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The Wzx/Wzy-dependent pathway is the predominant pathway for O-antigen production in Gram-negative bacteria. The O-antigen repeat unit (O unit) is an oligosaccharide that is assembled at the cytoplasmic face of the membrane on undecaprenyl pyrophosphate. Wzx then flips it to the periplasmic face for polymerization by Wzy, which adds an O unit to the reducing end of a growing O-unit polymer in each round of polymerization. Wzx and Wzy both exhibit enormous sequence diversity. It has recently been shown that, contrary to earlier reports, the efficiency of diverse Wzx forms can be significantly reduced by minor structural variations to their native O-unit substrate. However, details of Wzy substrate specificity remain unexplored. The closely related galactose-initiated Salmonella O antigens present a rare opportunity to address these matters. The D1 and D2 O units differ only in an internal mannose-rhamnose linkage, and D3 expresses both in the same chain. D1 and D2 polymerases were shown to be specific for O units with their respective α or β configuration for the internal mannose-rhamnose linkage. The Wzy encoded by D3 gene cluster polymerizes only D1 O units, and deleting the gene does not eliminate polymeric O antigen, both observations indicating the presence of an additional wzy gene. The levels of Wzx and Wzy substrate specificity will affect the ease with which new O units can evolve, and also our ability to modify O antigens, capsules or secreted polysaccharides by glyco-engineering, to generate novel polysaccharides, as the Wzx/Wzy-dependent pathway is responsible for much of the diversity.

O antigens are polysaccharides consisting of repeat units of three to eight sugars, generally assembled by genes in a discrete O antigen gene cluster. Salmonella enterica produces 46 forms of O antigen, and most of the variation is determined by genes in the gene cluster. However in some cases the structures are modified by enzymes encoded outside of the gene cluster, and several such modifications have been reported for Salmonella enterica group E, some with the genes on bacteriophages and one gene at a distant chromosomal site. We identified the enzyme, WbaK, that is responsible for O-acetylating the subgroup E1 O antigen, and found that the gene is located just downstream of the gene cluster as currently known. The wbaK gene appears to have been imported by a recombination event that also replaced the last 37 bp of the wbaP gene, indicating that homologous recombination was involved. Some of the group E strains we studied must have the original gene cluster, as they lack wbaK and the sequence downstream of wbaP is very similar to that in several other S. enterica O antigen gene clusters. In effect the gene cluster was extended by one gene in subgroup E1. It appears that a function that is usually encoded by a gene outside of the gene cluster has been added to the gene cluster, in this case giving an example of how such gene clusters can evolve.

Bacterial populations in clinical and laboratory settings contain a significant proportion of mutants with elevated mutation rates (mutators). Mutators have a particular advantage when multiple beneficial mutations are needed for fitness, as in antibiotic resistance. Nevertheless, high mutation rates potentially lead to increasing numbers of deleterious mutations and subsequently to the decreased fitness of mutators. To test how fitness changed with mutation accumulation, genome sequencing and fitness assays of nine Escherichia coli mutY mutators were undertaken in an evolving chemostat population at three time points. Unexpectedly, the fitness in members of the mutator subpopulation became constant despite a growing number of mutations over time. To test if the accumulated mutations affected fitness, we replaced each of the known beneficial mutations with wild-type alleles in a mutator isolate. We found that the other 25 accumulated mutations were not deleterious. Our results suggest that isolates with deleterious mutations are eliminated by competition in a continuous culture, leaving mutators with mostly neutral mutations. Interestingly, the mutator-non-mutator balance in the population reversed after the fitness plateau of mutators was reached, suggesting that the mutator-non-mutator ratio in populations has more to do with competition between members of the population than the accumulation of deleterious mutations.

