The isolation and cultivation of a spiral bacterium in the early 1980s (62) from the gastric biopsies of patients suffering from chronic gastritis and ulcers have since resulted in a wealth of new knowledge about one of the largest worldwide infectious diseases known to mankind. Helicobacter pylori colonizes the gastric mucosa of almost half of the world's population and is associated with gastroduodenal diseases ranging from superficial and chronic gastritis, duodenal and gastric ulcers, to gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma (18, 43, 55). There is also some evidence that infection with H. pylori may play a role in increasing the severity or risk of infection by other gastrointestinal pathogens and in childhood malnutrition, particularly in less developed countries (17, 20). Consistent with its global significance, a tremendous amount of attention in the last two decades has been directed toward understanding the biology and pathogenesis of H. pylori.
A shift in the paradigm of bacteriology came in 1995 with the release of the first complete genomic sequence of a free-living bacterium, Haemophilus influenzae (30). In the last 5 years, there has been an explosion of microbial genomic sequence data generated from a wide range of organisms, with almost 30 completed microbial genomes now published and over 100 microbial genomic projects under way. A milestone in microbial genomics was set when H. pylori became the first bacterial species to have its genome sequenced and compared from two independent isolates (8, 99). The subsequent comparison has provided the first detailed look at the physical chromosomal organization and has begun to identify a minimal set of common genes that can be used as candidates for therapeutic strategies.
Comparative Analysis of the H. pylori Genomes
Prior to the completion of the H. pylori genome sequences, many analyses were performed that addressed the genomic structure and diversity of H. pylori. Physical maps generated by pulsed-field gel electrophoresis (PFGE) suggested that there was extreme macrodiversity between independent H. pylori strains, even those isolated from similar geographical locations (95, 96). Studies that sampled the entire genome and addressed nucleotide differences, such as randomly amplified polymorphic DNA (RAPD)-PCR, also provided a picture of extensive nucleotide diversity and showed that any particular H. pylori strain could be distinguished from essentially all other strains (5, 14, 39, 53). Other techniques such as PCR-restriction fragment length polymorphism (RFLP), oligofingerprinting, restriction fragment end labeling, and ribotyping supported these observations (4, 32, 45, 63, 72, 87, 97, 101). Sequencing studies, where individual genes were sequenced from multiple strains, demonstrated that it was extremely rare for an orthologous gene from different strains to have the same sequence (1, 50). Analyses from geographically diverse regions identified two weak clonal groupings based on geographical separation. However, recombination between strains occurs frequently and has been used to explain the high degree of allelic diversity (1, 91, 94). Multilocus enzyme electrophoresis, a non-nucleotide-based technique, using several enzymes has also demonstrated a significant level of diversity between isolates and has been used to differentiate between H. pylori strains (36, 40).
Genome Sequencing and General Features
The two independent H. pylori genomes that have been completely sequenced have distinct origins. H. pylori 26695, sequenced by The Institute for Genomic Research, was isolated in the United Kingdom in the early 1980s from a patient suffering from gastritis (24). This strain has been passaged repeatedly in the laboratory prior to sequencing. In contrast, H. pylori J99, which was sequenced in a collaborative effort between Astra AB (now AstraZeneca PLC) and Genome Therapeutics Corporation, was isolated in the United States in 1994 from a patient suffering from a duodenal ulcer and duodenitis and had not been extensively passaged prior to sequencing (8). As with most microbial genome sequencing projects to date, J99 and 26695 were sequenced using a random shot-gun approach from libraries of cloned chromosomal fragments of ~2.5 kb. In the case of J99, approximately 45,000 sequence reads were assembled using PHRAP, which resulted in 68 nonredundant contigs, representing approximately 98% of the genome. Sequencing of PCR products spanning the gaps between the contigs subsequently closed the genome sequence. The assembly of the H. pylori J99 genome was confirmed by PFGE analysis and probe hybridization (8).
