OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica

Published online 19 December 2015 Nucleic Acids Research, 2016, Vol. 44, No. 8 3595–3609 doi: 10.1093/nar/gkv1483 OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica Ignacio Cota1, Boyke Bunk2,3, Cathrin Spr¨ oer2,3,J¨ org Overmann2,3, Christoph K ¨ onig4and Josep Casades´ us1,* 1Departamento de Gen´ etica, Universidad de Sevilla, Facultad de Biolog´ ıa, Apartado 1095, 41080 Sevilla, Spain, 2Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, 38124 Braunschweig, Germany, 3German Centre of Infection Research (DZIF), Partner Site Hannover-Braunschweig, 38124 Braunschweig, Germany and 4Pacific Biosciences, 1380 Willow Rd, Menlo Park, CA 94025, USA Received October 5, 2015; Revised December 7, 2015; Accepted December 8, 2015 ABSTRACT Phase variation of the Salmonella enterica opvAB operon generates a bacterial lineage with standard lipopolysaccharide structure (OpvABOFF) and a lineage with shorter O-antigen chains (OpvABON). Regulation of OpvAB lineage formation is transcriptional, and is controlled by the LysR-type factor OxyR and by DNA adenine methylation. The opvAB regulatory region contains four sites for OxyR binding (OBSA-D), and four methylatable GATC motifs (GATC1–4). OpvABOFF and OpvABON cell lineages display opposite DNA methylation patterns in the opvAB regulatory region: (i) in the OpvABOFF state, GATC1 and GATC3are non-methylated, whereas GATC2and GATC4are methylated; (ii) in the OpvABON state, GATC2and GATC4are non-methylated, whereas GATC1and GATC3are methylated. We provide evidence that such DNA methylation patterns are generated by OxyR binding. The higher stability of the OpvABOFF lineage may be caused by binding of OxyR to sites that are identical to the consensus (OBSAand OBSc), while the sites bound by OxyR in OpvABON cells (OBSBand OBSD) are not. In support of this view, amelioration of either OBSBor OBSDlocks the system in the ON state. We also show that the GATCbinding protein SeqA and the nucleoid protein HU are ancillary factors in opvAB control. INTRODUCTION For decades, bacteriological research was based on the study of large populations of bacterial cells in batch cultures. This experimental approach assumed that the value of any parameter measured in the population would reflect a unimodal distribution around the average value in individual cells. This may be true for many cellular parameters. However, in the last two decades single cell analysis has shown that clonal populations of bacteria, even when growing in homogeneous environments, can exhibit phenotypic heterogeneity between individual cells (1–3). In certain cases, phenotypic heterogeneity reflects the occurrence of bistability, the formation of two subpopulations with distinct patterns of gene expression (4,5). Phenotypic diversity can be also generated by reversible ON-OFF switching of gene expression at high frequencies, a phenomenon known as phase variation (6,7). In bacterial pathogens, phase variation often occurs at loci that encode envelope structures and may be viewed as a strategy to generate programmed polymorphism (6,7). Indeed, lineage formation can help to evade the host immune system and to protect bacterial subpopulations against bacteriophage infection, among other potential adaptive advantages (7). The molecular mechanisms of phase variation are diverse. Some are genetic, such as site-specific recombination (8) and slipped-strand mispairing in tracts of repetitive DNA sequences (9). In other cases, however, the formation of bacterial lineages has epigenetic origin, without alteration of the DNA sequence (10–12). Some of the best known examples of epigenetic phase variation involve the formation of heritable DNA adenine (Dam) methylation patterns (10–12). The list includes the pap operon of uropathogenic Escherichia coli, which encodes fimbrial adhesins for adherence to the urinary tract epithelium (10,13), the agn43 aggregation gene of E. coli (14) and the glycosyltransferase operon gtr of Salmonella enterica (15). In all these cases, a transcriptional regulator binds a regulatory region that contains GATC sites, which become nonmethylated because binding of the regulator hinders Dam methylase activity. The OFF and ON states of the phase variation locus thus differ in the methylation state of crit- *To whom correspondence should be addressed. Tel: +34 95 455 7105; Fax +34 95 455 7104; Email: [email protected] C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 3596 Nucleic Acids Research, 2016, Vol. 44, No. 8 ical GATC sites (12). Because DNA base methylation often prevents or restrains binding of proteins to DNA, nonmethylation can increase binding of the regulatory protein, thus generating a positive feedback loop that propagates the epigenetic state (12). However, in all phase variation systems both the OFF and ON states are metastable, which permits phenotypic switching after a number of generations (6,7). The switching frequencies are idiosyncratic for each