-borne OpvA-mCherry. D. Z-stacks of a single cell from panel C accompanied by an idealized representation of the ribbon-like protein distribution. Scale bar: 1 μm. doi:10.1371/journal.pgen.1005667.g001 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 3/18 fluorescent cells, OpvB was seen forming helical intertwining ribbons in the inner membrane (Fig 1A and 1B). The subcellular distribution of OpvA was examined using a plasmid-borne opvA::mCherry fusion. This experimental choice was based on the consideration that construction of an mCherry fusion in the upstream gene opvA would likely prevent opvB expression because of a polarity effect. In the strain carrying plasmid-borne opvA::mCherry, intense fluorescence was observed in all cells (Fig 1C), presumably because opvA::mCherry overexpression from the multicopy plasmid abolished phase variation. This construction was useful, however, to permit clear-cut observation of helical intertwining ribbons formed by OpvA (Fig 1D). The evidence that OpvA and OpvB may have a similar or identical distribution in the bacterial envelope is consistent with the physical interaction previously described between OpvA and OpvB [32]. Roles of OpvA and OpvB in control of O-antigen chain length Constitutive expression of opvAB leads to the production of a particular form of O-antigen in the S.enterica LPS, with a modal length of 3–8 repeat units [32]. A diagram of LPS structure is presented in S1 Fig, together with an electrophoretic separation of O-antigen chains and a diagram of the differences in LPS structure between opvAB OFF and opvAB ON pubpopulations. To investigate the role of individual OpvA and OpvB proteins in control of O-antigen chain length, non-polar mutations in opvA and opvB were constructed in the wild type and in an opvAB ON background. In the wild type, lack of either OpvA or OpvB did not alter the electrophoretic profile of LPS (Fig 2), an observation consistent with two known facts: the subpopulation of cells that express opvAB in wild type Salmonella is very small [32], and an OpvAB − mutant displays an LPS profile identical to that of the wild type [32]. In contrast, OpvA − opvB ON and opvA ON OpvB − mutants showed differences with the parental opvAB ON strain and also with the wild type: 1. Absence of OpvB (opvA ON ) yielded a seemingly disorganized LPS with no clear modal length (Fig 2), reminiscent of the LPS produced in the absence of the modal length regulators Wzz ST and Wzz fepE [34,35,36]. 2. Absence of OpvA (opvB ON ) yielded an LPS with the modal lengths typically conferred by Wzz ST and Wzz fepE [34] but also showed a preferred opvAB ON -like modal length in the lower weight band region (Fig 2). These observations suggested that the function of OpvA might be to prevent the formation of normal O-antigen so that OpvB could then impose its preferred modal length. To test this hypothesis, LPS structure was analyzed in an OpvA − opvB ON background in the absence of either Wzz ST or Wzz fepE . The results support the view that OpvB needs OpvA to prevent O- antigen formation by customary modal length regulators. In the absence of Wzz ST , OpvB alone was able to produce an O-antigen similar to that found in the opvAB ON strain (Fig 2). In contrast, lack of Wzz fepE did not seem to facilitate OpvB function, suggesting that OpvB may mainly compete with Wzz ST . This preference may be related to the fact that both Wzz ST and OpvB convey relatively short preferred modal lengths: 3–8 for OpvB [32] and 16–35 for Wzz ST [36,37,38] compared with >100 for Wzz fepE [34]. Selection of opvAB ON S.enterica cells upon bacteriophage challenge The LPS O-antigen is a typical receptor for bacteriophages [39] and modification of the O-anti- gen can confer bacteriophage resistance [40]. On these grounds, we tested whether opvAB expression increased Salmonella resistance to the virulent phages 9NA [41,42] and Det7 