Evolutionary history of Helicobacter pylori sequences reflect past human migrations in Southeast Asia

The human population history in Southeast Asia was shaped by numerous migrations and population expansions. Their reconstruction based on archaeological, linguistic or human genetic data is often hampered by the limited number of informative polymorphisms in classical human genetic markers, such as the hypervariable regions of the mitochondrial DNA. Here, we analyse housekeeping gene sequences of the human stomach bacterium Helicobacter pylori from various countries in Southeast Asia and we provide evidence that H. pylori accompanied at least three ancient human migrations into this area: i) a migration from India introducing hpEurope bacteria into Thailand, Cambodia and Malaysia; ii) a migration of the ancestors of Austro-Asiatic speaking people into Vietnam and Cambodia carrying hspEAsia bacteria; and iii) a migration of the ancestors of the Thai people from Southern China into Thailand carrying H. pylori of population hpAsia2. Moreover, the H. pylori sequences reflect iv) the migrations of Chinese to Thailand and Malaysia within the last 200 years spreading hspEasia strains, and v) migrations of Indians to Malaysia within the last 200 years distributing both hpAsia2 and hpEurope bacteria. The distribution of the bacterial populations seems to strongly influence the incidence of gastric cancer as countries with predominantly hspEAsia isolates exhibit a high incidence of gastric cancer while the incidence is low in countries with a high proportion of hpAsia2 or hpEurope strains. In the future, the host range expansion of hpEurope strains among Asian populations, combined with human motility, may have a significant impact on gastric cancer incidence in Asia.     

Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori

iceA1 in Helicobacter pylori is a homolog of nlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity.     

Evolutionary dynamics of insertion sequences in Helicobacter pylori

Prokaryotic insertion sequence (IS) elements behave like parasites in terms of their ability to invade and proliferate in microbial gene pools and like symbionts when they coevolve with their bacterial hosts. Here we investigated the evolutionary history of IS605 and IS607 of Helicobacter pylori, a genetically diverse gastric pathogen. These elements contain unrelated transposase genes (orfA) and also a homolog of the Salmonella virulence gene gipA (orfB). A total of 488 East Asian, Indian, Peruvian, and Spanish isolates were screened, and 18 and 14% of them harbored IS605 and IS607, respectively. IS605 nucleotide sequence analysis (n = 42) revealed geographic subdivisions similar to those of H. pylori; the geographic subdivision was blurred, however, due in part to homologous recombination, as indicated by split decomposition and homoplasy tests (homoplasy ratio, 0.56). In contrast, the IS607 populations (n = 44) showed strong geographic subdivisions with less homologous recombination (homoplasy ratio, 0.2). Diversifying selection (ratio of nonsynonymous change to synonymous change, >1) was evident in approximately 15% of the IS605 orfA codons analyzed but not in the IS607 orfA codons. Diversifying selection was also evident in approximately 2% of the IS605 orfB and approximately 10% of the IS607 orfB codons analyzed. We suggest that the evolution of these elements reflects selection for optimal transposition activity in the case of IS605 orfA and for interactions between the OrfB proteins and other cellular constituents that potentially contribute to bacterial fitness. Taken together, similarities in IS elements and H. pylori population genetic structures and evidence of adaptive evolution in IS elements suggest that there is coevolution between these elements and their bacterial hosts.     

Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori

Helicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation. Among the barriers to transformation are numerous strain-specific restriction-modification systems in H. pylori. We have developed a method to protect plasmid DNA from restriction by in vitro site-specific methylation using cell-free extracts of H. pylori before transformation. In two cases, plasmid DNA treated with cell-free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly increased compared with mock-treated plasmid DNA. The results indicate that the restriction barrier in H. pylori can be largely overcome by specific DNA methylation in vitro. The approach described should significantly enhance the ability to manipulate gene function in H. pylori and other organisms that have substantial restriction barriers to transformation.     