Although the 26695 genome was 24 kb larger, both the J99 and 26695 genomes possessed a total (G + C)% of 39% (Table 1). Several general features, including average lengths of coding sequences, coding density, and the bias of initiation codons, are similar in the two strains (Table 1). Consistent with the genome of strain 26695, the genome of J99 had no clearly identifiable origin of replication. Certain genes, including dnaA, dnaN, and gyrA, are often found near the origin of replication in prokaryotes, but these genes are not in close proximity either to each other or to the repeated heptamer that was designated as nucleotide number one in both published H. pylori sequences. Further evidence that this position may represent the replication origin came from using an algorithm that analyzes the bias of short oligomers whose orientation is preferentially skewed around the replication origin of prokaryotes (85).
Both genomes contain two copies of the 16S and 23S-5S rRNA loci in the same two relative locations, although 26695 possesses an extra orphan 5S rRNA copy in addition to those associated with each 23S-5S locus. The 23S-5S and the 16S rRNA loci are not contiguous in either 26695 or J99, contrary to the organization seen in the majority of the organisms whose genomes have been completely sequenced.
There have been 1,496 putative open reading frames (ORFs) identified in H. pylori J99 (with the addition of the recently identified secE gene  (Table 1). This is 94 less than that first predicted for strain 26695 (99), although only 57 less than strain 26695 after the removal of some of the smaller ORFs after comparative annotation (Table 1) (8). Both J99 and 26695 contain a subset of putative ORFs that are not found in the other strain, 89 and 117 for strains J99 and 26695, respectively. However, it must be cautioned that while these were defined as "strain-specific genes" (8), this is only in the context of the whole genome comparison between J99 and 26695, as other isolates of H. pylori are likely to also contain a subset of these genes. In both genomes, almost 60% of the ORFs were attributed a predicted function, whereas 24% were conserved in other bacterial species but were of unknown function and 17% were H. pylori specific with no known homologs in the current databases. The above numbers (24% and 17%) of ORFs with unknown function, either conserved or H. pylori specific, include a comparison with the recently completed Campylobacter jejuni NCTC11168 genome (76). There are 86 ORFs in H. pylori J99 that had previously been assigned as H. pylori specific but now have orthologous partners in C. jejuni NCTC11168. However, as these two genera are extremely closely related, it is still somewhat surprising that such a large percentage of genes have remained H. pylori specific, over 20% when including those with an unknown function or with a known or predicted function, such as many of the outer membrane proteins (7).
Two different insertion sequence elements, IS605 and IS606, have been identified in H. pylori strains, often in multiple copies and at different physical locations on the chromosomes (42, 52). H. pylori J99 lacks a complete copy of the IS605 element, whereas strain 26695 contains five complete copies, three of which lie in the hypervariable plasticity zone and the remaining two are associated with organizational differences between the two strains (see below). Both strains have complete copies of the IS606 element, although present at different locations (Fig. 1). Both strains also contain remnants of these insertion elements, and the location of the complete and partial IS elements in both strains is biased toward the half of the genome that surrounds the predicted replication origin (8) (Fig. 1). There are five cases of coincidence in the location of partial or complete copies of insertion elements in these two strains (Fig. 1), suggesting either that certain loci are more receptive to insertion sequences or that these elements have been maintained from a common progenitor strain.
Physical arrangement of the genome
PFGE has been used to demonstrate macrodiversity in the genome structures of H. pylori isolates from both Japanese (95) and North American (96) strains. These studies indicated that essentially all strains had a unique profile when cut with various restriction endonucleases. In only one case did two strains, UA800 and UA802, that were isolated from unrelated patients at different times, have identical restriction patterns (96). Furthermore, PFGE and subsequent hybridization with specific probes suggest that, independent of the source of the strain, the gene order among H. pylori isolates is highly variable (48, 96).
Several methods were employed to assess the macrodiversity of the genomes of H. pylori J99 and 26695. The nucleotide positions of all the orthologous genes on both genomes were plotted against each other (Fig. 2). This graph clearly demonstrates that the majority of the genes in these two strains lie at the same relative position. Indeed, only 10 regions of the J99 genome, ranging in size from 1 to 83 kb, needed to be artificially transposed and/or inverted to enable the orthologous genes to be mapped onto the 26695 genome (8) (Fig. 1), and these can be visualized in Fig. 2. Two of these rearrangements (2 and 3) are large inversions and translocations and result in the movement of a large number of genes to a different quadrant of the circular chromosome, as well as the inversion of their relative transcriptional direction (Fig. 1) (8). The remaining eight organizational differences represent the movement of a smaller number of genes. Interestingly, at the endpoints of these organizational differences, one can find insertion elements, repeated sequences, or restriction-modification genes in at least one of the genomes, consistent with these elements being involved in the genomic instability.
