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DNA Research Advance Access originally published online on April 27, 2008
DNA Research 2008 15(3):163-168; doi:10.1093/dnares/dsn006
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© The Author 2008. Kazusa DNA Research Institute
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A Variable Gene in a Conserved Region of the Helicobacter pylori Genome: Isotopic Gene Replacement or Rapid Evolution?

Armelle Ménard1,2, Antoine Danchin3, Sandrine Dupouy1,2, Francis Mégraud1,2 and Philippe Lehours1,2,*

1 INSERM U853, Laboratoire de Bactériologie, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, F-33076 Bordeaux cedex, France
2 Université Victor Segalen Bordeaux 2, Laboratoire de Bactériologie, Bordeaux F-33076, France
3 Institut Pasteur, Génétique des Génomes Bactériens - CNRS URA2171, Paris F-750015, France

Received 6 November 2007; accepted 3 April 2008.


   Abstract
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The present study concerns the identification of a novel coding sequence in a region of the Helicobacter pylori genome, located between JHP1069/HP1141 and JHP1071/HP1143 according to the numbering of the J99 and 26695 reference strains, respectively, and spanning three different coding DNA sequences (CDSs). The CDSs located at the centre of this locus were highly polymorphic, as determined by the analysis of 24 European isolates, 3 Asian, and 3 African isolates. Phylogenetic and molecular evolutionary analyses showed that the CDSs were not restricted to the geographical origin of the strains. Despite a very high variability observed in the deduced protein sequences, significant similarity was observed, always with the same protein families, i.e. ATPase and bacteriophage receptor/invasion proteins. Although this variability could be explained by isotopic gene replacement via horizontal transfer of a gene with the same function but coming from a variety of sources, it seems more likely that the very high sequence variation observed at this locus is the result of a strong selection pressure exerted on the corresponding gene product. The CDSs identified in the present study could be used as strain specific markers.

Key words: Helicobacter pylori; coding DNA sequence; genetic diversity; diversifying selection

Comparative analyses conducted on Helicobacter pylori genome sequences, i.e. from H. pylori strain J99 associated with peptic ulcer,1Go,2Go strain 266953Go associated with gastritis, and strain HPAG1 associated with atrophic gastritis,4Go revealed a significant macrodiversity (presence or absence of genes) and microdiversity (high polymorphism among orthologous genes).5Go,6Go,7Go The plasticity zones and the cag pathogenicity island (cag PAI) are considered to be the main variable genomic areas. The remaining variable genes are distributed throughout the H. pylori genome and some of them have been individualized in clusters of instability concerning blocks of 5–8 coding DNA sequences (CDSs).5Go,8Go,9Go

Subtractive hybridization is a powerful tool for comparative prokaryotic genomics and was validated on H. pylori by several authors.10Go,11Go In a previous study, we used subtractive hybridization to compare the genetic content of one H. pylori strain isolated from a gastric MALT lymphoma strain (strain B34) and one chronic gastritis only strain.12Go One original 1092 bp sequence was identified, with no significant nucleotide similarity in comparison to the H. pylori reference strains 26695 and J99 genomes which were available. The aim of the present study was to localize this sequence in the H. pylori genome, to determine its prevalence, and to analyze its genetic diversity in H. pylori.

Using an in-house genome walking method as previously described,13Go the original region was localized in the H. pylori genome and a new CDS was subsequently identified using the CDS finder website (http://www.ncbi.nlm.nih.gov/gCDS/CDSig.cgi). This new CDS, called CDS2, is located between two CDS homologous to JHP1069/HP1141 and JHP1071/HP1143 according to the numbering of the J99 and 26695 reference strains, respectively.1Go,3Go CDS2 replaced JHP1070/HP1142, called CDS1, in H. pylori reference strains J99 and 26695.

