Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori.

The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 - modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5'-Gm6ACC-3'. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ΔmodH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility.

A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N(6)-adenine DNA methyltransferases of Neisseria meningitidis.

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N(6)-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N(6)-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY M6A: G-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC M6A: CC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC M6A: GC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY M6A: G-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC M6A: GG-3' sites, to 100% at 5'-ACGT M6A: GG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.

ModM DNA methyltransferase methylome analysis reveals a potential role for Moraxella catarrhalis phasevarions in otitis media.

Moraxella catarrhalis is a significant cause of otitis media and exacerbations of chronic obstructive pulmonary disease. Here, we characterize a phase-variable DNA methyltransferase (ModM), which contains 5'-CAAC-3' repeats in its open reading frame that mediate high-frequency mutation resulting in reversible on/off switching of ModM expression. Three modM alleles have been identified (modM1-3), with modM2 being the most commonly found allele. Using single-molecule, real-time (SMRT) genome sequencing and methylome analysis, we have determined that the ModM2 methylation target is 5'-GAR(m6)AC-3', and 100% of these sites are methylated in the genome of the M. catarrhalis 25239 ModM2 on strain. Proteomic analysis of ModM2 on and off variants revealed that ModM2 regulates expression of multiple genes that have potential roles in colonization, infection, and protection against host defenses. Investigation of the distribution of modM alleles in a panel of M. catarrhalis strains, isolated from the nasopharynx of healthy children or middle ear effusions from patients with otitis media, revealed a statistically significant association of modM3 with otitis media isolates. The modulation of gene expression via the ModM phase-variable regulon (phasevarion), and the significant association of the modM3 allele with otitis media, suggests a key role for ModM phasevarions in the pathogenesis of this organism.

Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms.

Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host-bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of FucNAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component.

Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates.

O-glycosylation of proteins in Neisseria meningitidis is catalyzed by PglL, which belongs to a protein family including WaaL O-antigen ligases. We developed two hidden Markov models that identify 31 novel candidate PglL homologs in diverse bacterial species, and describe several conserved sequence and structural features. Most of these genes are adjacent to possible novel target proteins for glycosylation. We show that in the general glycosylation system of N. meningitidis, efficient glycosylation of additional protein substrates requires local structural similarity to the pilin acceptor site. For some Neisserial PglL substrates identified by sensitive analytical approaches, only a small fraction of the total protein pool is modified in the native organism, whereas others are completely glycosylated. Our results show that bacterial protein O-glycosylation is common, and that substrate selection in the general Neisserial system is dominated by recognition of structural homology.

Dual pili post-translational modifications synergize to mediate meningococcal adherence to platelet activating factor receptor on human airway cells.

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, twitching motility, auto-aggregation, and DNA transformation. Pili of N. meningitidis are subject to several different post-translational modifications. Among these pilin modifications, the presence of phosphorylcholine (ChoP) and a glycan on the pilin protein are phase-variable (subject to high frequency, reversible on/off switching of expression). In this study we report the location of two ChoP modifications on the C-terminus of N. meningitidis pilin. We show that the surface accessibility of ChoP on pili is affected by phase variable changes to the structure of the pilin-linked glycan. We identify for the first time that the platelet activating factor receptor (PAFr) is a key, early event receptor for meningococcal adherence to human bronchial epithelial cells and tissue, and that synergy between the pilin-linked glycan and ChoP post-translational modifications is required for pili to optimally engage PAFr to mediate adherence to human airway cells.

Investigations into genome diversity of Haemophilus influenzae using whole genome sequencing of clinical isolates and laboratory transformants.

BACKGROUND: Haemophilus influenzae is an important human commensal pathogen associated with significant levels of disease. High-throughput DNA sequencing was used to investigate differences in genome content within this species. RESULTS: Genomic DNA sequence was obtained from 85 strains of H. influenzae and from other related species, selected based on geographical site of isolation, disease association and documented genotypic and phenotypic differences. When compared by Mauve alignment these indicated groupings of H. influenzae that were consistent with previously published analyses; capsule expressing strains fell into two distinct groups and those of serotype b (Hib) were found in two closely positioned lineages. For 18 Hib strains representing both lineages we found many discrete regions (up to 40% of the total genome) displaying sequence variation when compared to a common reference strain. Evidence that this naturally occurring pattern of inter-strain variation in H. influenzae can be mediated by transformation was obtained through sequencing DNA obtained from a pool of 200 independent transformants of a recipient (strain Rd) using donor DNA from a heterologous Hib strain (Eagan). CONCLUSION: Much of the inter-strain variation in genome sequence in H. influenzae is likely the result of inter-strain exchanges of DNA, most plausibly through transformation.

