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Beneficial mutations in diversifying glucose-limited Escherichia coli populations are mostly unidentified. The genome of an evolved isolate with multiple differences from that of the ancestor was fully assembled. Remarkably, a single mutation in hfq was responsible for the multiple benefits under glucose limitation through changes in at least five regulation targets. doi: 10.1128/JB.00368-10

Despite longstanding vaccination programs, pertussis remains endemic to many industrialized countries, including Australia, Canada, Italy, Japan, the Netherlands, Switzerland, and the United States, all of which have reported recent increases [...]

The genome of an Escherichia coli MC4100 strain with a [lambda] placMu50 fusion revealed numerous regulatory differences from MG1655, including one that arose during laboratory storage. The 194 mutational differences between MC4100(MuLac) and other K-12 sequences were mostly allocated to specific lineages, indicating the considerable mutational divergence between K-12 strains.

Streptococcus pneumoniae (the pneumococcus) produces 1 of 91 capsular polysaccharides (CPS) that define the serolype. The cps loci of 88 pneumococcal serotypes whose CPS is synthesized by the Wzy-dependent pathway were compared with each other and with additional streptococcal polysaccharide biosynthetic loci and were clustered according to the proportion of shared homology groups (HGs), weighted for the sequence similarities between the genes encoding the shared HGs. The cps loci of the 88 pneumococcal serotypes were distributed into eight major clusters and 21 subclusters. All serotypes within the same serogroup fell into the same major cluster, but in six cases, serotypes within the same serogroup were in different subclusters and, conversely, nine subclusters included completely different serotypes. The closely related cps loci within a subcluster were compared to the known CPS structures to relate gene content to structure. The Streptococcus oralis and Streptococcus mitis polysaccharide biosynthetic loci clustered within the pneumococcal cps loci and were in a subcluster that also included the cps locus of pneumococcal serotype 21, whereas the Streptococcus agalactiae cps loci formed a single cluster that was not closely related to any of the pneumococcal cps clusters.

The sequences of the capsular biosynthetic (cps) loci of 90 serotypes of Streptococcus pneumoniae have recently been determined. Bioinformatic procedures were used to predict the general functions of 1,973 of the 1,999 gene products and to identify proteins within the same homology group, Pfam family, and CAZy glycosyltransferase family. Correlating eps gene content with the 54 known capsular polysaccharide (CPS) structures provided tentative assignments of the specific functions of the different homology groups of each functional class (regulatory proteins, enzymes for synthesis of CPS constituents, polymerases, flippases, initial sugar transferases, glycosyltransferases [GTs], phosphotransferases, acetyltransferases, and pyruvyltransferases). Assignment of the glycosidic linkages catalyzed by the 342 GTs (92 homology groups) is problematic, but tentative assignments could be made by using this large set of cos loci and CPS structures to correlate the presence of particular GTs with specific glycosidic linkages, by correlating inverting or retaining linkages in CPS repeat units with the inverting or retaining mechanisms of the GTs predicted from their CAZy family membership, and by comparing the CPS structures of serotypes that have very similar cps gene contents. These large-scale comparisons between structure and gene content assigned the linkages catalyzed by 72% of the GTs, and all linkages were assigned in 32 of the serotypes with known repeat unit structures. Clear examples where very similar initial sugar transferases or glycosyltransferases catalyze different linkages in different serotypes were also identified. These assignments should provide a stimulus for biochemical studies to evaluate the reactions that are proposed.

The O-antigen moiety of the LPS is one of the most variable cell surface components of the Gram-negative bacterial outer membrane. Variation is due to the presence of different sugars and sugar linkages. Here, it is reported that a group of Escherichia coli O serogroups (017, 044, 073, 077 and 0106), and the Salmonella enterica serogroup 0:6,14 (H), share a common four-sugar backbone O-subunit structure, and possess almost identical O-antigen gene clusters. Whereas the E. coli 077 antigen does not have any substitutions, the other O antigens in this group differ by the addition of one or two glucose side branches at various positions of the backbone. The O-antigen gene clusters for all members of the group encode only the proteins required for biosynthesis of the common four-sugar backbone. The identification of three genes within a putative prophage in the E. coli 044 genome is also reported; these genes are presumably involved in the glucosylation of the basic tetrasaccharide unit. This was confirmed by deletion of one of the genes, which encodes a putative glucosyltransferase. Structural analysis of the O antigen produced by the mutant strain demonstrated the absence of glucosylation. An O-antigen structure shared by five E. coli and one S. enterica serogroups, all of which have a long evolutionary history, suggests that the common backbone may be important for the survival of E. coli strains in the environment, or for their pathogenicity.