The presence of strain-specific genes in both J99 and 26695 does interrupt the continuity of the gene order identity. To examine the conservation of gene order, each J99 predicted gene and its corresponding strain 26695 ortholog, if present, were examined to determine whether their immediate neighbors were conserved. Of the 1,496 predicted genes in strain J99, 1,268 (85%) have the same neighboring gene on each side in both genomes; 161 (10.4%) have the same neighbor on one side and a strain-specific gene on the other side; and 40 (2.7%) have a strain-specific gene on both sides. Only 27 (1.9%) have the same neighbor on one side and an orthologous gene (present in both J99 and 26695) out of order on the other side due to a difference in the physical structure of the two genomes. There are several regions of highly conserved gene similarity across the H. pylori genomes. In nine cases there are strings of greater than 50 continuous orthologous genes (with the longest being 133 consecutive orthologs) representing 46% of the total orthologous genes (Fig. 1).
Nucleotide comparisons and allelic diversity
Sequencing studies of several genes (cagA, vacA, flaA, flaB, cysS, ureC/glmM, and tnpA/B genes from IS605) have demonstrated that the nucleotide diversity of orthologous genes between independent H. pylori isolates is relatively high and that it was extremely rare that orthologous genes from two strains of H. pylori would contain the same sequence (25, 33, 42, 50, 94). Indeed, it has been shown (1, 53, 94) that recombination between gene alleles within the H. pylori population is extremely common, and any geographically based clonal groupings are very weak. Methods that detect nucleotide diversity, other than direct sequencing, have also been used to measure the genetic diversity of H. pylori isolates, including RAPD-PCR and PCR-based RFLP (4, 5, 14, 39). These data, many of which sample the whole genome, also demonstrate that it is rare for two H. pylori isolates to possess the same pattern. For this reason, although the gene order was surprisingly highly conserved between the genomes of H. pylori J99 and 26695, it was not unexpected to observe significant nucleotide diversity between the orthologous genes. However, due to the redundancy in the genetic code, there was a higher level of divergence in the genes than in the respective encoded proteins. Consistent with literature studies, no two orthologous genes between J99 and 26695 shared 100% identity whereas 41 proteins (2.9%) were 100% identical. Indeed, only eight orthologous genes (0.6%) had an identity level greater than 98%, whereas 310 proteins (22.2%) shared this level of identity (21). The average nucleotide identity for all orthologous genes was 92.6%, although the average nucleotide identity for the orthologs with a predicted function is higher, at 94.0%. The expected increase in the predicted protein similarity due to silent nucleotide changes is evident in both classes, being 93.4 and 95.4% for all the orthologous proteins and those with a predicted function, respectively (9) The H. pylori-specific genes have the lowest level of identity at 88.4% (9). The lengths of all the orthologous proteins were also compared as a further measure of divergence. Of the 429 proteins classified in metabolic functional groups (amino acid, cofactor, nucleoside and fatty acid biosynthesis, DNA replication, energy metabolism, transcription, and translation), only 49 (11.4%) were different in size, and then by an average of 3.6 amino acid residues. In contrast, the length divergence of the orthologous proteins with no predicted function was an average of 29.4 residues and occurred in 28% of the proteins (157 out of 555), with the H. pylori-specific genes contributing the majority of the diversity (9). It is possible that the nucleotide differences are a result of DNA mismatch during replication as both J99 and 26695 lack an identifiable mutH or mutL ortholog, the mutator genes that, in combination with MutS, are responsible for efficient correction of purine/pyrimidine mismatches during replication (9). This lack of a complete MutHLS system has been suggested to be reason for the high frequency of transition mutations in orthologous H. pylori genes (102).