Simple sequence repeats in Haemophilus influenzae.

Simple sequence repeat (SSRs) of DNA are subject to high rates of mutation and are important mediators of adaptation in Haemophilus influenzae. Previous studies of the Rd KW20 genome identified the primacy of tetranucleotide SSRs in mediating phase variation (the rapid reversible switching of gene expression) of surface exposed structures such as lipopolysaccharide. The recent sequencing of the genomes of multiple strains of H. influenzae allowed the comparison of the SSRs (repeat units of one to nine nucleotides in length) in detail across four complete H. influenzae genomes and then comparison with a further 12 genomes when they became available. The SSR loci were broadly classified into three groups: (1) those that did not vary; (2) those for which some variation between strains was observed but this could not be linked to variation of gene expression; and (3) those that both varied and were located in regions consistent with mediating phase variable gene expression. Comparative analysis of 988 SSR associated loci confirmed that tetranucleotide repeats were the major mediators of phase variation and extended the repertoire of known tetranucleotide SSR loci by identifying ten previously uncharacterised tetranucleotide SSR loci with the potential to mediate phase variation which were unequally distributed across the H. influenzae pan-genome. Further, analysis of non-tetranucleotide SSR in the 16 strains revealed a number of mononucleotide, dinucleotide, pentanucleotide, heptanucleotide, and octanucleotide SSRs which were consistent with these tracts mediating phase variation. This study substantiates previous findings as to the important role that tetranucleotide SSRs play in H. influenzae biology. Two Brazilian isolates showed the most variation in their complement of SSRs suggesting the possibility of geographic and phenotypic influences on SSR distribution.

Sequence and functional analyses of Haemophilus spp. genomic islands.

BACKGROUND: A major part of horizontal gene transfer that contributes to the diversification and adaptation of bacteria is facilitated by genomic islands. The evolution of these islands is poorly understood. Some progress was made with the identification of a set of phylogenetically related genomic islands among the Proteobacteria, recognized from the investigation of the evolutionary origins of a Haemophilus influenzae antibiotic resistance island, namely ICEHin1056. More clarity comes from this comparative analysis of seven complete sequences of the ICEHin1056 genomic island subfamily. RESULTS: These genomic islands have core and accessory genes in approximately equal proportion, with none demonstrating recent acquisition from other islands. The number of variable sites within core genes is similar to that found in the host bacteria. Furthermore, the GC content of the core genes is similar to that of the host bacteria (38% to 40%). Most of the core gene content is formed by the syntenic type IV secretion system dependent conjugative module and replicative module. GC content and lack of variable sites indicate that the antibiotic resistance genes were acquired relatively recently. An analysis of conjugation efficiency and antibiotic susceptibility demonstrates that phenotypic expression of genomic island-borne genes differs between different hosts. CONCLUSION: Genomic islands of the ICEHin1056 subfamily have a longstanding relationship with H. influenzae and H. parainfluenzae and are co-evolving as semi-autonomous genomes within the 'supragenomes' of their host species. They have promoted bacterial diversity and adaptation through becoming efficient vectors of antibiotic resistance by the recent acquisition of antibiotic resistance transposons.

Selection for efficient translation initiation biases codon usage at second amino acid position in secretory proteins.

The definition of a typical sec-dependent bacterial signal peptide contains a positive charge at the N-terminus, thought to be required for membrane association. In this study the amino acid distribution of all Escherichia coli secretory proteins were analysed. This revealed that there was a statistically significant bias for lysine at the second codon position (P2), consistent with a role for the positive charge in secretion. Removal of the positively charged residue P2 in two different model systems revealed that a positive charge is not required for protein export. A well-characterized feature of large amino acids like lysine at P2 is inhibition of N-terminal methionine removal by methionyl amino-peptidase (MAP). Substitution of lysine at P2 for other large or small amino acids did not affect protein export. Analysis of codon usage revealed that there was a bias for the AAA lysine codon at P2, suggesting that a non-coding function for the AAA codon may be responsible for the strong bias for lysine at P2 of secretory signal sequences. We conclude that the selection for high translation initiation efficiency maybe the selective pressure that has led to codon and consequent amino acid usage at P2 of secretory proteins.

Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli.

Pili (type IV fimbriae) of Neisseria meningitidis are glycosylated by the addition of O-linked sugars. Recent work has shown that PglF, a protein with homology to O-antigen 'flippases', is required for the biosynthesis of the pilin-linked glycan and suggests pilin glycosylation occurs in a manner analogous to the wzy-dependent addition of O-antigen to the core-LPS. O-Antigen ligases are crucial in this pathway for the transfer of undecraprenol-linked sugars to the LPS-core in Gram-negative bacteria. An O-antigen ligase homologue, pglL, was identified in N. meningitidis. PglL mutants showed no change in LPS phenotypes but did show loss of pilin glycosylation, confirming PglL is essential for pilin O-linked glycosylation in N. meningitidis.

Whole genome analysis reveals a high incidence of non-optimal codons in secretory signal sequences of Escherichia coli.

Translational pausing may occur due to a number of mechanisms, including the presence of non-optimal codons, and it is thought to play a role in the folding of specific polypeptide domains during translation and in the facilitation of signal peptide recognition during sec-dependent protein targeting. In this whole genome analysis of Escherichia coli we have found that non-optimal codons in the signal peptide-encoding sequences of secretory genes are overrepresented relative to the "mature" portions of these genes; this is in addition to their overrepresentation in the 5'-regions of genes encoding non-secretory proteins. We also find increased non-optimal codon usage at the 3' ends of most E. coli genes, in both non-secretory and secretory sequences. Whereas presumptive translational pausing at the 5' and 3' ends of E. coli messenger RNAs may clearly have a general role in translation, we suggest that it also has a specific role in sec-dependent protein export, possibly in facilitating signal peptide recognition. This finding may have important implications for our understanding of how the majority of non-cytoplasmic proteins are targeted, a process that is essential to all biological cells.

Analysis of the role of pglI in pilin glycosylation of Neisseria meningitidis.

Pilin is the major subunit of the essential virulence factor pili and is glycosylated at Ser63. In this study we investigated the gene pglI to determine whether it is involved in the biosynthesis of the pilin-linked glycan of Neisseria meningitidis strain C311#3. A N. meningitidis C311#3pglI mutant resulted in a change of apparent molecular weight in SDS-PAGE and altered binding of antisera, consistent with a role in the biosynthesis of the pilin-linked glycan. These data, in conjunction with homology with well-characterised acyltransferases suggests a specific role for pglI in the biosynthesis of the basal 2,4-diacetamido-2,4,6-trideoxyhexose residue of the pilin-linked glycan.

Genetic characterization of pilin glycosylation in Neisseria meningitidis.

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of an O-linked trisaccharide, Gal(beta1-4)Gal(alpha1-3)2,4-diacetimido-2,4,6-trideoxyhexose++ +. In a previous study the authors identified and characterized a gene, pglA, encoding a galactosyltransferase involved in pilin glycosylation. In this study a set of random genomic sequences from N. meningitidis strain MC58 was used to search for further genes involved in pilin glycosylation. Initially, an open reading frame was identified, and designated pglD (pilin glycosylation gene D), which was homologous to genes involved in polysaccharide biosynthesis. The region adjacent to this gene was cloned and nucleotide sequence analysis revealed two further genes, pglB and pglC, which were also homologous with genes involved in polysaccharide biosynthesis. Insertional mutations were constructed in pglB, pglC and pglD in N. meningitidis C311#3, a strain with well-defined LPS and pilin-linked glycan structures, to determine whether these genes had a role in the biosynthesis of either of these molecules. Analysis of these mutants revealed that there was no alteration in the phenotype of LPS in any of the mutant strains as judged by SDS-PAGE gel migration. In contrast, increased gel migration of the pilin subunit molecules of pglB, pglC and pglD mutants by Western analysis was observed. Pilin from each of the pglB, pglC and pglD mutants did not react with a terminal-galactose-specific stain, confirming that the gel migration differences were due to the alteration or absence of the pilin-linked trisaccharide structure in these mutants. In addition, antisera specific for the C311#3 trisaccharide failed to react with pilin from the pglB, pglC, pglD and galE mutants. Analysis of nucleotide sequence homologies has suggested specific roles for pglB, pglC and pglD in the biosynthesis of the 2,4-diacetimido-2,4,6-trideoxyhexose structure.