phase variation locus, and may vary depending on culture conditions (12). The opvAB locus of S. enterica serovar Typhimurium, previously annotated as STM2209-STM2208,isa Salmonella-specific locus that encodes cytoplasmic membrane proteins involved in control of O-antigen chain length (16). The opvA and opvB genes form a bicistronic transcriptional unit, which is transcribed from a canonical, ␴70-dependent promoter under the control of the LysR-type factor OxyR (16). Expression of opvAB is phase variable, and S. enterica batch cultures contain subpopulations of OpvABOFF and OpvABON cells. Each subpopulation harbors a distinct type of O-antigen, and OpvAB-mediated modification renders OpvABON cells resistant to bacteriophages that use the O-antigen as receptor (17). However, the OpvABON subpopulation shows sensitivity to serum, reduced capacity to proliferate in macrophages and attenuation in the mouse model (16,17). In this work we describe the epigenetic mechanism responsible for the formation of OpvABOFF and OpvABON cell lineages in S. enterica. Each lineage shows a distinct pattern of GATC methylation at the opvAB regulatory region. We present evidence that such patterns are generated by differential OxyR binding at the opvAB regulatory region. We also show that the GATC-binding protein SeqA and the nucleoid protein HU are ancillary factors for opvAB lineage formation. Finally, we present a model of opvAB phase variation, partly based upon experimental evidence, partly inspired by literature data and containing some speculative elements as well. MATERIALS AND METHODS Bacterial strains, bacteriophage, media and culture conditions The strains of S. enterica used in this study (Supplementary Table S1) belong to serovar Typhimurium, and originate from strain ATCC 14028. For simplicity, S. enterica serovar Typhimurium is routinely abbreviated as S. enterica.E. coli CC118 ␭pir [phoA20 thi-1 rspE rpoB argE(Am) recA1 (␭pir)]andE. coli S17–1 ␭pir [recA pro hsdR RP4–2Tc::Mu-Km::Tn7(␭pir)] were used for directed construction of point mutations. E. coli M15 [pREP4] (Qiagen, Valencia, CA, USA) was used for 6×His-OxyRC199S production. Plasmid pTP166 (18) was kindly provided by Martin G. Marinus, University of Massachusetts, Worcester, MA, USA. Bertani’s lysogeny broth (LB) was used as standard liquid medium. Solid LB contained agar at 1.5% final concentration. Green plates (19) contained methyl blue (Sigma-Aldrich, St Louis, MO, USA) instead of aniline blue. The indicator for monitoring ␤-galactosidase activity in plate tests was 5-bromo-4-chloro-3-indolyl-␤-Dgalactopyranoside (X-gal; Sigma-Aldrich, 40 ␮g/ml). Antibiotics were used at the concentrations described previously (20). To grow oxyR strains on LB agar, 75 ␮lofa 10 mg/ml catalase solution (Sigma-Aldrich, St Louis, MO, USA) were spread on the surface of the plates. Transductional crosses using phage P22 HT 105/1int201 (21) were used for construction of strains with altered chromosomal markers. The transduction protocol has been described elsewhere (22). To obtain phage-free isolates, transductants were purified by streaking on green plates. Phage sensitivity was tested by cross-streaking with the clearplaque mutant P22 H5. The oligonucleotides used in this study have either been described previously (16) or are listed in Supplementary Table S2. Gene disruption was achieved using plasmids pKD3, pKD4 and pKD13 (23) and oligonucleotides PS1,PS2 or PS4. Verification of the constructs was achieved using oligonucleotides E1 and E2. Antibiotic resistance cassettes introduced during strain construction were excised by recombination with plasmid pCP20 (23). Directed construction of point mutations Mutation of GATC sites within the opvAB regulatory region was achieved using previously described procedures and oligonucleotides (16). Additional primers are included in Supplementary Table S2. Antibiotic resistance cassettes from pKD3 and pKD4 were introduced in opvAB::lac and opvAB::gfp backgrounds using oligonucleotides delGATCPS1 and delGATC-PS2, respectively (16). The resulting strains were used as intermediates in the construction of point mutations. Mutation of OxyR binding sites was achieved in the same way using primers labeled OxyRB and OxyRD (Supplementary Table S2). ␤-galactosidase assays Bacterial cultures were grown in LB until stationary phase (O.D.600 ∼4). Levels of ␤-galactosidase activity were assayed using the CHCl3-sodium dodecyl sulfate permeabilization procedure (24). All data are averages and standard deviations from more than three independent experiments. Calculation of phase transition frequencies Phase transition rates were estimated as described by Eisenstein (25). Briefly, a strain harboring an opvAB::lac fusion wasplatedonLB+X-gal.After16hgrowthat37 oC, colonies displaying ON and OFF phenotypes were chosen, resuspended in phosphate buffered saline (PBS) and respread on new plates. Phase transition frequencies were calculated using the formula (M/N)/gwhere M is the number