Shigella dysenteriae type 1 is the most virulent serotype of Shigella. Enterotoxigenic Escherichia coli O148 is pathogenic and can cause diarrhoea. The following structure was established for the tetrasaccharide repeating unit of the E. coli O148 O antigen: [right arrow] 3)-[alpha](-L-Rhap-(1 [right arrow] 3)-[alpha](-L-Rhap-(1 [right arrow] 2)-[alpha]-D-Glcp-(1[right arrow] 3)-[alpha]-D-GlcpNAc-(1 [right arrow]. This differs from the structure reported earlier for S. dysenteriae type 1 by having a glucose (GIc) residue in place of a galactose (Gal) residue. The two bacteria also have the same genes for O antigen synthesis, with the same organization and high level of DNA identity, except that in S. dysenteriae type 1 wbbG is interrupted by a deletion, and a galactosyltransferase gene wbbP located on a plasmid is responsible for the transfer of galactose to make a novel antigenic epitope of the O antigen. The S. dysenteriae type 10 antigen was reconstructed by replacing the E. coli O148 wbbG gene with the wbbP gene, and it had the LPS structure and antigenic properties of S. dysenteriae type 1, indicating that the S. dysenteriae type 1 O antigen evolved from that of E. coli O148. It was also confirmed that wbbG of E. coli O148 is a glucosyltransferase gene, and two serotype-specific genes of E. coli O148 and S. dysenteriae type 1 were identified.

Serovar Typhimurium of Salmonella enterica is a model organism for studies of pathogenesis that exhibits phage-type variation and variation in host range and virulence, but in a recent study showed no sequence variation in four genes, indicating the clonal nature of this serovar. We determined the relationships of 46 Typhimurium isolates of nine phage types using mutational changes detected either by matching AFLP (amplified fragment length polymorphism) fragments to computer-modeled LT2 AFLP fragments or by sequencing intergenic regions. Fifty-one polymorphic sites were detected, which gave a single phylogenetic tree. Comparison with genome sequences of five other serovars, Typhi, Paratyphi A, Gallinarum, Enteritidis, and Pullorum, enabled determination of the root of the tree. Only two parallel events were observed, giving high confidence in the tree branching order. The mutation-based tree provided a high level of consistency and a clear lineage for the Typhimurium isolates studied. This enabled us to show that for seven of the nine phage types used, the isolates studied have a single origin, but that two phage types clearly have more than one independent origin. We found that sequencing intergenic regions provides a good strategy for detection of mutational polymorphisms and study of phylogenetic relationships of closely related isolates and would be applicable to many other species.

Shigella strains are nonmotile. The master operon of flagellar synthesis, flhDC, was analyzed for genetic damage in 46 Shigella strains representing all known serotypes. In 11 strains (B1, B3, B6, B8, B10, B18, D5, F1B, D10, F3A, and F3C) the flhDC operon was completely deleted. PCR and sequence analysis of the flhDC region of the remaining 35 strains revealed many insertions or deletions associated with insertion sequences, and the majority of the strains were found to be defective in their flhDC genes. As these genes also play a role in regulation of nonflagellar genes, the loss may have other consequences or be driven by selection pressures other than those against flagellar motility. It has been suggested that Shigella strains fall mostly into three clusters within Escherichia coli, with five outlier strains, four of which are also within E. coli (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). The distribution of genetic changes in the flhDC region correlated very well with the three clusters and outlier strains found using housekeeping gene DNA sequences, enabling us to follow the sequence of mutational change in the flhDC locus. Two cluster 2 strains were found to have unique flhDC sequences, which are most probably due to recombination during the exchange of the adjacent O-antigen gene c lusters.