Background: Evolutionary divergence is common within bacterial species and populations, even during a single bacterial infection. We use large-scale genomic and phenotypic analysis to identify the extent of diversification in controlled experimental populations and apply these data to differentiate between several potential mechanisms of evolutionary divergence., Results: We defined testable differences between five proposed mechanisms and used experimental evolution studies to follow eight glucose-limited Escherichia coli chemostat populations at two growth rates. Simple phenotypic tests identified 11 phenotype combinations evolving under glucose limitation. Each evolved population exhibited 3 to 5 different combinations of the 11 phenotypic clusters. Genome sequencing of a representative of each phenotypic cluster from each population identified 193 mutations in 48 isolates. Only two of the 48 strains had evolved identically. Convergent paths to the same phenotype occurred, but two pleiotropic mutations were unique to slow-growing bacteria, permitting them greater phenotypic variance. Indeed, greater diversity arose in slower-growing, more stressed cultures. Mutation accumulation, hypermutator presence and fitness mechanisms varied between and within populations, with the evolved fitness considerably more uniform with fast growth cultures. Negative frequency-dependent fitness was shown by a subset of isolates., Conclusions: Evolutionary diversity is unlikely to be explained by any one of the available mechanisms. For a large population as used in this study, our results suggest that multiple mechanisms contribute to the mix of phenotypes and evolved fitness types in a diversifying population. Another major conclusion is that the capacity of a population to diversify is a function of growth rate.

Salmonella enterica serovar Typhimurium is one of the leading causes of gastroenteritis in humans. Phage typing has been used for the epidemiological surveillance of S. Typhimurium for over 4 decades. However, knowledge of the evolutionary relationships between phage types is very limited. In this study, we used single nucleotide polymorphisms (SNPs) as molecular markers to determine the relationships between common S. Typhimurium phage types. Forty-four SNPs, including 24 identified in a previous study and 20 from 6 available whole-genome sequences, were used to analyze 215 S. Typhimurium isolates belonging to 45 phage types. Altogether, 215 isolates and 6 genome strains were differentiated into 33 SNP profiles and four distinctive phylogenetic clusters. Fourteen phage types, including DT9, one of the most common phage types in Australia, were differentiated into multiple SNP profiles. These SNP profiles were distributed into different phylogenetic clusters, indicating that they have arisen independently multiple times. This finding suggests that phage typing may not be useful for long-term epidemiological studies over long periods (years) and diverse localities (different countries or continents). SNP typing provided a discriminative power similar to that of phage typing. However, 12 SNP profiles contained more than one phage type, and more SNPs would be needed for further differentiation. SNP typing should be considered as a replacement for phage typing for the identification of S. Typhimurium strains.

Neisseria meningitidis is a leading pathogen of epidemic bacterial meningitis and fulminant sepsis worldwide. Twelve different N. meningitidis serogroups have been identified to date based on antigenic differences in the capsular polysaccharide. However, more than 90% of human cases of N. meningitidis meningitis are the result of infection with just five serogroups, A, B, C, W135, and Y. Efficient methods of detection and genogrouping of N. meningitidis isolates are needed, therefore, in order to monitor prevalent serogroups as a means of disease control and prevention. The capsular gene complex regions have been sequenced from only seven out of the 12 serogroups. In this study, the capsular gene complexes of the remaining five serogroups were sequenced and analyzed. Primers were designed that were specific for N. meningitidis species and for the 12 individual serogroups, and a multiplex PCR assay using these specific primers was developed for N. meningitidis detection and genogrouping. The assay was tested using 15 reference strains covering all 12 serogroups, 143 clinical isolates, and 21 strains from closely related species or from species that cause meningitis. The assay could detect N. meningitidis serogroups and was shown to be specific, with a detection sensitivity of 1 ng of genomic DNA (equivalent to ∼4 × 10(5) genomes) or 3 × 10(5) CFU/ml in noncultured mock cerebrospinal fluid (CSF) specimens. This study, therefore, describes for the first time the development of a molecular protocol for the detection of all N. meningitidis serogroups. This multiplex PCR-based assay may have use for the clinical diagnosis and epidemiological surveillance of N. meningitidis.

Although over 50 complete Escherichia coli/Shigella genome sequences are available, it is only for closely related strains, for example the O55:H7 and O157:H7 clones of E. coli, that we can assign differences to individual evolutionary events along specific lineages. Here we sequence the genomes of 14 isolates of a uropathogenic E. coli clone that persisted for 3 years within a household, including a dog, causing a urinary tract infection (UTI) in the dog after 2 years. The 20 mutations observed fit a single tree that allows us to estimate the mutation rate to be about 1.1 per genome per year, with minimal evidence for adaptive change, including in relation to the UTI episode. The host data also imply at least 6 host transfer events over the 3 years, with 2 lineages present over much of that period. To our knowledge, these are the first direct measurements for a clone in a well-defined host community that includes rates of mutation and host transmission. There is a concentration of non-synonymous mutations associated with 2 transfers to the dog, suggesting some selection pressure from the change of host. However, there are no changes to which we can attribute the UTI event in the dog, which suggests that this occurrence after 2 years of the clone being in the household may have been due to chance, or some unknown change in the host or environment. The ability of a UTI strain to persist for 2 years and also to transfer readily within a household has implications for epidemiology, diagnosis, and clinical intervention.