Codon usage was analyzed to determine whether the drift in the amino acid coding triplets endowed J99 and 26695 with a different bias toward particular amino acids. While it was evident that H. pylori does possess an overall bias toward certain amino acids, this bias did not differ significantly between the two strains at a genomic level although it was evident in individual orthologous genes (10). The bias toward (G + C) nucleotides in the third "wobble" position of the coding triplets was higher than the entire genome at 42.7 and 42.0% for J99 and 26695, respectively. This was unexpected as the bias in this position is usually more extreme to compensate for the fixed positions in the coding triplet. For example, in low (G + C)% organisms like Borrelia burgdorferi (29%) or Campylobacter jejuni (31%), the (G + C) bias in the wobble position is considerably lower at around 20%.
The rate of nucleotide sequence variation observed between J99 and 26695 would provide the individuality that has been detected with different fingerprinting methods such as RAPD-PCR, repetitive sequence element (REP)-PCR, and oligofingerprinting (4, 5, 14, 32, 39, 45, 53, 63, 72, 87, 97, 101). Similarly, this variation would affect physical PFGE maps of the genomes due to alterations in the restriction endonuclease recognition sites. For example, a single silent nucleotide change is responsible for six of the seven additional NotI restriction sites found in the J99 genome compared to 26695 (8). These changes coupled with the possibility of large chromosomal rearrangements may provide a distorted impression of the macrodiversity level of H. pylori genomes. However, all of the techniques that sample the nucleotide variation evident between H. pylori isolates will remain invaluable as a rapid and precise method for the epidemiological identification and fingerprinting of H. pylori strains.
Comparative functional analyses
Central intermediary and general metabolism
Detailed analyses of the predicted metabolic function of H. pylori annotated from the genomic sequences have recently been reviewed (21, 60). Analyses of the genomes of J99 and 26695 show that H. pylori does not appear capable of using complex carbohydrates as energy sources. Glucose appears to be the only carbohydrate utilized by H. pylori and is metabolized via the Entner-Douderoff pathway. The glycolytic/gluconeogenic pathway is likely to be used for anabolic biosynthesis rather than catabolic energy production. These predictions from genomic analysis are supported by experimental evidence from several laboratories (21).
Both literature reports and genomic analysis suggest that the primary sources of pyruvate in H. pylori are lactate, l-alanine, l-serine, and d-amino acids rather than glucose or malate. Pyruvate can be converted to acetyl coenzyme A by a pyruvate oxidoreductase. Acetate formation by fermentation of pyruvate has been reported for H. pylori, and the genome of J99 contains homologs to the pta (phosphate acetyltransferase) and ackA (acetate kinase) genes. Interestingly, in strain 26695 pta has a frameshift mutation that would inactivate the gene product. The reverse reaction that would convert acetate to acetyl-coA can be carried out in 26695 by an acetyl-coA synthetase (HP1045) (Table 2), a gene that is not found in H. pylori J99. H. pylori has the ability to ferment pyruvate to ethanol (83, 84) via an alcohol dehydrogenase although J99 contains a second unique paralogous enzyme (JHP1429) (Table 2), suggesting a redundancy in this pathway. Genomic analysis identifies homologs to enzymes of the tricarboxylic acid cycle in H. pylori, which suggests that it possesses a branched noncyclic pathway. However, literature reports of experimentally derived enzymatic activities do not correlate well with the genome annotations (21). Further efforts are needed before the metabolic features of H. pylori are completely understood.
All the enzymes required for the biosynthesis of biotin, folate, heme, molybdopterin, pantothenate, pyridoxyl phospate, riboflavin, and thioredoxin are present in both H. pylori genomes. De novo pyrimidine biosynthesis has been experimentally shown in H. pylori, and all the genes encoding the necessary enzymes have been identified in the genome. In the case of de novo purine biosynthesis from formate, glycine, or serine, most of the necessary enzymes do not appear to be present, but homologs encoding putative salvage and interconversion pathways have been identified. H. pylori contains orthologs of all the enzymes required for the initiation and elongation of fatty acid biosynthesis. However, in addition to the acyl-carrier protein contained in both genomes, strain 26695 contains a unique second paralog (HP0962) (Table 2) that possesses an extended N-terminal domain. Genomic analyses predict that H. pylori J99 and 26695 can synthesize eight amino acids, while the remaining amino acids are likely to be transported into the bacterium using the large number of transporters encoded within the genome (21).