of cells that underwent a phase transition, N the total number of cells scored, and gthe total number of generations that gave rise to the colony. Flow cytometry Bacterial cultures were grown in LB at 37◦C until exponential phase (O.D.600 ∼0.3). Cells were then diluted in PBS to a final concentration of ∼107/ml. Data acquisition and Nucleic Acids Research, 2016, Vol. 44, No. 8 3597 analysis were performed using a Cytomics FC500-MPL cytometer (Beckman Coulter, Brea, CA, USA). Data were collected for 100 000 events per sample, and were analyzed with CXP and FlowJo8.7 software. Data are represented by a dot plot (forward scatter [cell size] versus fluorescence intensity [opvAB::gfp expression]). Construction of plasmid pIZ1885 (pQE30::oxyRC199S) A DNA fragment containing oxyRC199S (16) was amplified using oligonucleotides His-oxyR-BamHI-5 and HisoxyR-SalI-3, and cloned into pQE30 (Qiagen, Valencia, CA, USA) using the BamHI and SalI sites. The recombinant plasmid (pIZ1885) was verified by restriction analysis and DNA sequencing. Purification of OxyR protein For 6×His-OxyRC199S purification, plasmid pIZ1885 was transformed into E. coli M15 [pREP4] (Qiagen, Valencia, CA, USA). M15/pIZ1885 was grown in LB broth containing ampicillin, and expression of 6×His-OxyRC199S was induced with 1 mM isopropyl ␤-D-thiogalactopyranoside (IPTG). After 3 h of induction, cells were centrifuged and resuspended in 10 ml of lysis buffer (20 mM Tris, 300 mM NaCl, 10 mM imidazole) per g of pelleted cells, and were lysed by sonication. The suspension was centrifuged at 10 000 rpm for 30 min and the supernatant containing the soluble fraction of 6×His-OxyRC199S was transferred to a HisTrap HP nickel affinity chromatography column (GE Healthcare, Wauwatosa, WI, USA). The column was washed with 4 ml of lysis buffer, 4 ml of washing buffer (20 mM Tris, 300 mM NaCl, 30 mM imidazole) and 4 ml of the same buffer with 50 mM imidazole. Protein elution was performed with 3 ml of elution buffer (20 mM Tris, 300 mM NaCl, 300 mM imidazole). Elution fractions enriched in 6×His-OxyRC199S were selected and combined. Imidazole was removed by transferring to an Amicon R ultra centrifugal filter (Merck Millipore, Darmstadt, Germany) and washing with storage buffer (20 mM Tris, 300 mM NaCl, 10% glycerol) or by dialyzing in cellulose membranes (Sigma-Aldrich, St Louis, MO, USA). 6×His-OxyRC199S was either used immediately or frozen in liquid nitrogen and stored at −80oC. Gel mobility shift assay A DNA fragment containing predicted OxyR binding sites in the opvAB regulatory region and labeled with 6-carboxyfluorescein (6-FAM) was prepared by polymerase chain reaction (PCR) amplification using primers FAMGATClargo-5 and FAMGATClargo-3 (Supplementary Table S2). The PCR product was purified with the Wizard R  SV Clean-Up System (Promega). The envR control fragment was prepared using primers envR-For-Dnase and envR-Rev-Dnase (26), and was kindly provided by Elena Espinosa. Thirty five nanogram were used for each reaction. The FAM-labeled probe was incubated at room temperature for 30 min with increasing concentrations of purified 6×His-OxyRC199S in a final volume of 20 ␮l with 1×OxyR binding buffer [25 mM Tris–HCl pH 7.5, 50 mM KCl, 5 mM MgCl2,5%glycerol,50␮g/ml bovine serum albumin (BSA), 1 mM DTT, 1 ␮g/ml poly(dI-dC)]. Protein–DNA complexes were subjected to electrophoresis at 4◦C in a 5% non-denaturing polyacrylamide gel in Trisglycine-ethylenediaminetetraacetic acid (EDTA) buffer (25 mM Tris–HCl pH 7.5, 380 mM glycine, 1.5 mM EDTA). The gel was then analyzed in a FLA-5100 Scanner (Fujifilm, Tokyo, Japan). DNA methylation in vitro PCR fragments were methylated in vitro using Dam methylase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions and subsequently digested with MboI (New England Biolabs). The undigested product was purified using the Wizard R SV Clean-Up system (Promega, Madison, WI, USA). DNase I footprinting DNA probes containing the opvAB promoter and the upstream regulatory region, labeled with 6carboxyfluorescein (6-FAM) at the opposite ends, were prepared by PCR amplification using the primer pairs FAMGATClargo-5 +FAMGATClargo-3 and seqGATC-5 +FAMGATClargoconFAM-3. Dam-methylated versions of the probes were prepared as described above. DNase I footprinting was performed as described elsewhere (27) with minor modifications. DNase I footprinting reactions were performed in 15 ␮l reaction volumes containing 1× OxyR binding buffer and 2 ␮M6×His-OxyRC199S.The binding reaction was allowed to equilibrate at room temperature for 30 min. A total of 1 ␮l (0.05 units) of DNase I (Roche Farma, Barcelona, Spain) was then added, mixed gently and incubated at 37◦C for 5 min. The reaction was stopped by addition of 2 ␮l EDTA 100 mM followed by vigorous vortexing and thermal denaturation at 95◦Cfor 10 min. Digestion products were desalted using MicroSpin G-25 columns (GE Healthcare, Wauwatosa, WI, USA) and analyzed on an ABI 3730 DNA Analyzer along with GeneScan 500-LIZ size standards (Applied Biosystems, Foster City, CA, USA). SMRT R sequencing Cultures of S. enterica were enriched for OpvABON cells if needed (17). SMRTbellTM template libraries were prepared according to the instructions from Pacific Biosciences (Menlo Park, CA, USA), following the procedure and checklist for 1 kb template preparation and sequencing. Briefly, for preparation of 600 bp libraries, 4 ␮g of genomic DNA were sheared in microTubes using adaptive focused acoustics (Covaris, Woburn, MA, USA). Size range was monitored on an Agilent 2100 Bioanalyzer from Agilent Technologies, Santa Clara, CA, USA. DNAs were end-repaired and ligated to hairpin adapters applying components from the DNA Template Prep, Pacific Biosciences, Menlo Park, CA, USA. SMRTbellTM templates were exonuclease-treated for removal of incomplete reaction products. Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbellTM 3598 Nucleic Acids Research, 2016, Vol. 44, No. 8 templates were assessed with the Pacific Biosciences’ Binding Calculator. Six movies were taken for both states on the PacBio RSII (Pacific Biosciences, Menlo Park, CA, USA) using P4-C2 chemistry at 2 h collection time. Secondly, stationary phase cultures were enriched for OpvABON cells and libraries were prepared as given above. In this case five movies were taken using P4-C2 chemistry at 3 h collection time. Resulting data were mapped to the complete genome sequence (GenBank accession number CP001363.1) of S. enterica subsp. enterica serovar Typhimurium strain ATCC 14028, using the BLASR algorithm (28) as implemented in Pacific Biosciences’ SMRT R Portal 2.1.0 within the ‘RS Modification and Motif Analysis.1’ protocol applying default parameter settings. According to the setup of the experiment the secondary analysis jobs were named ‘OpvABOFF’ and ‘OpvABON’. Besides the global methylation pattern, the methylation status of four GATC sites upstream of the opvAB operon was inferred using SMRT R  View, investigating the chromosomal positions 2 361 489 and 2 361 490 (GATC1), 2 361 439 and 2 361 440 (GATC2), 2 361 416 and 2 361 417 (GATC3) and 2 361 366 and 2 361 367 (GATC4). Results are shown in supplementary .csv files S1 and S2 (OpvAB-OFF basemod summary and OpvABON basemod summary, respectively). Southern blot Genomic DNA was isolated by phenol extraction and ethanol precipitation from stationary cultures in LB (O.D.600 ∼4). A total of 16 ␮g of each DNA sample were digested with HaeIII and AccI (New England Biolabs, Ipswich, MA, USA), purified and divided into four fractions, three of which were subsequently digested with DpnI, MboI or Sau3AI (New England Biolabs). After digestion the samples were run in a 2% TAE-agarose gel at 100 V for 2 h. After electrophoresis, the DNA was denatured by treatment of the gel in acid conditions (0.25 M HCl, two washes 15 min each), followed by alkalinization (0.5 M NaOH, 1.5 M NaCl) and neutralization (0.5 M Tris, 1.5 M NaCl, pH 7.5; two washes, 30 min each). The gel was then washed in SSC 10×buffer (1.5 M NaCl, 150 mM trisodium citrate, pH 7) and the DNA was transferred by vacuum to an Amersham Hybond-N+ membrane (GE Healthcare, Wauwatosa, WI, USA) using a model 785 Vacuum Blotter (Bio-Rad, Hercules, CA, USA). The DNA in the membrane was then immobilized by UV crosslinking. A radioactive probe was prepared by PCR using dCTP [␣-32P] (Perkin Elmer, Waltham, MA, USA) and oligonucleotides 2208mut1DIRnuevo and 2208mut4INVnuevo (16). After the PCR reaction, nonincorporated nucleotides were removed by treatment in a Sephadex G-25 column (illustra MicroSpin G-25 columns, GE Healthcare, Wauwatosa, WI, USA) following the instructions of the manufacturer. Prior to hybridization the double-stranded DNA probe was denatured by heating at 95◦C for 3 min, followed by incubation on ice. Hybridization with the probe was performed overnight at 42◦Cinhybridization buffer (0.5 M sodium phosphate pH 7.2, 10 mM EDTA, 7% sodium dodecylsulphate (SDS)). Excess probe was removed with washing buffer (40 mM sodium phosphate pH 7.2, 1% SDS) at 38◦C (three washes, 30 min each). The membrane was developed using a FLA-5100 Scanner (Fujifilm, Tokyo, Japan). RESULTS Both the absence and the overexpression of Dam methylase increase opvAB expression and abolish phase variation Genes under Dam methylation control fall into two categories. One includes genes in which methylation and nonmethylation provide opposite signals (12). An example is the traJ gene of the Salmonella virulence plasmid, which is repressed by GATC methylation (29). In this class of genes, expression of the dam gene from a multicopy plasmid does not alter the wild-type phenotype (29). In other genes, however, a plasmid-borne dam gene does alter the gene expression pattern. This phenomenon is usually an indication that Dam dependent transcriptional control is more complex, and involves the formation of Dam methylation patterns (combinations of methylated and non-methylated GATC sites) (12). To ascertain whether opvAB belonged to the ‘simple’ or the ‘complex’ class of Dam methylationdependent genes, the effect of introducing a dam gene carried on plasmid pTP166 was assayed. The results were as follows: (i) In a wild-type background, an opvAB::lac translational fusion showed phase variation, and formed white (OpvABOFF) and blue (OpvABON) colonies in the presence of X-gal. In a dam background, phase variation was abolished, and all colonies were Lac+ (OpvABON). Plasmid pTP166 yielded an intermediate phenotype (Figure 1A), suggesting that formation of the OpvABOFF and OpvABON subpopulations might involve the establishment of a DNA methylation pattern in the GATC sites of the opvAB control region, rather than methylation or non-methylation of the full set of GATC sites. A similar phenomenon occurs in the gtr operon (15)whichisrepressedinadam background while introduction of a cloned dam gene results in an intermediate phenotype. (ii) Expression of opvAB::lac was also monitored by ␤- galactosidase assays (Figure 1B). Lack of Dam methylation increased expression of the opvAB operon as previously described (16). Introduction of the dam gene carried on the pTP166 plasmid yielded an intermediate opvAB expression level, as in the colonies described above. (iii) Expression of an opvAB::gfp transcriptional fusion was monitored by fluorescence analysis (Figure 1C). A major OpvABOFF subpopulation and a minor OpvABON subpopulation were detected in the wildtype. In a dam background, a single population in the ON state was observed, in accordance with the results obtained with a opvAB::lac fusion. In the presence of a cloned dam gene (pTP166), a single population with intermediate levels of expression was detected and a shift toward the ON state remained visible (Figure 1C). Altogether, the above observations suggested that DNA methylation patterns might be formed at the opvAB control region. This region, located upstream of the opvAB pro- Nucleic Acids Research, 2016, Vol. 44, No. 8 3599 Figure 1. Regulation of opvAB expression by Dam methylation and formation of OpvAB subpopulations. (A) Visual observation of phase variation on LB + X-gal plates in Salmonella enterica strains carrying an opvAB::lac fusion in the wild-type, a dam mutant and a strain that overproduced Dam methylase (ATCC 14028/pTP166). (B) Averages and standard deviations of ␤-galactosidase activity of the same strains. (C) GFP fluorescence distribution in a strain carrying an opvAB::gfp fusion in the same backgrounds. Data are represented by a dot plot (forward scatter [cellular size] versus fluorescence intensity [opvAB::gfp expression]). All data were collected for 100 000 events per sample. moter, contains four GATC sites separated by 46, 19 and 46 nt and centered at the −172.5, −122.5, −99.5 and −49.5 positions upstream of the transcription start site (Figure 2A and Supplementary Figure S1). From now on, these GATC sites will be referred to as GATC1to GATC4, the latter being closest to the −35 module of the opvAB promoter (Supplementary Figure S1). Roles of individual opvAB GATC sites in the formation of OpvABOFF and OpvABON cell lineages To study the contribution of each GATC site to opvAB regulation, mutations were introduced by site-directed mutagenesis. The mutations were designed to change GATC sites so that they would no longer be a substrate for Dam methylation. Because OxyR is essential for opvAB expression (16), alteration of consensus sequences was avoided inside putative OxyR binding sites. CATC sites were thus introduced in place of GATC sites, and every combination of mutated and non-mutated GATC sites was produced. The effect of GATC mutations on opvAB expression was first analyzed by comparing the ␤-galactosidase activity of an opvAB::lac translational fusion in dam+and dam backgrounds (Figure 2B). Relevant observations were as follows: (i) Mutation of GATC1and GATC3hadasmalleffect on regulation by Dam methylation, although the absolute values of ␤-galactosidase activity were higher. Mutation of GATC2resulted in diminished regulation by Dam methylation. When GATC4was mutated, control by Dam methylation showed an inverted pattern (expression was higher in a dam+background). (ii) As a general rule, combinations of two or more mutations seemed to have an additive effect. A remarkable case was the combination of mutated GATC2and GATC4which exacerbated the inversion of regulation by Dam methylation caused by mutation of GATC4 alone. It is noteworthy that mutations in GATC1, GATC2and GATC3together did not abolish Damdependent regulation, whereas a single mutation in GATC4inverted the pattern of Dam-dependent regulation. The overall conclusion from these experiments was that all four GATC sites are involved in Dam-dependent control of opvAB expression, and that the GATC4site may have an especially prominent role. Even though disruption of OxyR binding sites had been avoided, GATC mutations affected opvAB expression irrespective of the presence or absence of DNA methylation, as observed in a dam background (Figure 2B). In the absence of Dam methylation, mutations in GATC1and GATC3increased opvAB expression whereas mutations in GATC2 and GATC4resulted in lower opvAB expression. To separate such effects from those of Dam methylation itself, the ␤-galactosidase activity of opvAB::lac in a wild-type background was put in relation to the ␤-galactosidase activity in a dam background (Figure 2C). This representation leads to the interesting conclusion that mutations in GATC2and GATC4activate opvAB expression. Mutations in GATC1and GATC3show little effect on their own because opvAB expression is low in the wild-type, but they re- 3600 Nucleic Acids Research, 2016, Vol. 44, No. 8 Figure 2. Effect of mutations in the opvAB GATC sites on opvAB expression. (A) Diagram of the opvAB regulatory region, with the GATC sites and the OxyR binding sites outlined. (B) Averages and standard deviations of ␤-galactosidase activity of strains carrying an opvAB::lac fusion in a wild-type background (black bars) and in a dam background (white bars). Mutated GATC sites are indicated by numbers 1–4. (C)Relative␤- galactosidase activity of the opvAB::lac fusion in the same strains (activity in the wild-type divided by activity in a dam background). press opvAB expression when combined with activating mutations in GATC2and/or GATC4. Hence, the GATC sites in the opvAB regulatory region can be tentatively divided in two pairs: methylation of pair GATC1+GATC 3seems to be associated with the OpvABON state while methylation of pair GATC2+GATC 4seems to be associated with the OpvABOFF state. Analysis of fluorescence using an opvAB::gfp transcriptional fusion (Figure 3) allowed us to distinguish whether the differences in opvAB expression in GATC mutant backgrounds reflected differences in gene expression or differences in the sizes of the OpvABON and OpvABOFF subpopulations. The main observations were as follows: (i) In the wild-type, the OpvABON subpopulation comprised ∼0.18% cells. (ii) Mutation of GATC4caused a drastic increase in the size of the OpvABON subpopulation. Mutations in GATC1,GATC 2and GATC3had a smaller effect, which was more clearly seen when they were combined with each other and/or with a mutation in GATC4. (iii) Two subpopulations were still distinguished when three GATC sites were mutated, provided that either GATC3or GATC4remained unaltered. The relative size of the OpvABOFF and OpvABON subpopulations was however different in each case, with a predominant OpvABOFF subpopulation when GATC4remained unaltered and a predominant OpvABON subpopulation when GATC3remained unaltered. (iv) Mutation of both GATC3and GATC4eliminated subpopulation formation regardless of the presence of mutations in GATC1and GATC2, and yielded an OpvABON population. These observations are consistent with the gene expression analyses reported above, and permit to interpret the gene expression results in terms of subpopulation formation. Mutation of GATC4caused the most drastic increase in the proportion of OpvABON cells, thereby confirming that methylation of the GATC4site may have a relevant role in the formation of the OpvABOFF subpopulation. Increase of OpvABON subpopulation was likewise observed when a mutated GATC4was combined with other mutated GATC sites (Figure 3). OxyR binds the opvAB regulatory region Four putative OxyR binding half-sites are found in the regulatory region of opvAB centered in the −148, −116, −75 and −43 positions (Supplementary Figure S1), and sharing 10, 8, 10 and 7 nt respectively with the 10-nt consensus sequence (16). The OxyR binding half-sites upstream of the opvAB promoter will be from now on referred to as OBSAto OBSD, the latter being immediately upstream of the opvAB −35 promoter module (Figure 2A). Assuming a helical periodicity of 10.5 bp (30), the OBS are predicted to be spaced by one, two and one helical turns, which means that all the OxyR binding sites may be on the same face of the DNA helix. The distance between OBSAand OBSB, and between OBSCand OBSDas well, is canonical for binding of the reduced form of OxyR (31). GATC2and GATC4overlap with OBSBand OBSD, respectively (Figure 2A). To test whether OxyR binds the opvAB regulatory region, an electrophoretic mobility shift assay (EMSA) was carried out using purified OxyR protein (Figure 4A). To avoid uncontrolled oxidation of OxyR and because it was previously shown that the oxidation state of OxyR is not relevant for opvAB regulation (16), we used a mutant version of the OxyR protein, OxyRC199S, which cannot be oxidized but retains the properties of the reduced form of OxyR (31,32). Purified 6×His-OxyRC199S protein (henceforth named OxyR for simplicity) was thus used. A DNA fragment containing the four regulatory GATC sites and the four OxyR binding half-sites was produced using a 6FAM-labeled oligonucleotide and was incubated with increasing concentrations of OxyR. Binding was unambiguously detected. A DNA fragment from the regulatory region of an unrelated gene (envR) was used as a negative