Spontaneous mutations in the Helicobacter pylori rpsL gene

Several studies have revealed that the Helicobacter pylori genome differs markedly from strain to strain, perhaps as a result of mutations arising during persistent infection and/or related to the observed variation in virulence. The development of a detection system for mutations in H. pylori genes might therefore help us to develop a better understanding of its mutability, and in this way help us to develop plans for investigating the relationship between its genomic variability and the pathogenesis of various gastric and duodenal diseases associated with the long-term H. pylori infections. We have therefore begun a study of H. pylori mutability using the endogenous rpsL gene as a marker. Spontaneous mutant frequencies were measured and compared among H. pylori strains, after incubation on plates containing 50 microg/ml of streptomycin for 10 days as a selection procedure. The rpsL gene of each streptomycin-resistant (Str(r)) mutant was amplified by polymerase-chain-reaction (PCR) and sequenced. All of the mutations we characterized were localized at codons 43 or 88 of the rpsL gene and were base transitions from A to G, replacing lysine with arginine. This is in contrast to the spontaneous Str(r) mutants isolated from Escherichia coli, which resulted from either A to G transitions at lysine codons 42 and 87, or A to T or C transversions at lysine codon 42. The spontaneous mutant frequencies of the rpsL gene in H. pylori were of the order of 10(-9), and there were significant differences in spontaneous mutant frequencies among the strains tested. This mutation detection system might be of value in screening clinical isolates for H. pylori mutator phenotypes.     

Evolutionary history of the enolase gene family

The enzyme enolase [EC 4.2.1.11] is found in all organisms, with vertebrates exhibiting tissue-specific isozymes encoded by three genes: alpha (alpha), beta (beta), and gamma (gamma) enolase. Limited taxonomic sampling of enolase has obscured the timing of gene duplication events. To help clarify the evolutionary history of the gene family, cDNAs were sequenced from six taxa representing major lineages of vertebrates: Chiloscyllium punctatum (shark), Amia calva (bowfin), Salmo trutta (trout), Latimeria chalumnae (coelacanth), Lepidosiren paradoxa (South American lungfish), and Neoceratodus forsteri (Australian lungfish). Phylogenetic analysis of all enolase and related gene sequences revealed an early gene duplication event prior to the last common ancestor of living organisms. Several distantly related archaebacterial sequences were designated as 'enolase-2', whereas all other enolase sequences were designated 'enolase-1'. Two of the three isozymes of enolase-1, alpha- and beta-enolase, were discovered in actinopterygian, sarcopterygian, and chondrichthian fishes. Phylogenetic analysis of vertebrate enolases revealed that the two gene duplications leading to the three isozymes of enolase-1 occurred subsequent to the divergence of living agnathans, near the Proterozoic/Phanerozoic boundary (approximately 550Mya). Two copies of enolase, designated alpha(1) and alpha(2), were found in the trout and are presumed to be the result of a genome duplication event.     

The secE gene of Helicobacter pylori

Despite extensive annotation by two independent teams, the Helicobacter pylori genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing secE component of the major secretion machinery of Helicobacter pylori.     

Diversity of restriction-modification gene homologues in Helicobacter pylori

The complete genome sequences of two Helicobacter pylori strains have recently become available. We have searched them for homologues of restriction-modification genes. One strain (26695) carried 52 such homologues, and the other (J99) carried 53. Their sequence alignments were arranged in the form of a phylogenetic tree and compared with the tree based on rRNA. The trees showed that the homologues are scattered among diverse groups of bacteria. They also revealed high polymorphism within the species--there are 42 pairs with high homology, 10 specific to 26695, and 11 specific to J99. Many of the restriction-modification homologues were characterized by a GC content lower than that of the average gene in the genome. Some of the restriction-modification homologues showed a different codon use bias from the average genes. These observations are interpreted in terms of horizontal transfer of the restriction-modification genes.     