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 4/18 [43,44]. We also tested the historic phage P22, using a virulent mutant to avoid lysogeny [45]. Three strains (wild type, opvAB ON and ΔopvAB) were challenged with 9NA, Det7, and P22, which belong to different bacteriophage families and use the O-antigen as receptor. The experiments shown in Fig 3 were carried out by inoculating an exponential culture of S.enterica with an aliquot of a phage suspension at a multiplicity of infection (MOI) >10, and monitoring bacterial growth afterwards. The results can be summarized as follows: 1. Growth of the opvAB ON strain was not affected by the presence of 9NA, Det7, or P22, suggesting that the strain was resistant to these bacteriophages (S2 Fig). 2. A culture of the wild type strain became clear 1–2 hours after P22 infection, suggesting that cell lysis had occurred. However, bacterial growth was observed around 4 hours after infection, and was interpreted as occurrence of P22 resistance. Infection with either 9NA or Det7 did not cause clearing but growth retardation, and growth resumed 4 hours after infection (Fig 3). Fig 2. Analysis of the roles of OpvA and OpvB in the control of O-antigen chain length. LPS profiles of S.enterica strains carrying mutations in opvAB and O-antigen length regulator genes wzz ST and wzz fepE ,as observed by electrophoresis and silver staining. doi:10.1371/journal.pgen.1005667.g002 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 5/18 3. Cultures of the ΔopvAB strain infected with 9NA, Det7, or P22 became clear or almost clear. Growth was detected later, albeit with significant delay compared with the wild type. Because the wild type, the opvAB ON strain and the ΔopvAB strain show similar or identical growth rates in LB broth (S2 Fig), the explanation of this phenomenon is that growth of the ΔopvAB strain in the presence of 9NA, Det7, or P22 selects phage-resistant mutants (see below). A tentative interpretation of these observations was that the wild type strain contained a subpopulation of opvAB ON cells that survived phage challenge. Because opvAB phase variation is skewed towards the OFF state [32], the small size of the opvAB ON subpopulation and the regular formation of phage-sensitive opvAB OFF cells caused growth retardation (albeit to different degrees depending on the phage). In contrast, the opvAB ON strain grew normally, an observation consistent with the occurrence of phage resistance in the entire bacterial population. This Fig 3. Effect of phage challenge on S.enterica growth and LPS structure. Growth of the wild type strain (blue squares), an opvAB ON strain (green triangles) and a ΔopvAB strain (red circles) in LB + P22 (A), LB + 9NA (B), and LB + Det7 (C). Values are averages and standard deviations from 6 independent experiments. D. LPS structure of the wild type and opvAB ON strains after growth in LB, LB + P22, LB + 9NA, and LB + Det7. doi:10.1371/journal.pgen.1005667.g003 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 6/18 interpretation was supported by analysis of the LPS profiles of wild type and opvAB ON strains grown in the presence of P22, 9NA, and Det7 until stationary phase (OD 600 ~4) (Fig 3). After phage challenge, the wild type contained an LPS different from the LPS found in LB (Fig 3D), and similar or identical to the LPS found in the opvAB ON strain (Fig 2; see also [32]). In contrast, the LPS from the opvAB ON strain did not change upon phage challenge (Fig 3D). Confirmation that challenge of the wild type with P22, 9NA, and Det7 selected opvAB ON S. enterica cells was obtained by flow cytometry analysis (Fig 4). Expression of opvAB was monitored using a green fluorescent protein (gfp) fusion constructed downstream opvB (so that the strain remains OpvAB + ). In the absence of phage, most S.enterica cells expressed opvAB at low levels; however, a small subpopulation that expressed opvAB at high levels was also