The percentage of identity between the nucleotide sequences of CDS1 and CDS2 was determined using the LALIGN software,14Go which identifies multiple matching subsegments in two sequences (http://www.ch.embnet.org/software/LALIGN_form.html). CDS2 showed 54.9% identity in a 2046 nucleotides overlap with JHP1070 and 55.5% identity in a 2083 nucleotides overlap with HP1142. CDS2 encodes a putative polypeptide of 820 residues (Genbank accesion number EF492441 [GenBank] , EMBL Nucleotide Sequence AM902682 [GenBank] ). Regarding the protein homology, CDS2 shared 23.6% identity with JHP1070 in a 628 amino acid overlap and 24.4% identity with HP1142 in a 630 amino acid overlap. Finally, a strong nucleotide identity was found with the HPAG1_1080 sequence4Go with 89.3% identity in a 2469 nucleotides overlap.

The prevalence and the genetic diversity of the identified genomic locus were first determined for 24 H. pylori strains: 13 H. pylori strains isolated from gastric MALT lymphoma patients obtained from two multicentre French protocols and 11 strains isolated from French chronic gastritis only patients, as previously described12Go,15Go by PCR amplification using primers hybridizing to the conserved sequence of the flanking genes (JHP1069/HP1141 and JHP1071/HP1143) according to the numbering of the J99 and 26695 strains, respectively. The primers were designed using the web Primer3 software (http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi).16Go Direct sequencing was carried out on both strands, and nucleotide and deduced protein sequences were compared with the NCBI Blast program (http://www.ncbi.nlm.nih.gov/BLAST/). A CDS was always present at this locus: CDS1 was found in 54% of the strains, CDS2 in 29% of the strains, and an additional CDS, called CDS3, was identified in 17% of the strains. In the chronic gastritis only H. pylori strain G2, CDS3 had a 53.4% identity in a 2005 nucleotide overlap with CDS1 and a 52.9% identity in a 2063 nucleotides overlap with CDS2, and it encodes a putative polypeptide of 861 residues (GenBank accesion number EF492442 [GenBank] , EMBL Nucleotide Sequence AM902683 [GenBank] ). CDS3 still has no counterpart in databases. Considering the three CDSs, no significant association with a virulence factor was found, nor with a pathology (data not shown). The presence or absence of these CDSs was also verified by dot blot hybridization, as previously described.12Go It showed that the presence of one of these three CDSs was exclusive (no local duplication, data not shown).

We first focused on the role of the genes present around the polymorphic locus. According to the revised annotation of the H. pylori genome,17Go JHP1069/HP1141 encodes a methionyl-tRNA formyltransferase (fmt) and JHP1071/HP1143, a conserved hypothetical protein. fmt is considered to be an essential gene which links general metabolism with the translation process (protein biosynthesis).18Go,19Go As shown in Fig. 1, JHP1069/HP1141 and JHP1071/HP1143 are surrounded by genes of hypothetical function. Considering the G + C% content of the region, all of the CDSs contained a G + C% similar to the rest of the H. pylori genome (~39%) except for these variable regions: CDS1, CDS2, and CDS3 had 29, 30, and 31% G + C% content, respectively. The lower G + C% content suggests an external origin of these CDSs or a rapid adaptation.20Go Indeed, Saunders et al.21Go, using a tetranucleotide and hexanucleotide signature analysis, identified substantial differences between JHP1070 and HP1142 genes and hypothesized that they were horizontally transferred.


Figure 1
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  		Figure 1. Representation of the genomic area of interest, according to the genome sequences of the two Helicobacter pylori reference strains J99 and 26695, which contain the variable CDS identified in the present study. Each CDS is represented by an arrow with the direction indicating the translational direction. The numbering under each CDS corresponds to the number of CDS in H. pylori J99 (top line) and 26695 (bottom line). The function of each CDS is indicated according to the revised annotation of Boneca et al.17 Fmt, methionyl-tRNA formyltransferase; BirA, biotin ligase bifunctional protein; ParB, replication/partition-related protein; ParA, chromosome partition protein (Soj).