Comparison of the O antigens of Shigella boydii types 10 and 6 by chemical analysis and nuclear magnetic resonance spectroscopy showed that their structures are similar, with the only difference being the presence or absence of D-ribofuranose, which is the immunodominant sugar in S. boydii type 10. In S. boydii type 6, a residue previously reported as [alpha]-D-GlcpA, was shown to be [beta]-D-GlcpA as in S. boydii type 10. S. boydii types 10 and 6 are reported not to cross-react serologically, and the role of D-ribofuranose in the specificity of S. boydii was confirmed by making a mutant of type 10 that lacked D-ribofuranose. However, S. boydii type 11, which has a D-ribofuranose but with different linkage does show cross-reaction with type 10. The O-antigen gene loci of S. boydii types 10 and 6 were shown to be virtually identical except that orf8 (wbaM), which was confirmed as the ribofuranosyltransferase gene, is interrupted by IS629 in type 6. Therefore, it is proposed that the O-antigen gene cluster of S. boydii type 6 was derived from type 10 by an IS element insertion.

Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-n-glucose (GlcNAc), 2-acetamidoylaminn-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Sahnonella enterica serovar Touera and S. enterica subsp, arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes.

The molecular basis of the loss of tryptophan utilization (indole-negative phenotype) of Shigella strains, in effect clones of Escherichia coli, was investigated. Analysis of the tna operon of 23 Shigella strains representing each of the indole-negative serotypes revealed that insertion sequence-mediated insertion and/or deletions damaged the tna operon, leading to inability to convert tryptophan to indole. These events differ for cluster 1, cluster 3, and the outlier Shigella strains, confirming our previous observation of independent origins of these lineages from within E. coli. Parallel loss of the trait and prevalence of indole-negative strains suggest that the trait is deleterious in Shigella strains and advantages those without it.

Escherichia coli O157, Salmonella enterica 030, and Citrobacterfreundii F90 have identical O-antigen structures, as do E. coli 055 and S. enterica O511. The O-antigen gene cluster sequences for E. coli O157 and E. coli 055 have been published, and the genes necessary for O-antigen biosynthesis have been identified, although transferase genes for glycosidic linkages are only generic and have not been allocated to specific linkages. We determined sequences for S. enterica 030 and C. freundii F90 O-antigen gene clusters and compared them to the sequence of the previously described E. coil 0157 cluster. We also determined the sequence of the S. enterica 050 O-antigen gene cluster and compared it to the sequence of the previously described E. coli 055 cluster. For both the S. enterica O30-C. freundii F90-E. coli O157 group and the S. enterica O50-E. coli 055 group of O antigens, the gene clusters have identical or nearly identical organizations. The two sets of gene clusters had comparable overall levels of similarity in their genes, which were lower than the levels determined for housekeeping genes for these species, which were 55 to 65% for the genes encoding glycosyltransferases and O-antigen processing proteins and 75 to 93% for the nucleotide-sugar pathway genes. Nonetheless, the similarity of the levels of divergence in the five gene clusters required us to consider the possibility that the parent gene cluster for each structure was in the common ancestor of the species and that divergence is faster than expected for the common ancestor hypothesis. We propose that the identical O-antigen gene clusters originated from a common ancestor, and we discuss some possible explanations for the increased rate of divergence that is seen in these genes.

The structural and genetic organization of the Escherichia coli O52 O antigen was studied. As identified by sugar and methylation analysis and nuclear magnetic resonance spectroscopy, the O antigen of E. coli O52 has a partially O-acetylated disaccharide repeating unit (O unit) containing D-fucofuranose and 6-deoxy-D-manno. heptopyranose, as well as a minor 6-deoxy-3-O-methylhexose (most likely, 3-O-methylfucose). The O-antigen gene cluster of E. coli O52, which is located between the galF and grid genes, was found to contain putative genes for the synthesis of the O-antigen constituents, sugar transferase genes, and ABC-2 transporter genes. Further analysis confirmed that O52 employs an ATP-binding cassette (ABC) transporter-dependent pathway for translocation and polymerization of the O unit. This is the first report of an ABC transporter being involved in translocation of a heteropolysaccharide O antigen in E. coil Genes specific for E. coli O52 were also identified.