Sequence and organization of coelacanth neurohypophysial hormone genes: Evolutionary history of the vertebrate neurohypophysial hormone gene locus

Background  

The mammalian neurohypophysial hormones, vasopressin and oxytocin are involved in osmoregulation and uterine smooth muscle contraction respectively. All jawed vertebrates contain at least one homolog each of vasopressin and oxytocin whereas jawless vertebrates contain a single neurohypophysial hormone called vasotocin. The vasopressin homolog in non-mammalian vertebrates is vasotocin; and the oxytocin homolog is mesotocin in non-eutherian tetrapods, mesotocin and [Phe²]mesotocin in lungfishes, and isotocin in ray-finned fishes. The genes encoding vasopressin and oxytocin genes are closely linked in the human and rodent genomes in a tail-to-tail orientation. In contrast, their pufferfish homologs (vasotocin and isotocin) are located on the same strand of DNA with isotocin gene located upstream of vasotocin gene separated by five genes, suggesting that this locus has experienced rearrangements in either mammalian or ray-finned fish lineage, or in both lineages. The coelacanths occupy a unique phylogenetic position close to the divergence of the mammalian and ray-finned fish lineages.     

Phasevarion mediated epigenetic gene regulation in Helicobacter pylori

Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis.     

Acid-induced expression of an LPS-associated gene in Helicobacter pylori

To investigate urease-independent mechanisms by which Helicobacter pylori resists acid stress, subtractive RNA hybridization was used to identify H. pylori genes whose expression is induced after exposure to acid pH. This approach led to the isolation of a gene that encoded a predicted 34.8 kDa protein (WbcJ), which was homologous to known bacterial O-antigen biosynthesis proteins involved in the conversion of GDP-mannose to GDP-fucose. An isogenic wbcJ null mutant strain failed to express O-antigen and Lewis X or Lewis Y determinants and was more sensitive to acid stress than was the wild-type strain. Qualitative differences in LPS profiles were observed in H. pylori cells grown at pH 5 compared with pH 7, which suggests that H. pylori may alter its LPS structure in response to acidic pH. This may be an important adaptation facilitating H. pylori colonization of the acidic gastric environment.     

Protein Evolutionary Rates Correlate with Expression Independently of Synonymous Substitutions in Helicobacter pylori

In free-living microorganisms, such as Escherichia coli and Saccharomyces cerevisiae, both synonymous and nonsynonymous substitution frequencies correlate with expression levels. Here, we have tested the hypothesis that the correlation between amino acid substitution rates and expression is a by-product of selection for codon bias and translational efficiency in highly expressed genes. To this end, we have examined the correlation between protein evolutionary rates and expression in the human gastric pathogen Helicobacter pylori, where the absence of selection on synonymous sites enables the two types of substitutions to be uncoupled. The results revealed a statistically significant negative correlation between expression levels and nonsynonymous substitutions in both H. pylori and E. coli. We also found that neighboring genes located on the same, but not on opposite strands, evolve at significantly more similar rates than random gene pairs, as expected by co-expression of genes located in the same operon. However, the two species differ in that synonymous substitutions show a strand-specific pattern in E. coli, whereas the weak similarity in synonymous substitutions for neighbors in H. pylori is independent of gene orientation. These results suggest a direct influence of expression levels on nonsynonymous substitution frequencies independent of codon bias and selective constraints on synonymous sites. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Regulation of Helicobacter pylori adherence by gene conversion

Genetic diversification of Helicobacter pylori adhesin genes may allow adaptation of adherence properties to facilitate persistence despite host defences. The sabA gene encodes an adhesin that binds sialyl‐Lewis antigens on inflamed gastric tissue. We found variability in the copy number and locus of the sabA gene and the closely related sabB and omp27 genes due to gene conversion among 51 North American paediatric H. pylori strains. We determined that sabB to sabA gene conversion is predominantly the result of intra‐genomic recombination and RecA, RecG and AddA influence the rate at which it occurs. Although all clinical strains had at least one sabA gene copy, sabA and sabB were lost due to gene conversion at similar rates in vitro, suggesting host selection to maintain the sabA gene. sabA gene duplication resulted in increased SabA protein production and increased adherence to sialyl‐Lewis antigens and mouse gastric tissue. In conclusion, gene conversion is a mechanism for H. pylori to regulate sabA expression level and adherence.     