 
CDS1 has been annotated as a predicted coding region JHP1070 with no homolog in the databases. It codes for a putative polypeptide of 759 residues. Using a Blastp search, significant homologies were found with (i) Rlo proteins (R-linked ORF) from Campylobacter (e.g. RloG, E = e-11 in Campylobacter jejuni strain RM1167, or RloC, E = 7e-13 in C. jejuni strain RM11221),22Go,23Go (ii) an ATP/GTP binding-site (GXXXXGKT), and (iii) a putative phage murein transglycosylase YomI (SPbeta phage protein; lytic transglycosylase, E = 4.52e-05 in Bacillus subtilis).24Go The same significant homologies with Rlo and YomI were also found in CDS2 and CDS3. Finally, another interesting point to consider is that the three CDSs shared significant homology with chromosome partition protein SMC: for example, Treponema denticola ATCC 35405 chromosome NC_002967 [GenBank] E = 1.11e-11 for CDS1, Fusobacterium nucleatum subsp. nucleatum ATCC 25586 E = 8.66e-10 for CDS2, Fusobacterium nucleatum subsp. nucleatum ATCC 25586 E = 3.86e-08 for CDS3.25Go We did not find any significant motifs indicating that the proteins could be secreted and/or present in the membrane, but this does not preclude an association with the membrane via interaction with an integral membrane protein partner.

How can one explain the apparent variability of the locus identified in the present study? One potential hypothesis is that the region is a hot spot for gene insertion/deletion, with a specific selection pressure maintaining a particular function at that precise location in the genome. Suerbaum and Josenhans26Go recently reviewed the current data on the genetic diversity of H. pylori and argued that this bacterium uses mutation and recombination processes to adapt to its individual host by modifying molecules that interact with the host.26Go Because the three CDSs retain the same similarities, it is likely that (i) these proteins share the same function or (ii) the gene is submitted to specific selection pressure making it evolve at a very rapid rate. We proposed that such a protein could be a phage receptor/translocator or that it could allow the DNA phage to enter host cells by remodelling the cell wall.27Go–29Go Indeed, as already described in Escherichia coli, this kind of protein is subjected to a strong positive selection.30Go

Helicobacter pylori genotypes vary markedly with their geographical region, and this is particularly the case for genes under positive selection. Therefore, the corresponding genes were looked for in three East Asian strains and three African strains. All three CDS were found: CDS1 was found in one Asian (strain 8038) and one African strain (strain TALLAN), CDS2 in two Asian strains (strains 12001 and strain 8033), and CDS3 in one Asian (strain 19A) and one African strain (strain BAPOOI) (Fig. 2). A phylogenetic analysis was conducted on the deduced amino acid sequences of CDS. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4.31Go Phylogenetic trees were generated by the neighbour-joining method.32Go Molecular distances were determined using the Kimura two-parameter model.33Go The tree showed three independent clusters which were clearly separated and corresponded to CDS1, CDS2, and CDS3, respectively (Fig. 2). However, the exact organization of these different CDS cannot be determined since this consensus tree cannot be rooted to other species. Indeed, no CDS with significant homology has ever been found in other species (in databases). Interestingly, even though the testing was performed on a limited number of non-European strains, these results indicate that the presence of one of the three CDS cannot be restricted to the geographical origin of the strains.


Figure 2
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  		Figure 2. Phylogenetic analysis of CDS, CDS1, CDS2, and CDS3, proteins generated with the neighbour-joining method. The phylogeny presented is based on the alignment of the entire deduced protein. The bootstrap values are indicated next to each node. Nucleotide and protein sequences are available in GenBank (EMBL) for the Helicobacter pylori strains B34, G2, 8038, TALLAN, 12001, 8033, 19A, and BAPOO1 under the accession numbers EF492441 [GenBank] (AM902682 [GenBank] ), EF492442 [GenBank] (AM902683 [GenBank] ), EU553483 [GenBank] (AM946633 [GenBank] ), EU553485 [GenBank] (AM946634 [GenBank] ), EU553505 (AM946635 [GenBank] ), EU553482 [GenBank] (AM946636 [GenBank] ), EU553481 [GenBank] (AM946637 [GenBank] ), and EU556504 [GenBank] (AM946638 [GenBank] ), respectively. Nucleotide and protein sequences of H. pylori reference strains 26695, J99, and HPAG1 are available in GenBank under the accession numbers AE000511 [GenBank] , AE001439 [GenBank] , and ABF85147 [GenBank] , respectively.