The genetic variation in the dTDP-L-rhamnose pathway genes (rmlA, rmlB, rmlC and rmlD) in Vibrio cholerae was investigated. The genes are part of the O antigen gene cluster and the aim was to study lateral gene transfer of O antigen gene clusters. The rml genes of an O6 strain were cloned using an Escherichia coli K-12 strain designed for selecting cloned rml genes. Thirty-three strains carrying the known rhamnose-containing O antigens were probed with O6-based rml gene probes, and 19 were positive with from one to all four of the gene probes. Nine rml gene sets from this group were sequenced and found to be in the order rmlBADC, at the 5' end of the gene clusters. A gradient in the level of variation was observed, with highly similar sequences at the 5' end rmlB gene, but very divergent and strain-specific sequences at the 3' end of the rml gene set. The change in level of similarity varied in position, but was always abrupt and coincided with a change in GC content, indicating that the 5' and 3' parts are of different origin, and that recombination within rml genes has occurred. The rml gene sets of two of the strains that did not hybridize with any O6 rml gene probes were also cloned and sequenced. Both gene sets were in the middle of the O antigen gene cluster and were very divergent from each other and all other rml gene sets. This supports the hypothesis that presence of rml genes at the end of the O antigen gene cluster facilitates lateral gene transfer of rml-containing O antigen gene clusters in V. cholerae. The sequence relationships make it possible to identify sites of recombination and to distinguish DNA that has long been in V. cholerae and DNA that probably came into the species with the O antigen gene cluster.

Research has been conducted on Escherichia coli O55, an antigen associated with enteropathogenic E. coli clones. The genetics of this antigen has been investigated via sequencing of the O55 antigen genes and the results are described.

Research has been conducted on Salmonella enterica serogroup. Results indicate that these serogroups have different O-antigens, each with a specific O-antigen cluster form.

Results demonstrate that several Salmonella enterica strains exhibit a manB gene, which display a 93 to 99% DNA identity to the colanic acid manB gene of the bacterium. However, the manC genes differ from those of manB genes indicating that these two gene sets have evolved independently.

Researchers show that the O antigen of Salmonella has been transferred between species and subspecies of S. enterica. The O antigen is a surface component of Gram-negative bacteria.

Remnant chromosomal O-antigen genes from the Escherichia coli clone Sonnei were cloned and characterized. The results suggested that Sonnei used to have a O-antigen gene cluster, a large portion of which disappeared. The selective advantage provided by the O antigen encoded by the plasmid-borne genes for adaptation to new environments and the eventual inactivation were proposed to explain the deletion. Moreover, a possible relative of the E. coli Sonnei was determined through sequencing and analysis of the polymerase chain reaction.

The molecular basis of variation in Vibrio cholera ribotypes is investigated in 47 isolates over 33 years through the analysis of specific enzyme markers and the intergenic spacers between the genes for RNA located at the 16S and 23S ribosomal subunits. Results reveal at least eight recombination events and deletion of tandem operons giving rise to a high degree of ribotype variability. Further, the recombination rate is very high as evidenced by the discovery of new ribotypes within the study period.

The model system for Escherichia coli with O111 genes was used to determine the chain length specificity of the wzz genes from O1, O2, O7, and O157 strains. wzz genes from Escherichia coli strains O1, O2, O7, and O157 were cloned and transferred to P4657 which lacks O antigen genes. The results indicate that the O antigen chain length of the Escherichia coli O1, O2, O7, and O157 strains were altered by replacing specific amino acids in the wzz, suggesting that amino acid variation in wzz is responsible for the varying chain length of the O antigen.

Sequencing of the spacer region between the 16S and 23S ribosomal operons, rrnA and rrnE, respectively, was undertaken to determine variation and investigate phylogeny in Salmonella enterica subspecies. The variation in this DNA segment was found to be due to insertions and deletions and the degree of variability is much higher compared to that in Escherichia coli. The results of phylogenetic analyses are consistent with the accepted phylogenetic relationships in S. enterica subspecies.