detected. Phage challenge yielded mostly S.enterica cells with high levels of opvAB expression. These observations provide additional evidence that phages P22, 9NA, and Det7 kill the opvAB OFF subpopulation, and that opvAB ON cells overtake the culture. Reversibility of OpvAB-mediated bacteriophage resistance If the above model was correct, we reasoned, cessation of phage challenge should permit resuscitation of a phage-sensitive subpopulation as a consequence of opvAB phase variation. This prediction was tested by isolating single colonies from cultures in LB + phage. After removal of phage by streaking on green plates, individual isolates were cultured in LB and re-challenged with P22, 9NA, and Det7 (20 isolates for each phage). All were phage-sensitive and their LPS profile was identical to that obtained before phage challenge. Representative examples are shown in Fig 5. Unlike the wild type, individual isolates of the ΔopvAB strain remained phageresistant after single colony isolation and were considered mutants (see below). Mutational bacteriophage resistance in the absence of OpvAB Challenge of a ΔopvAB strain with phages P22, 9NA, and Det7 prevented growth for 5–6h, and growth resumed afterwards (Figs 3and 5). To investigate the cause(s) of phage resistance in the absence of OpvAB, individual colonies were isolated from stationary cultures of a ΔopvAB strain in LB + P22, LB + 9NA, and LB + Det7. Phage was removed by streaking on green plates. Independent isolates (each from a different culture) were then tested for phage resistance. Sixty seven out of 72 independent isolates turned out to be phage-resistant, thus confirming that they were mutants. Analysis of LPS in independent phage-resistant mutants revealed that a large fraction of such mutants displayed visible LPS anomalies (Fig 6). The few mutant isolates (5/67) that did not show LPS alterations may have LPS alterations that cannot be detected in gels or carry mutations that confer phage resistance by mechanisms unrelated to the LPS. Whatever the case, these experiments support the conclusion that resistance of S. enterica to phages P22, 9NA, and Det7 in the absence of OpvAB is mutational. To determine whether isolates resistant to one phage were also resistant to other phages that target the O-antigen, cross-resistance was tested by growth in LB upon phage inoculation. Sixty seven mutants (24 P22-resistant, 24 9NA-resistant, and 19 Det7-resistant) were tested (S1 Table). The main conclusions from these experiments were as follows: 1. Major alteration of LPS conferred resistance to all three phages. 2. More subtle LPS alteration (as observed in 16/24 P22-resistant mutants) conferred incomplete resistance to 9NA but did not confer resistance to Det7. Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 7/18 Effect of opvAB expression on Salmonella enterica virulence Because the LPS plays roles in the interaction between S.enterica and the animal host [34,46,47], we tested whether OpvAB-mediated alteration of O-antigen chain length affected Salmonella virulence. For this purpose, competitive indexes (CI's) [48] were calculated in the following experiments: (i) oral and intraperitoneal inoculation of BALB/c mice; (ii) infection of Fig 4. Flow cytometry analysis of opvAB OFF and opvAB ON subpopulations. A. GFP fluorescence distribution in an ATCC 14028 derivative carrying an opvB::gfp fusion before (t = 0) and after growth in LB, LB + P22, LB + 9NA, and LB + Det7 (t = 8h). Data are represented by a dot plot (forward scatter [cellular size] versus fluorescence intensity [opvB::gfp expression]). All data were collected for 100,000 events per sample. B. Medians of GFP fluorescence from the same experiments. doi:10.1371/journal.pgen.1005667.g004 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 8/18 Fig 5. Reversibility of the phage-resistant phenotype in the wild type strain. Left: Growth of the wild type strain (blue squares), an opvAB ON strain (green triangles) and a ΔopvAB strain (red circles) in LB + P22 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 9/18 2. Kaiser D (1986) Control of multicellular development: Dictyostelium and Myxococcus. Annu Rev Genet 20: 539–566. PMID: 3028248 3. Stragier P, Losick R (1996) Molecular genetics of sporulation in Bacillus subtilis. Annu Rev Genet 30: 297–241. PMID: 8982457 4. Andrewes FW (1922) Studies in group agglutination. I. The Salmonella group and its antigenic structure. J Path Bact 25: 505–524. 