Antimutator role of the DNA glycosylase mutY gene in Helicobacter pylori

Helicobacter pylori has a highly variable genome with ongoing diversification via inter- and intragenomic recombination and spontaneous mutation. DNA repair genes modulating mutation and recombination rates that influence diversification have not been well characterized for H. pylori. To examine the role of putative base excision repair ung and mutY glycosylase and xthA apurinic/apyrimidinic endonuclease genes in H. pylori, mutants of each were constructed in strain JP26 by allelic exchange. Spontaneous mutation frequencies of JP26 mutY mutants, assessed by rifampin resistance, were consistently higher (26-fold) than that of the wild type, whereas the ung and xthA mutants showed smaller increases. In trans complementation of the JP26 mutY mutant restored spontaneous mutation frequencies to wild-type levels. In cross-species studies, H. pylori mutY complemented an Escherichia coli mutY mutant and vice versa. In contrast, the ung and mutY mutants did not show higher frequencies of intergenomic recombination or greater sensitivity to UV-induced DNA damage than the wild type. The H. pylori mutY open reading frame contains an eight-adenine homonucleotide tract; we provide evidence that this is subject to slipped-strand mispairing, leading to frameshifts that eliminate gene function. Our findings indicate that H. pylori possesses phase-variable base excision repair, consistent with a tension between repair and mutation.     

Identification of a D-glycero-D-manno-heptosyltransferase gene from Helicobacter pylori

We have identified a Helicobacter pylori d-glycero-d-manno-heptosyltransferase gene, HP0479, which is involved in the biosynthesis of the outer core region of H. pylori lipopolysaccharide (LPS). Insertional inactivation of HP0479 resulted in formation of a truncated LPS molecule lacking an alpha-1,6-glucan-, dd-heptose-containing outer core region and O-chain polysaccharide. Detailed structural analysis of purified LPS from HP0479 mutants of strains SS1, 26695, O:3, and PJ1 by a combination of chemical and mass spectrometric methods showed that HP0479 likely encodes alpha-1,2-d-glycero-d-manno-heptosyltransferase, which adds a d-glycero-d-manno-heptose residue (DDHepII) to a distal dd-heptose of the core oligosaccharide backbone of H. pylori LPS. When the wild-type HP0479 gene was reintegrated into the chromosome of strain 26695 by using an "antibiotic cassette swapping" method, the complete LPS structure was restored. Introduction of the HP0479 mutation into the H. pylori mouse-colonizing Sydney (SS1) strain and the clinical isolate PJ1, which expresses dd-heptoglycan, resulted in the loss of colonization in a mouse model. This indicates that H. pylori expressing a deeply truncated LPS is unable to successfully colonize the murine stomach and provides evidence for a critical role of the outer core region of H. pylori LPS in colonization.     

Evolutionary history of introns in a multidomain globin gene

TheArtemia hemoglobin contains two sub-units that are similar or different chains of nine globin domains. The domains are ancestrally related and are presumed to be derived from copies of an original single-domain parent gene. Since the gene copies have remained in the same environment for several hundred million years they provide an excellent model for the investigation of intron stability. The cDNA for one of the two types of nine-domain subunit (domains T1–T9) has been sequenced. Comparison with the corresponding genomic DNA reveals a total of 17 intradomain introns. Fourteen of the introns are in locations on the protein that are conventional in globins of other species. In eight of the nine domains an intron corresponds to the B helix, amino acid B12, following the second nucleotide (phase 2), and in six domains a G-helix intron is located between G6 and G7 (phase 0). The consistency of this pattern is supportive of the introns having been inherited from a single-domain parent gene. The remaining three introns are in unconventional locations. Two occur in the F helix, either in amino acid F3 (phase 1) in domain T3, or between F2 and F3 (phase 0) in domain T6. The two F introns strengthen an interpretation of intron inheritance since globin F introns are rare, and in domains T3 and T6 they replace rather than supplement the conventional G introns, as though displacement from G to F occurred before that part of the gene became duplicated. It is inferred that one of the F introns subsequently moved by one nucleotide. Similarly, the third unconventional intron location is the G intron in domain T4 which is in G6, phase 2, one nucleotide earlier than the other G introns. Domain T4 is also unusual in lacking a B intron. The pattern of introns in theArtemia globin gene supports a concept of general positional stability but the exceptions, where introns have moved out of reading frame, or have moved by several codons, or have been deleted, suggest that intron displacements can occur after inheritance from an ancient source. Correspondence to: C.N.A. Trotman