Transcription and translation
The transcription and translation processes in H. pylori appear to bear a lot of similarities to those of other gram-negative bacteria. One significant difference is the fusion of the rpoB and rpoC genes encoding the β and β′ subunits of RNA polymerase that is evident in both H. pylori genomes (21). The same is true in Wolinella, but not in Campylobacter and other related genera, where the coding regions overlap (106). The fusion of the two subunits in H. pylori and Wolinella sp. may have been the result of a frameshift mutation, and recently the separation of rpoB and rpoC by insertion mutagenesis to yield two polypeptides results in viable H. pylori that retains the ability to colonize and proliferate in C57BL/6 mice (81). Analyses of the genomes of both J99 and 26695 indicate that there are only three sigma factors present in H. pylori, rpoD, rpoN, and fliA. No homologs to the stationary-phase sigma factor, RpoS, or the heat shock-specific RpoH are evident. Two-component systems are a highly conserved mechanism of controlling bacterial gene expression, often in response to external stimuli. Both J99 and 26695 genomes have four orthologs of histidine kinase sensor proteins and seven distinct orthologs of DNA-binding response regulators. Both genomes contain homologs to the termination factors NusA, NusB, and Rho, and the paucity of transcriptional termination stem-loop structures is evidence that termination in H. pylori is likely to be Rho-dependent (21).
Both H. pylori strains have 36 tRNA genes in the same relative physical location and all the expected tRNA synthetases except for glutaminyl- and asparaginyl-tRNA synthetases (8). Two copies of the gene encoding glutamyl-tRNA synthetase, gltX, are present in J99 and 26695, one of which may function as a glutaminyl-tRNA synthetase. Alternatively, H. pylori may perform an in situ transamidation of glutamate to glutamine (and possibly also aspartate to asparagine) via the gatABC gene products, which in Bacillus subtilis encode a glutamyl-tRNAGln amidotransferase, demonstrated to functionally replace glutaminyl-tRNA synthetase activity (19).
DNA replication, restriction, and modification
H. pylori contains only five identifiable genes (dnaE, dnaN, dnaQ, dnaX, and holB) that encode core subunits of DNA polymerase III, which although representing a less complex holoenzyme than E. coli, is consistent with the complexity observed in many of the sequenced organisms to date. Both H. pylori J99 and 26695 contain multiple homologs to DNA topoisomerase I (topA). The orthologous topA gene lies adjacent to the flagellin B subunit (92) whereas the two J99-unique and the single 26695-unique topA genes are all located within the plasticity zones of the respective genomes (Table 2). The plasticity zone in both strains also contains an orthologous gene that resembles the xerCD family of integrases/recombinases, and J99 contains an additional unique xerCD homolog.
H. pylori possesses an unusually large number of genes with homology to restriction and modification enzymes. Interestingly, many of these genes appear to be strain-specific. This is consistent with subtractive hybridization studies between H. pylori J166 and 26695 that demonstrated that many of the unique genes that were found in strain J166 encoded putative DNA restriction/modification enzymes (2). This result, taken together with the analysis of the presence or absence of the J99 or 26695 unique genes in additional strains, suggests that every H. pylori isolate may contain its own specific complement of these genes (8). Genomic analysis allows these gene products to be classified as type I, II, or III restriction or modification enzymes, and recent studies have begun to look at the nucleotide sequence specificity of these enzymes as well as at their expression levels. Recently, efforts have begun to clone and express the DNA restriction and modification genes from H. pylori 26695 and J99 and test for activity. Many of the restriction genes do not appear to be active, although the cognate methylases were. H. pylori 26695 has four active restriction genes with specificities representing isoschizomers of AhdI, MboI, HinfI, and MboII. H. pylori J99 has four active restriction genes with specificities representing isoschizomers of Tsp4SI, BsaJI, HhaI, and a novel specificity (GCWGC) called Hpy99I (57). Interestingly, all the active genes identified were strain-specific. While both H. pylori J99 or 26695 contained many unique DNA restriction-modification genes, which comprised approximately 60% of the functionally assigned strain-specific genes, there were 25 genes that did appear to be orthologous (21). Among these were 10 type II methyltransferases, all of which lacked an immediately recognizable, tandemly oriented cognate restriction enzyme partner. However, all were flanked on at least one side by a gene of unknown function, often that was specific to H. pylori. If these methyltransferases are found to be conserved between other H. pylori strains, it may support the hypothesis that one method of gene regulation in H. pylori is by methylation (8).