 
The type of selection operating at the amino acid level was also evaluated by comparing non-synonymous substitutions (Ka) and synonymous substitutions (Ks).34Go The overall mean of Ks and Ka substitutions was determined using the Nei–Gojobori method.35Go The codon based Z-test of selection36Go was used to evaluate the significance of Ka/Ks substitution values. Bootstrap confidence levels were determined by randomly resampling the sequencing data 1000 times. The results are indicated for each CDS in Table 1. Since Ka/Ks was <1 for the three CDSs analyzed, the purifying selection hypothesis was tested and the significant P-value obtained supports the hypothesis of conservation at the protein level for each CDS (Z-test P < 0.001).


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  		Table 1. Analysis of molecular distances and synonymous and non-synonymous nucleotide substitutions within CDSs, CDS1 (n = 4), CDS2 (n = 4), and CDS3 (n = 3), in different Helicobacter pylori strains

 
Finally, we propose that the very high variation observed in the protein sequences reflects the permanent selection pressure exerted by phages or other elements interacting with the organism's cell envelope. If this is the case, this locus could be used as a marker for constraints operating in the environmental niches in which particular H. pylori strains evolve. The presence of phages in H. pylori has been rarely described.37Go For example, Marsich et al.38Go postulated that H. pylori lysozyme gene (lys) had a prophage origin. Numerous other explanations cannot be excluded, such as bacterial mammalian host interaction, protozoan predation, or porin specificity. Indeed several publications have focused on cases of genes that vary markedly among H. pylori isolates. One example is the replacement of babA by babB as reported by Solnick et al.39Go Helicobacter pylori BabA is the ABO blood group antigen binding adhesin, which has a closely related paralogue (BabB) whose function is unknown. An extensive genotypic diversity in babA and babB across different strains, as well as within a strain colonizing an individual patient has been shown in line with the hypothesis that diverse profiles of babA and babB reflect selective pressures for adherence, which may differ across different hosts and within an individual over time.40Go

In summary, a novel polymorphic locus comprised of a single gene was identified in the H. pylori genome. Although this variation could be explained by isotopic gene replacement via horizontal transfer of a gene with the same function but coming from a variety of sources, it seems more likely that the very high sequence variation observed at this locus is the result of a strong selection pressure exerted on the corresponding gene product. We propose that the evolution of CDS1, CDS2, and CDS3 is due to the occurrence of a specific environmental event, such as interaction with a biological structure, e.g. bacteriophage which are involved in surface cell secretion. The genes identified in the present study could be used as strain specific markers for particular niches. The predicted function of the gene products, although highly speculative, should encourage investigators to explore the presence of phages in the H. pylori environment and study their relationship regarding pathogenicity.


   Acknowledgments
 
The authors want to thank Dr Monica Oleastro from the Departamento de Doenças Infecciosas of the Instituto Nacional Saúde Dr Ricardo Jorge (Lisbon, Portugal) for the comparison of non-synonymous substitutions and synonymous substitutions among the three CDSs described in this study and Dr Jorge M. B. Vítor and Dr Vale Filipa from the Faculty of Pharmacy in Lisbon for strains. The study was financially supported by the Institut de Recherche des Maladies de l'Appareil Digestif (IRMAD), the Association pour la Recherche contre le Cancer (ARC), and the Conseil Régional d'Aquitaine, France.


   Footnotes
 
* To whom correspondence should be addressed. Tel. +33 5-57-57-12-86. Fax. 33 5-56-51-41-82. E-mail: philippe.lehours{at}labhel.u-bordeaux2.fr

Edited by Masahira Hattori


   References
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