5. Legroux R, Magrou J (1920) État organisé des colonies bactériennes. Ann Inst Pasteur (Paris) 34: 417–431. 6. Shapiro JA, Higgins NP (1989) Differential activity of a transposable element in Escherichia coli colonies. J Bacteriol 171: 5975–5986. PMID: 2553666 7. Shapiro JA (1998) Thinking about bacterial populations as multicellular organisms. Annu Rev Microbiol 52: 81–104. PMID: 9891794 8. Zgur-Bertok D (2007) Phenotypic heterogeneity in bacterial populations. Acta Agric Slovenica 90: 17– 24. 9. Dhar N, McKinney JD (2007) Microbial phenotypic heterogeneity and antibiotic tolerance. Curr Opin Microbiol 10: 30–38. PMID: 17215163 10. Davidson CJ, Surette MG (2008) Individuality in bacteria. Annu Rev Genet 42: 253–268. doi: 10.1146/ annurev.genet.42.110807.091601 PMID: 18652543 11. Veening JW, Smits WK, Kuipers OP (2008) Bistability, epigenetics, and bet-hedging in bacteria. Annu Rev Microbiol 62: 193–210. doi: 10.1146/annurev.micro.62.081307.163002 PMID: 18537474 12. Casadesus J, Low DA (2013) Programmed heterogeneity: epigenetic mechanisms in bacteria. J Biol Chem 288: 13929–13935. doi: 10.1074/jbc.R113.472274 PMID: 23592777 13. Thattai M, van Oudenaarden A (2004) Stochastic gene expression in fluctuating environments. Genetics 167: 523–530. PMID: 15166174 14. Kussell E, Leibler S (2005) Phenotypic diversity, population growth, and information in fluctuating environments. Science 309: 2075–2078. PMID: 16123265 15. Wolf DM, Vazirani VV, Arkin AP (2005) Diversity in times of adversity: probabilistic strategies in microbial survival games. J Theor Biol 234: 227–253. PMID: 15757681 16. Hernandez SB, Cota I, Ducret A, Aussel L, Casadesus J (2012) Adaptation and preadaptation of Salmonella enterica to bile. PLoS Genet 8: e1002459. doi: 10.1371/journal.pgen.1002459 PMID: 22275872 17. Sanchez-Romero MA, Casadesus J (2014) Contribution of phenotypic heterogeneity to adaptive antibiotic resistance. Proc Natl Acad Sci U S A 111: 355–360. doi: 10.1073/pnas.1316084111 PMID: 24351930 18. Ni M, Decrulle AL, Fontaine F, Demarez A, Taddei F, et al. (2012) Pre-disposition and epigenetics govern variation in bacterial survival upon stress. PLoS Genet 8: e1003148. doi: 10.1371/journal.pgen. 1003148 PMID: 23284305 19. Silverman M, Zieg J, Hilmen M, Simon M (1979) Phase variation in Salmonella: genetic analysis of a recombinational switch. Proc Natl Acad Sci U S A 76: 391–395. PMID: 370828 20. Moxon ER, Rainey PB, Nowak MA, Lenski RE (1994) Adaptive evolution of highly mutable loci in pathogenic bacteria. Curr Biol 4: 24–33. PMID: 7922307 21. Moxon R, Bayliss C, Hood D (2006) Bacterial contingency loci: the role of simple sequence DNA repeats in bacterial adaptation. Annu Rev Genet 40: 307–333. PMID: 17094739 22. Dubnau D, Losick R (2006) Bistability in bacteria. Mol Microbiol 61: 564–572. PMID: 16879639 23. Hernday A, Krabbe M, Braaten B, Low D (2002) Self-perpetuating epigenetic pili switches in bacteria. Proc Natl Acad Sci U S A 99: 16470–16476. PMID: 12202745 24. Casadesus J, Low D (2006) Epigenetic gene regulation in the bacterial world. Microbiol Mol Biol Rev 70: 830–856. PMID: 16959970 25. van der Woude MW (2011) Phase variation: how to create and coordinate population diversity. Curr Opin Microbiol 14: 205–211. doi: 10.1016/j.mib.2011.01.002 PMID: 21292543 26. van der Woude MW, Baumler AJ (2004) Phase and antigenic variation in bacteria. Clin Microbiol Rev 17: 581–611, table of contents. PMID: 15258095 27. van der Woude MW (2006) Re-examining the role and random nature of phase variation. FEMS Microbiol Lett 254: 190–197. PMID: 16445745 28. Kim M, Ryu S (2012) Spontaneous and transient defence against bacteriophage by phase-variable glucosylation of O-antigen in Salmonella enterica serovar Typhimurium. Mol Microbiol 86: 411–425. doi: 10.1111/j.1365-2958.2012.08202.x PMID: 22928771 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 16 / 18 29. Zaleski P, Wojciechowski M, Piekarowicz A (2005) The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection. Microbiology 151: 3361– 3369. PMID: 16207918 30. Srikhanta YN, Maguire TL, Stacey KJ, Grimmond SM, Jennings MP (2005) The phasevarion: a genetic system controlling coordinated, random switching of expression of multiple genes. Proc Natl Acad Sci U S A 102: 5547–5551. PMID: 15802471 31. Hoskisson PA, Smith MC (2007) Hypervariation and phase variation in the bacteriophage 'resistome'. Curr Opin Microbiol 10: 396–400. PMID: 17719266 32. Cota I, Blanc-Potard AB, Casadesus J (2012) STM2209-STM2208 (opvAB): a phase variation locus of Salmonella enterica involved in control of O-antigen chain length. PLoS One 7: e36863. doi: 10.1371/ journal.pone.0036863 PMID: 22606300 33. Ghosh AS, Young KD (2005) Helical disposition of proteins and lipopolysaccharide in the outer membrane of Escherichia coli. J Bacteriol 187: 1913–1922. PMID: 15743937 34. Murray GL, Attridge SR, Morona R (2003) Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Mol Microbiol 47: 1395–1406. PMID: 12603743 35. Goldman RC, Hunt F (1990) Mechanism of O-antigen distribution in lipopolysaccharide. J Bacteriol 172: 5352–5359. PMID: 1697578 36. Bastin DA, Stevenson G, Brown PK, Haase A, Reeves PR (1993) Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length. Mol Microbiol 7: 725–734. PMID: 7682279 37. Daniels C, Morona R (1999) Analysis of Shigella flexneri Wzz (Rol) function by mutagenesis and crosslinking: Wzz is able to oligomerize. Mol Microbiol 34: 181–194. PMID: 10540296 38. Batchelor RA, Alifano P, Biffali E, Hull SI, Hull RA (1992) Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation. J Bacteriol 174: 5228–5236. PMID: 1379582 39. Lindberg AA (1973) Bacteriophage receptors. Annu Rev Microbiol 27: 205–241. PMID: 4584686 40. Kintz E, Davies MR, Hammarlof DL, Canals R, Hinton JC, et al. (2015) A BTP1 prophage gene present in invasive non-typhoidal Salmonella determines composition and length of the O-antigen of the lipopolysaccharide. Mol Microbiol 96: 263–275. doi: 10.1111/mmi.12933 PMID: 25586744 41. Wilkinson RG, Gemski P Jr., Stocker BA (1972) Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J Gen Microbiol 70: 527–554. PMID: 4556257 42. Casjens SR, Leavitt JC, Hatfull GF, Hendrix RW (2014) Genome sequence of Salmonella phage 9NA. Genome Announc 2: e00531–14 doi: 10.1128/genomeA.00531-14 PMID: 25146133 43. Walter M, Fiedler C, Grassl R, Biebl M, Rachel R, et al. (2008) Structure of the receptor-binding protein of bacteriophage det7: a podoviral tail spike in a myovirus. J Virol 82: 2265–2273. PMID: 18077713 44. Casjens SR, Jacobs-Sera D, Hatfull GF, Hendrix RW (2015) Genome sequence of Salmonella enterica phage Det7. Genome Announc 3: e00279–00215. doi: 10.1128/genomeA.00279-15 PMID: 25953168 45. Smith HO, Levine M (1964) Two sequential repressions of DNA synthesis in the establishment of lysogeny by phage P22 and Its mutants. Proc Natl Acad Sci U S A 52: 356–363. PMID: 14206603 46. Raetz CR, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 635–700. PMID: 12045108 47. Bravo D, Silva C, Carter JA, Hoare A, Alvarez SA, et al. (2008) Growth-phase regulation of lipopolysaccharide O-antigen chain length influences serum resistance in serovars of Salmonella. J Med Microbiol 57: 938–946. doi: 10.1099/jmm.0.47848-0 PMID: 18628492 48. Beuzon CR, Holden DW (2001) Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo. Microbes Infect 3: 1345–1352. PMID: 11755424 49. Williams GC (1966) Natural selection, the costs of reproduction, and a refinement of Lack's principle. Am Nat 100: 687–690. 