The bacterial species Vibrio cholerae includes harmless aquatic strains as well as strains capable of causing epidemics and global pandemics of cholera. While investigating the relationship between pathogenic and nonpathogenic strains, we identified a chromosomal pathogenicity island (PAI) that is present in epidemic and pandemic strains but absent from nonpathogenic strains. Initially, two ToxR-regulated genes (aldA and tagA) were studied and were found to be associated with epidemic and pandemic strains but absent in nontoxigenic strains. The region containing aldA and tagA comprises 13 kb of previously unidentified DNA and is part of a PAI that contains a regulator of virulence genes (ToxT) and a gene cluster encoding an essential colonization factor and the cholera toxin phage receptor (toxin-coregulated pilus; TCP). The PAI is 39.5 kb in size, has low %G+C (35%), contains putative integrase and transposase genes, is flanked by att sites, and inserts near a 10Sa RNA gene (ssrA), suggesting it may be of bacteriophage origin. We found this PAI in two clinical non-O1/non-O139 cholera toxin-positive strains, suggesting that it can be transferred within V. cholerae. The sequence within this PAI includes an ORF with homology to a gene associated with the type IV pilus gene cluster of enteropathogenic Escherichia coli, a transposase from Vibrio anguillarum, and several ORFs with no known homology. As the PAI contains the CTX[Phi] receptor, it may represent the initial genetic factor required for the emergence of epidemic and pandemic cholera. We propose to call this island VPI (V. cholerae pathogenicity island).

Research was conducted to examine the O-antigen gene cluster of Salmonella enterica group D3 strain M840. In this study, specific attributes of the D3 O antigen were identified through the examination of the said gene cluster using techniques such as Southern blotting. Results show the existence of novel beta-mannosyl transferase and wzy genes in the central region of the M840 strain.

A study is conducted to identify a fucose synthetase gene found in the K-12 colanic acid gene cluster of Escherichia coli. This gene is believed to encode a protein that facilitates the epimerase and reductase reaction components for the synthesis of GDP-L-fucose. GDP-L-fucose, a derivative of GDP-D-mannose, is a substrate that plays a role in the mixture of fucose to polysaccharides or glycoproteins.

The sequence of K-12 chromosome in Escherichia coli, which includes a 19-gene cluster responsible for the production of colanic acid, has been determined. The cluster includes genes for GDP-L-fucose synthesis and for the enzyme, GDP-D-mannose 4,6-dehydratase involved in this pathway. The first three genes encode a phosphatase, an outer and an inner membrane lipoprotein, and have homologs in other extracellular polysaccharide gene clusters. These help in the transport of polysaccharide from the cell.

The C-terminal end of the Salmonella enterica wbaP gene controls the initial galactosyltransferase (GT) step of O-antigen synthesis as wbaP with an intact C-terminal and a mutant N-terminal retains GT activity. The C-terminal controls the transfer of galactosyl-1-phosphate from GDP-galactose to undecaprenyl phosphate. The N-terminal controls the later stage (T) of the synthesis which involves the release of undecaprenyl pyrophosphate-Gal from WbaP. The T stage occurs before the O-antigen flips from the cytoplasmic end to the periplasmic end of the cytoplasmic membrane.

The wzx (rbfX) gene, which is characteristic of the major class of O-antigen clusters in gram-negative bacteria, encodes a hydrophobic protein with 12 potential transmembrane segments. The wzx mutant accumulates undecaprenol pyrophosphate-linked O units which seem to be on the cytoplasmic side of the cytoplasmic membrane. This indicates that the wzx gene encodes a flippase for O-unit translocation across that membrane.

Escherichia coli O8 and O9 strains of wzz genes which are identical to and have sequences within the range of variation of those which determine the chain length of typical O-antigens, were discovered. The wzz genes which have previously been found to affect O-antigen chain length, when cloned and transferred to O8 and O9 strains, affected the chain length of a capsule-relate form of LPS, K(sub LPS).

The genes encoding abequosyltransferase and acetyltransferase, utilized for O-antigen-specific biosynthesis in Salmonella enterica groups B and C2, have been identified by in vitro assay. From S. enterica groups A and D, the paratosyl-, tyvelosyl- and abequosyltransferase genes and, from Yersinia pseudotuberculosis, serovar IIA have also been detected. The genes involved in O-antigen synthesis are grouped in the rfb gene cluster. Comparison of dideoxyhexosyltransferases and acetyltransferases is also discussed.