Outer membrane protein genes and lipopolysaccharide biosynthesis
The outer membrane protein profile of H. pylori strains differs significantly from other gram-negative species as no major outer membrane proteins (OMPs) predominate, but rather multiple lower-abundance OMPs are observed (7). Analysis of the H. pylori J99 and 26695 genomes have identified five paralogous gene families ranging in size from 3 to 33 members, and two of these families contained members that were specific for either H. pylori J99 or 26695 (7). These gene families comprise approximately 5.5% of the coding capacity of each strain. Some of the proteins in the large family (Hop/Hor proteins) (7) have been shown to be porins (22, 26) and/or adhesins for gastric epithelial cells (44, 71), and this unusual set of OMPs may be a reflection of the adaptation of H. pylori to the unique gastric environment where it is found. Intriguingly, both H. pylori J99 and 26695 contain two pairs of duplicated OMP genes that are essentially identical to each other, and yet differ significantly between the strains (7). This high intrastrain identity coupled with significant interstrain diversity strongly suggests that DNA uptake from surrounding cells and homologous recombination between the duplicated loci keeps the paralogous genes essentially identical while allowing the orthologous genes in independent strains to diverge (7), perhaps providing a mechanism for host defense evasion or determination of host specificity. Significantly, these duplicated gene pairs were also found, at the same relative chromosomal location, in other H. pylori strains examined, although the N-terminal region had been deleted in a few cases (7, 53).
H. pylori contains homologs of all genes necessary for 2-keto-3-deoxyoctulosonic acid (KDO)-lipid A biosynthesis, a common structure found in the lipopolysaccharide (LPS) of the majority of gram-negative bacteria. Synthesis of the LPS core requires the sequential addition of sugar moieties, carried out by glycosyltransferases. While H. pylori 26695 and J99 have seven putative glycosyltransferase genes in common, they contain one and two unique genes, respectively, that have been identified as glycosyltransferases that may be involved in the specific addition of LPS core sugars (21). The O-antigen chain of the LPS of H. pylori is composed of Lewis acids (Lex and Ley), which are identical to those found on host tissues and have been implicated in colonization and persistence of H. pylori and may also play a role in autoimmunity (12, 13). Both H. pylori genomes encode two α-(1,3)-fucosyltransferase, one α-(1,2)-fucosyltransferase, and a β-1,4-galactosyltransferase gene that are involved in this biosynthesis of the O-antigen (34, 58, 64, 103).
Phase variation of genes responsible for altering the bacterial cell surface, the interface for interaction with the host immune system, is being identified as a common mode for pathogens to adapt and avoid immune recognition (47, 76, 77). Phase variation is often mediated by a slipped-strand repair mechanism at simple repeats within the coding sequence that places the gene in and out of frame, or in regulatory sequences that affect the expression level. These simple repeats can be mononucleotide up to tetranucleotide and can vary greatly in their length, with the longer repeats being more susceptible to mutation (15). A number of genes in H. pylori have been identified as being subject to this phase-variable regulation at both mono- and dinucleotide repeats (8, 86, 99), with almost half affecting the makeup of the cell envelope. The slipped-strand repair on these genes would be able to play a significant role in altering the composition of the outer membrane of the bacterium and may play a role in host adaptation or evasion of the host immune system. Among the six phase-variable genes involved in LPS biosynthesis, the length of a mononucleotide C repeat in the fucosyltransferases has been shown to be responsible for the variable expression of the Lewis antigens on the O-antigen chain of the LPS in H. pylori strains (11, 103).