Despite high vaccine coverage, pertussis incidence has increased substantially in recent years in many countries. A significant factor that may be contributing to this increase is adaptation to the vaccine by Bordetella pertussis, the causative agent of pertussis. In this study, we first assessed the genetic diversity of B. pertussis by microarray-based comparative genome sequencing of 10 isolates representing diverse genotypes and different years of isolation. We discovered 171 single nucleotide polymorphisms (SNPs) in a total of 1.4 Mb genome analyzed. The frequency of base changes was estimated as one per 32 kb per isolate, confirming that B. pertussis is one of the least variable bacterial pathogens. We then analyzed an international collection of 316 B. pertussis isolates using a subset of 65 of the SNPs and identified 42 distinct SNP profiles (SPs). Phylogenetic analysis grouped the SPs into six clusters. The majority of recent isolates belonged to clusters I-IV and were descendants of a single prevaccine lineage. Cluster I appeared to be a major clone with a worldwide distribution. Typing of genes encoding acellular vaccine (ACV) antigens, ptxA, prn, fhaB, fim2, and fim3 revealed the emergence and increasing incidence of non-ACV alleles occurring in clusters I and IV, which may have been driven by ACV immune selection. Our findings suggest that B. pertussis, despite its high population homogeneity, is evolving in response to vaccination pressure with recent expansion of clones carrying variants of genes encoding ACV antigens.

The O antigen, consisting of many repeats of an oligosaccharide unit, is part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. It is on the cell surface and appears to be a major target for both immune system and bacteriophages, and therefore becomes one of the most variable cell constituents. The variability of the O antigen provides the major basis for serotyping schemes of Gram-negative bacteria. The genes responsible for the synthesis of O antigen are usually in a single cluster known as O antigen gene cluster, and their location on the chromosome within a species is generally conserved. Three O antigen biosynthesis pathways including Wzx/Wzy, ABC-transporter and Synthase have been discovered. In this chapter, the traditional and molecular O serotyping schemes are compared, O antigen structures and gene clusters of well-studied species are described, processes for formation and distribution of the variety of O antigens are discussed, and finally, the role of O antigen in bacterial virulence.

The genus Salmonella consists of two species S. enterica and S. bongori. S. enterica has a well defined subspecies structure with seven subspecies consistently delineated by sequence variation. Frequency of recombination between subspecies and within a subspecies is markedly different. Subspecies I undergoes frequent recombination as demonstrated recently, demystifying the long-held belief that Salmonella is a highly clonal organism. The majority of disease causing serovars are from subspecies I with the most important serovars in human health being Typhimurium and Typhi. Typhimurium has developed considerable diversity and may be a very old serovar. The majority of the isolates belong to a single clonal complex by multilocus sequence typing. Typhimurium isolates are divided into phage types and some of the phage types do not have a single origin as determined using mutational changes. Phage type DT104 is heterogeneous and represented in multiple sequence types, with its multidrug-resistant variant most successful causing epidemics in many parts of the world. Typhi, a human restricted serovar, is relatively young compared to Typhimurium, and has a low level of sequence variation. Single nucleotide polymorphisms (SNPs) have been shown to be very useful for typing and resolving relationships within Typhi. Genome sequences of 19 isolates revealed more than 1700 SNPs. The fully resolved phylogenetic tree allows one to trace the mutational changes occurred during clonal diversification. Genome wide SNPs have greatly enhanced our understanding of the evolution of Salmonella clones.

Expression of olive flounder hepcidin I (HepI) fused with truncated OmpA signal peptides (OmpASP(tr)) as directional signals does not produce soluble fusion proteins. However, by inserting amino acid segments (xxx) varying in pI and hydrophobicity/hydrophilicity into a leader sequence containing a truncated OmpASP (OmpASP(tr)) and a factor Xa cleavage site (Xa) [OmpASP(tr)|(xxx)|Xa], we were able in some cases to express soluble recombinant HepI. Soluble expression of the recombinant protein strongly correlated with (xxx) insertions of high pI and hydrophilicity. Therefore, we modified the OmpASP(tr)|(xxx)|Xa sequence by inserting Arg and Lys into (xxx) to increase the hydrophilicity of the signal peptide region. These modifications enhanced the expression of soluble recombinant HepI. Hydropathic profile analysis of the OmpASP(tr)|(xxx)|Xa| HepI fusion proteins revealed that the transmembrane-like domains derived from the OmpASP(tr)|(xxx)|Xa sequence were larger than the internal positively charged domain native to HepI. It should therefore be possible to overcome the obstacle of internal positively charged domains to obtain soluble expression of recombinant proteins by monitoring the hydrophilicity and hydropathic profile of the signal peptide region using a computer program.

