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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

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.

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.

Shigella is an important human pathogen. It is generally agreed that Shigella and Escherichia coil constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E. coil and other Shigella forms and seems to represent another species. This gives S. boydii type 13 an important status in evolution. O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gramnegative bacteria and plays an important role in pathogenicity. The chemical structure and genetic organization of the S. boydii type 13 O antigen were investigated. The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected [sup.1]H, [sup.13]C and [sup.1]H, [sup.31]P HMQC experiments, along with chemical methods. The O antigen gene cluster of S. boydii type 13 was located and sequenced. Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified. Seven genes were found to be specific to S. boydii type 13. The S. boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters.

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.

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 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.

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.

DNA sequence analysis of the asd genes from 45 isolates of Vibrio cholerae revealed variation between the two forms of environmental nontoxigenic isolates and among 19 clinical O1 isolates, but no differences in the sixth- and seventh-pandemic isolates. Phylogenetic trees of Vibrio cholerae with V. mimicus as the outgroup showed that sixth- and seventh-pandemic and US Gold isolates evolved from the different lineages of environmental nontoxigenic O1 isolates.

Examination of 58 Vibrio cholerae epidemiologically unassociated isolates and isolated from patients of different countries from 1931 to 1993 by ribotyping helps analyze the genetic variation and molecular evolution of the seventh-pandemic clone of Vibrio cholerae and its relationship to other pandemic and epidemic V. cholerae isolates. Major differences in ribotypes exist between sixth and pre-seventh-pandemic isolates. O139 Bengal isolates are similar to isolates from the early seventh-pandemic isolates. Majority of seventh-pandemic isolates fall into two groups and O139 strains evolved from seventh-pandemic isolates have changed twice, in the 1970s and in 1992.

rfbT is not a separate gene and is not involved in ligation. Mutations in the rfbP gene are referred to as rfbP(T) mutations. The rfbP gene encodes bifunctional proteins and the galactosyl-1-phosphate transferase gene. The T function is associated with the flipping of the UndPP-linked single O unit from one face to the other face of the inner membrane.

The group D2 strain of Salmonella enterica is used to clone the rfb gene cluster. Mapping, hybridization and sequential analysis show that the group D2 rfb gene is similar to the D1 group in which the mannosyl transferase gene rfbO is replaced with the E1-specific mannosyl transferase rfbO. An insertion sequence-mediated recombination event between groups D1 and E1 results in the group D2 rfb gene organization.

E. coli K-12 gene organization belongs to the congregation of rfb gene cluster, which has segments homologous to those of Dysenteriae and Flexneri. Study of DNA sequences and comparison with other sequences reveal the presence of conserved, powerful regulatory regions within E. coli and S. enterica clones K-12 O16. O antigen synthesis is rfb-dependent for the conversion of Glc NAc to UndP and resembles that of Dysentriae and Flexneri.

Presence of a specific phage such as P22 is a prerequisite for effecting transduction in Escherichia coli. P22's characteristic properties render it indispensable to transductional crosses within a particular bacterial species. P22 maintains its functionality when present in aggregates of phages, and facilitates the production of high-titer stocks. It also aids the isolation of high-frequency transduction and mutants lacking integration. Its receptor is identified as a group B polymerized O antigen of Salmonella enterica.

Exploration of the genetic variation of O antigens of Salmonella enterica involved the study of 164 strains belonging to 45 serogroups. Twenty-five single-gene rfb DNA probes used for colony hybridization revealed that the strains in the serogroups were identical to the rfb genes. A minimum of three of the four rhamnose genes were discovered in all the 17 serogroups which contained rhamnose and at least one was discovered in three others. Mannose pathway genes were less common, and of the 27 groups studied, only nine hybridized to the rfbM and rfbK probes.

The rfb gene cluster codes for the nucleotide sugar biosynthetic pathway and the transferases required for assembly of the repeating units of the O antigen in lipopolysaccharides. Three rhamnosyl tranferase and four mannosyl transferase genes of the O serogroups S, C2 and E1 in Salmonella enterica were identified and characterized. The nucleotide sequences of the different transferase genes were used to analyze the evolution of the rfb gene cluster.

The region of the Yersinia pseudotuberculosis serogroup IIA genome coding for the enzymes for abequose biosynthesis, rfb, was sequenced and compared with the segment of Salmonella enterica genome containing the genes for dideoxyhexose biosynthesis. Results indicate that the two segments have similar gene sequence and remarkable similarities with respect to base composition. However, conserved base and amino acid sequences differ between the two groups suggesting that the two pathways have undergone independent evolution.

A bacterial strain is developed to facilitate the determination of glycosyltransferase (GT) specificities for O antigens of known structure and gene cluster sequence. The strain is used for determining the specificity of the Escherichia coli O111 O-antigen GT genes.

Gene cloning and expression studies reveal that 43 of the 53 flagellar H-antigen types are encoded by genes at the filC locus and the other 10 mapping to separate loci in Escherichia coli. Sequence comparison indicates that both horizontal DNA transfer and fixation of mutations give rise to polymorphism in the filC locus.