control, and binding was not detected (Figure 4A). Nucleic Acids Research, 2016, Vol. 44, No. 8 3601 Figure 3. GFP fluorescence distribution in Salmonella enterica strains carrying an opvAB::gfp fusion and mutations in the opvAB GATC sites. Mutated GATC sites are indicated by numbers 1–4. Data are represented by a dot plot, and were collected for 100 000 events per sample. OxyR protects the opvAB regulatory region To define the binding pattern of OxyR to the opvAB regulatory region, purified OxyR was used in a footprinting assay performed using 6-FAM-labeled DNA fragments and DNase I (Figure 4B). The same DNA fragment used in the EMSA assays, containing both the GATC sites and predicted OxyR binding sites, was labeled at the alternate ends and used in parallel experiments. Methylated and nonmethylated DNA probes were used, as well as a probe in which GATC sites 1–4 had been converted to CATC sites by site-directed mutagenesis. The analysis confirmed the ability of OxyR to bind the opvAB regulatory region in vitro (Figure 4and Supplementary Figure S2). Relevant observations were as follows: (i) Protection from DNase I digestion was detected in a 133 bp DNA span, albeit with regional differences. GATC1, GATC2, GATC3are located in the protected region. Fragment-specific binding patterns were detected and the overall protection was less efficient when the DNA probe was either methylated or GATC-less. (ii) The OBSAand OBSCsites were fully protected, while OBSBwas partially protected. (iii) OBSD, which contains the GATC4site, was not protected. The relevance of these observations may be limited as methylated and non-methylated DNA probes were used, and evidence presented above had suggested that opvAB regulation involved both methylated and non-methylated GATC sites (Figure 1). With this caveat, footprinting experiments confirmed the ability of OxyR to bind the opvAB regulatory region and defined the DNA region protected by OxyR binding. An additional, interesting observation was that OxyR protection extended outside the OxyR binding sites, as previously described for other LysR-type factors (33–36)(seebelow). 3602 Nucleic Acids Research, 2016, Vol. 44, No. 8 Figure 4. Binding of 6xHis-OxyRC199S to the opvAB promoter region. (A) Electrophoretic mobility shift assay of 6xHis-OxyRC199S binding to a DNA fragment containing the opvAB promoter and the upstream regulatory region. The regulatory region of envR was used as a negative control. (B) DNase I footprinting of 6xHis-OxyRC199S binding to DNA fragments containing the opvAB promoter and regulatory region with a 6-FAM label in either the top or the bottom strand. Methylated, non-methylated and GATC-less versions of the fragment were used. OpvABOFF and OpvABON subpopulations are characterized by inverse patterns of Dam methylation Single-molecule real-time (SMRT R ) sequencing results showed that >97% of the total of 38 458 GATC sites present in the genome of S. enterica serovar Typhimurium are methylated, and that non-methylated sites are the exception. Within this set, several non-methylated GATC sites were detected upstream of the opvAB operon. In order to analyze them in more detail, position-specific base modification analyses were performed. Addition of the virulent P22 H5 phage to a culture of S. enterica results in selection of the OpvABON subpopulation (17). Using this procedure, a culture was enriched in OpvABON cells and the methylation state of the opvAB GATC sites was analyzed using SMRT R sequencing (37). An ordinary culture, which contains >99% OpvABOFF cells (16,17), was also subjected to SMRT R sequencing. A total of 246 373 (430 408) polymerase reads with a mean polymerase read length of 10 010 (8516) bp and mean sequence coverage of 178×(329×) were obtained for the OpvABON (OpvABOFF) SMRT sequencing. The results from position-specific base modification analysis are shown in supplementary .csv files S1 and S2, and can be summarized as follows: (i) In an ordinary OpvABOFF culture, GATC1and GATC3 were non-methylated, whereas GATC2and GATC4 were methylated (Table 1). (ii) In the OpvABON culture, an inverse DNA methylation pattern was found: non-methylation of GATC2and GATC4and methylation of GATC1and GATC3(Table 1). These observations confirm that establishment of the OFF and ON states of the opvAB locus involves the formation of DNA methylation patterns, as in other phase variation loci under Dam methylation control (10,13,15). OxyR protects GATC sites from Dam methylation in vivo OxyR has been previously described as a DNA methylation-blocking factor, able to induce the formation of non-methylated GATC sites (15,38). To test whether OxyR has a similar DNA methylation-blocking ability in the opvAB operon, the methylation state of the GATC sites in the opvAB regulatory region was tested in vivo. For this purpose, a Southern blot was performed using genomic DNA extracted from the wild-type strain and from an oxyR mutant. The methylation state of individual GATC sites was