We have sequenced the O-antigen gene clusters for the Escherichia coli O98 and Yersinia kristensenii O11 O antigens. The basic structures of these O antigens are identical, and the sequence data indicate that Y. kristensenii O11 gained its O-antigen gene cluster by lateral gene transfer (LGT). Escherichia coli O98 has a typical O-antigen gene cluster between galF and gnd as is usual in E. coli. However, the O-antigen gene cluster of Y. kristensenii O11 is not located at the traditional Yersinia O-antigen gene cluster locus, between hemH and gsk, but at a novel chromosomal locus between aroA and cmk where it is flanked by remnant galF and gnd genes that indicate the probable source of the gene cluster. Phylogenetic analysis indicated that the source was not E. coli itself but a species in the Escherichia, Salmonella, and Klebsiella group of genera. Although other O-antigen studies imply LGT on the basis of the hypervariability of the loci and GC content, this report also identifies a potential donor and provides evidence for the mechanism involved. Remnant insertion sequence (IS) sequences flank the galF and gnd remnants and suggest that LGT of the gene cluster was IS mediated.

Amplified fragment length polymorphism (AFLP) is a powerful PCR-based fingerprinting method and has the capacity to reveal variation around the whole genome by selectively amplifying a subset of restriction fragments for comparison. The restriction fragments analyzed are small, and even mutation of 1 bp can be detected. The use of different sets of restriction enzymes or different primer combinations can generate large numbers of different AFLP fingerprints. AFLP is of particular value for studies of closely related strains, such as analysis of variation within a serovar of Salmonella enterica. We present here protocols for both radioactively labeled and fluorescent dye-labeled AFLP analyses that are also applicable to other bacterial species. Fluorescent AFLP has proved to be reproducible and capable of standardization.

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.

Shigella, which still stands as a genus with four species today, in reality belongs to the extremely diverse species Escherichia coli. There are several lineages of Shigella strains derived through independent acquisition of the pINV virulence plasmid. The chromosomally determined phenotypic properties of Shigella result from convergent evolution during niche adaptation, most due to loss of function, some from negative selection pressure.

The evolutionary history of the GDP-mannose pathway in Salmonella enterica was studied via sequencing manB and manC genes from 13 representative strains for O antigens containing mannose and/or sugar derivatives of GDP-D-mannose. In addition, colanic acid (CA) manB and manC genes were sequenced from selected strains, as the basis for a detailed comparison. Interestingly, including the eight previously characterized O antigen gene clusters, 12 of the 21 S. enterica strains studied in total (each representing a different O antigen structure) possess a manB gene which displays DNA identity, ranging from 93 to 99%, to the CA manB gene of S. enterica LT2. Furthermore, the CA-like manB genes (as well as the CA manB and manC genes) display subspecies specificity, and the CA and CA-like manB genes (for individual strains) appear to be evolving in concert via gene conversion events. In comparison, the manC genes were generally not CA-like, a situation also apparent in Escherichia coli,and therefore most strongly reflected the evolutionary history of the S. enterica O antigen GDP-mannose pathway. It appears that, in relatively recent times, gene capture from a distant source has occurred infrequently, and that groups of manB and manC genes have been maintained and are continuing to evolve within S. enterica and more closely related species.

Individual rrn operons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone of Vibrio cholerae. The genome of an early isolate of the seventh pandemic clone had nine rrn operons of which two were in tandem with other rrn operons. The site for BglI, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate. This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation. Additional rrn recombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon. Spacers of 431, 509, 607 and 711 bp were found. A total of at least eight rrn recombination events were detected. Three rrn loci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G. In addition there was variation due to deletion of tandem operons. The frequency of recombination between rrn operons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study. This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study. Recombination between rrn operons may be a factor in ribotype variation in all systems. The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved.