The O-antigen of Salmonella lipopolysaccharide is a major antigenic determinant and its chemical composition forms the basis for Salmonella serotyping. Modifications of the O-antigen that can affect the serotype include those carried out by the products of glycosyltransferase operons (gtr), which are present on specific Salmonella and phage genomes. Here we show that expression of the gtr genes encoded by phage P22 that confers the O1 serotype is under the control of phase variation. This phase variation occurs by a novel epigenetic mechanism requiring OxyR in conjunction with the DNA methyltransferase Dam. OxyR is an activator or a repressor of the system depending on which of its two binding sites in the gtr regulatory region is occupied. Binding is decreased by methylation at Dam target sequences in either site, and this confers heritability of the expression state to the system. Most Salmonella gtr operons share the key regulatory elements that are identified here as essential for this epigenetic phase variation.

The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions.

The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the ␣-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.Eubacterial genomes can encode DNA methyltransferases that are not part of restriction-modification systems, also referred to as orphan or solitary DNA methyltransferases. The best studied of these enzymes are two adenine methyltransferases: Dam, present in the ␥ subdivision of the Proteobacteria, and CcrM (for "cell cycle-regulated methyltransferase"), found in species of the ␣ subdivision. These methyltransferases are known to be involved in the control of many cellular events, including gene expression, cell cycle regulation, and DNA repair (reviewed in references 7 and 14). Where it has been examined, CcrM is essential for viability, whereas Dam and its homologs are essential in only a few species. In Escherichia coli and Salmonella enterica, Dam methyltransferase mutants have pleiotropic defects but are viable.The E. coli yhdJ gene, or b3262, was annotated as a putative methyltransferase-encoding gene (10), and the predicted protein contains conserved methyltransferase motifs for S-adenosyl-L-methionine (SAM) binding and catalysis. The order of these domains places YhdJ in the beta group of methyltransferases, along with CcrM and its homologs (12). E. coli YhdJ has identity with the adenine methyltransferases M. AvaIII (55.8%) and CcrM from Caulobacter crescentus (34.3%). Like CcrM but unlike M. AvaIII, yhdJ does not appear to be part of a restriction-modification system, as there is no gene encoding the former in close proximity.The existence of an essential DNA methyltransferase in E. coli could have significant implications for our understanding of DNA replication and cell cycle control. The recently published collection of in-frame E. coli deletions contains a yhdJ mutant, suggesting that YhdJ is not essential (1), whereas a previous report concluded that a gene deletion could not be obtained and that yhdJ expression was essential for viability (6). In this study, we therefore reexamine the role and function of the yhdJ gene product.The yhdJ gene is not essential, and its overexpression does not alter cell morphology. To address whether yhdJ is essential, the yhdJ coding sequence (except for the first and last codons) was replaced (9) with a chloramphenicol resistance gene (cat) in E. coli strain AB1157 (4), resulting in isolate GM8494. The ⌬yhdJ::cat mutation was transduced with P1vir (8) from GM8494 into E. coli WA802 (15) and designated GM8613. E. coli strain BW25113 and its yhdJ deletion derivative JW5543, from the E. coli gene knockout collection (1), were also analyzed. The yhdJ gene was also disrupted in S. enterica serovar Typhimurium LT2 by the procedure of Dats...