inferred from restriction analysis using enzymes that cut GATC sequences depending on their methylation state (MboI, DpnI and Sau3AI). GATC1and GATC3were found to be non-methylated while GATC2 and GATC4were found to be methylated in the wild-type strain (Figure 5). In contrast, in an oxyR background, all four GATC sites were found to be methylated (Figure 5). These observations confirmed that OxyR has DNA methylation-blocking ability in vivo at the opvAB regulatory region. Mutations in the OBSBand OBSDOxyR binding sites abolish phase variation Of the four OxyR binding half-sites in the opvAB regulatory region, OBSAand OBSCare an absolute match (10 out of 10 nt) to the consensus sequences defined for OxyR binding (31). In contrast, OBSBand OBSDshare only 8 and 7 out of 10 nt with the consensus sequence, respectively. The fact that opvAB phase variation is skewed toward the OFF state led us to hypothesize that the degree of OxyR binding site perfection played a role in such bias. To test our hypothesis, 1 nt change was introduced in OBSBand two nucleotide changes in OBSDso that their mutated versions would share 9 out of 10 nt with the consensus sequence. Construction of a perfect consensus sequence was avoided since it would inevitably destroy GATC2and GATC4. The consequences of OBSBand OBSDDNA sequence amelioration were analyzed using opvAB::gfp (Figure 6A) and opvAB::lac fusions (Figure 6B). Mutations in either OBSBor OBSDabolished opvAB phase variation, yielding a uniform OpvABON population. In the case of OBSB,a single nucleotide change led also to full expression of the Nucleic Acids Research, 2016, Vol. 44, No. 8 3603 Table 1. DNA modification status according to SMRT R View for position specific base-modification analysis upstream of the opvAB operona Site Genome position OpvABOFF OpvABON GATC12 361 489+ unmodified (1.35, 31) m6A (2.76, 59) 2 361 490−unmodified (1.22, 31) m6A (3.99, 46) GATC22 361 439+ m6A (4.55, 55) unmodified (0.93, 49) 2 361 440−m6A (2.85, 54) unmodified (0.85, 37) GATC32 361 416+ unmodified (0.78, 55) m6A (2.29, 45) 2 361 417−unmodified (0.43, 55) m6A (2.15, 36) GATC42 361 366+ m6A (2.79, 45) unmodified (1.02, 52) 2 361 367−m6A (3.14, 37) unmodified (0.59, 47) aInter pulse duration ratios as well as strand-specific coverage values are given in parentheses. Figure 5. Methylation state of GATC sites in the opvAB regulatory region in wild-type and oxyR backgrounds. (A) Southern blot of genomic DNA obtained from wild-type and oxyR cultures and digested with HaeIII and with AccI (control) and DpnI, MboI or Sau3AI. Fragment sizes are indicated in base pairs. (B) Diagram of the HaeIII-AccI fragment and pattern of fragments obtained. operon. The mutation in OBSDcaused a smaller increase in expression and was epistatic to the mutation in OBSB. An interpretation of these observations is that OBSBand OBSDDNA sequence amelioration may ‘trap’ OxyR in the OpvABON configuration. In support of this view, absence of Dam methylation had no effect on opvAB expression in these mutant backgrounds (Figure 6). Hence, the preference of OxyR for certain OxyR-binding sites may be a key factor in regulation of opvAB phase variation, and alternative binding of OxyR upstream of the opvAB promoter may generate the OpvABOFF and OpvABON subpopulations. SeqA contributes to the small size of the OpvABON subpopulation SeqA was considered a potential ancillary candidate for regulation of opvAB since it binds GATC sites (39) and is involved in regulation of other phase variation loci (40,41). Thus we analyzed the effect of a seqA mutation on opvAB expression and its influence on the formation of OpvAB subpopulations. A strain carrying a seqA null allele and an opvAB::lac fusion formed darker (Lac+) colonies on LB + X-gal than the wild-type, and displayed frequent sectoring. Nonetheless, two groups of differently colored colonies (light blue and dark blue) were still distinguishable (Figure 7A), which allowed calculation of phase transition frequencies. The OFF→ON transition rate was found to be 50-fold higher in a seqA background (3.0 ×10−3compared with 6.1 ×10−5in the wild-type), whereas the ON→OFF transition rates were similar (3.1 ×10−2compared to 3.7 ×10−2in the wild-type). Not surprisingly, the ␤-galactosidase activity of an opvAB::lac fusion was ∼10-fold higher in a seqA background (Figure 7B). Fluorescence assays showed that mutation of seqA caused an increase in the size of the OpvABON subpopulation (Figure 7C). The effect was stronger in the presence of mutations in GATC1and/or GATC2, and to a lesser extent in GATC3(Supplementary Figure S1). Interestingly, when GATC4was mutated, a mutation in seqA had an effect opposite to that observed in the wild-type: the OpvABON subpopulation was reduced (Supplementary Figure S1). When both GATC3and GATC4were mutated, the seqA mutation did not have a significant effect (Supplementary Figure S1). These results seem to indicate that the main role of SeqA in the regulation of opvAB is the maintenance of a