50. Nystrom T (2004) Growth versus maintenance: a trade-off dictated by RNA polymerase availability and sigma factor competition? Mol Microbiol 54: 855–862. PMID: 15522072 51. Shoval O, Sheftel H, Shinar G, Hart Y, Ramote O, et al. (2012) Evolutionary trade-offs, Pareto optimality, and the geometry of phenotype space. Science 336: 1157–1160. doi: 10.1126/science.1217405 PMID: 22539553 52. Bailly-Bechet M, Benecke A, Hardt WD, Lanza V, Sturm A, et al. (2011) An externally modulated, noise-driven switch for the regulation of SPI1 in Salmonella enterica serovar Typhimurium. J Math Biol 63: 637–662. doi: 10.1007/s00285-010-0385-1 PMID: 21107576 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 17 / 18 53. Ferenci T, Spira B (2007) Variation in stress responses within a bacterial species and the indirect costs of stress resistance. Ann N Y Acad Sci 1113: 105–113. PMID: 17483210 54. Andersson DI, Hughes D (2010) Antibiotic resistance and its cost: is it possible to reverse resistance? Nat Rev Microbiol 8: 260–271. doi: 10.1038/nrmicro2319 PMID: 20208551 55. De Paepe M, Gaboriau-Routhiau V, Rainteau D, Rakotobe S, Taddei F, et al. (2011) Trade-off between bile resistance and nutritional competence drives Escherichia coli diversification in the mouse gut. PLoS Genet 7: e1002107. doi: 10.1371/journal.pgen.1002107 PMID: 21698140 56. Vincent BM, Lancaster AK, Scherz-Shouval R, Whitesell L, Lindquist S (2013) Fitness trade-offs restrict the evolution of resistance to amphotericin B. PLoS Biol 11: e1001692. doi: 10.1371/journal.pbio. 1001692 PMID: 24204207 57. Leon M, Bastias R (2015) Virulence reduction in bacteriophage resistant bacteria. Front Microbiol 6: 343. doi: 10.3389/fmicb.2015.00343 PMID: 25954266 58. Taylor VL, Udaskin ML, Islam ST, Lam JS (2013) The D3 bacteriophage alpha-polymerase inhibitor (Iap) peptide disrupts O-antigen biosynthesis through mimicry of the chain length regulator Wzz in Pseudomonas aeruginosa. J Bacteriol 195: 4735–4741. doi: 10.1128/JB.00903-13 PMID: 23955007 59. Susskind MM, Botstein D (1978) Molecular genetics of bacteriophage P22. Microbiol Rev 42: 385– 413. PMID: 353481 60. Whitfield C, Trent MS (2014) Biosynthesis and export of bacterial lipopolysaccharides. Annu Rev Biochem 83: 99–128. doi: 10.1146/annurev-biochem-060713-035600 PMID: 24580642 61. Wilson GG, Murray NE (1991) Restriction and modification systems. Annu Rev Genet 25: 585–627. PMID: 1812816 62. Lee DJ, Bingle LE, Heurlier K, Pallen MJ, Penn CW, et al. (2009) Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiol 9: 252. doi: 10.1186/ 1471-2180-9-252 PMID: 20003185 63. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645. PMID: 10829079 64. Hautefort I, Proenca MJ, Hinton JC (2003) Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells. Appl Environ Microbiol 69: 7480–7491. PMID: 14660401 65. Chan RK, Botstein D, Watanabe T, Ogata Y (1972) Specialized transduction of tetracycline by phage P22 in Salmonella typhimurium. II. Properties of a high frequency transducing lysate. Virology 50: 883– 898. PMID: 4565618 66. Torreblanca J, Casadesús J (1996) DNA adenine methylase mutants of Salmonella typhimurium and a novel Dam-regulated locus. Genetics 144: 15–26. PMID: 8878670 67. Buendia-Claveria AM, Moussaid A, Ollero FJ, Vinardell JM, Torres A, et al. (2003) A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide. Microbiology 149: 1807–1818. PMID: 12855732 68. Segura I, Casadesus J, Ramos-Morales F (2004) Use of mixed infections to study cell invasion and intracellular proliferation of Salmonella enterica in eukaryotic cell cultures. J Microbiol Methods 56: 83– 91. PMID: 14706753 Epigenetic Control of O-Antigen Chain Length PLOS Genetics | DOI:10.1371/journal.pgen.1005667 November 19, 2015 18 / 18