Five members of the largest outer membrane protein superfamily in both strains J99 and 26695 contain dinucleotide CT repeats in the coding region for the signal sequence, which has been predicted to allow modulation of expression by slipped-strand repair (8). The same orthologs in both H. pylori J99 and 26695 possess these repeats, and in each case, the number of CT dinucleotide repeats in the two strains differs, but the predicted expression status is, nevertheless, unaffected (8). Three of the genes are out of frame, and two, including the Lewis babB adhesin (44), are in frame. Preliminary analysis suggested that the pattern of varying numbers of CT repeats with the same relative expression status held true when we compared additional H. pylori isolates. For example, we sequenced the signal sequence region from the hopH gene (7) in a panel of seven geographically diverse H. pylori isolates. While all of the hopH genes appeared in frame, only one other had nine CT repeats like J99, whereas five strains had six CT repeats like 26695. The hopH gene in the remaining strain had a single TT repeat flanked upstream and downstream by two and three CT repeats, respectively. Recently, however, the variable region of this gene has been sequenced by others (104) who found the gene in the off-position with five or seven CT repeats in several H. pylori isolates and also some that possessed a TT dinucleotide interruption. The interruption of CT dinucleotide repeats by a TT dinucleotide pair was also seen in the hopM and hopN duplicated genes in both J99 and 26695 that are surrounded by seven and six CT repeats, respectively, although all the genes remain in-frame. Whether this TT dinucleotide represents a subtle way to decrease the rate of slippage at these loci remains to be determined. Interestingly, in the cases of the hopZ and hopO genes, which appear out of frame in J99 and 26695, the number of CT repeats varied more widely, including examples that should have resulted in an in-frame gene but failed to due to other nucleotide variations in the adjacent area. For example, the hopZ gene from the different strains contained 6, 7, 8, 9, or 11 CT repeats, and yet due to other nucleotide variations, including changes in the mononucleotide A string that encodes the lysine residues beginning at the +2 position, all the genes were out of frame.
Comparative analyses between the genomes of H. pylori J99 and 26695 have identified that between 6 and 7% of the coding capacity of each strain contains genes that are not found in the other strain. Over 70% of these genes in both strains have no predicted function (Table 2), with the majority also being specific to H. pylori. Indeed, only 25 of the 89 and 26 of the 117 strain-specific genes were assigned a function in strains J99 and 26695, respectively. The chromosomal location of these strain-specific genes is intriguing. Almost half of these genes in each strain are located in a hypervariable region termed the "plasticity zone." This region is continuous in strain J99 but is split into two regions in 26695 due to one of the two large inversions (8) (Fig. 1 and 2). However, the position of the remaining strain-specific genes does not appear to be randomly located. Of the 35 and 39 strain-specific loci in J99 and 26695, there are 17 relative locations (18 if both ends of the plasticity zone, split by the inversion, are counted) where both the J99 and 26695 genomes contain genes absent in the other genome (Fig. 1). In both strains, the majority of these genes appear as singletons (Fig. 1). In one case, the unique gene encoding the outer membrane protein HomB (jhp870) in J99 appears partially present at the same location in 26695 as 219 bp encoding residues 496 to 569 (of a 668-residue protein) are present at 96.8% identity, suggesting that it was once present but has been deleted (7).
The hypervariable plasticity zone has a lower (G + C)% than the remainder of the genome, 35% compared to 39%, and in strain 26695 contains four of the seven complete insertion elements (8). Several of the genes, including IS605 elements, have been identified in the same genetic order on self-replicating plasmids in independent H. pylori strains (8). Taken together, these data suggest that this locus may have arisen from horizontal acquisition. While the plasticity zones contain almost half of both the J99 and 26695 strain-specific genes, there are a few genes that are conserved in other bacterial species but of unknown function as well as some with a predicted function. All of the unique genes assigned to the "DNA replication" (topA genes) and "cellular processes" (virB4 and virD4 genes) categories, with the exception of HP0548 in 26695 that encodes the cag-omega putative helicase, are located in the respective plasticity zones (8). While these genes were named strain specific, that is only in the context of strains J99 and 26695. Indeed, preliminary analysis of additional strains demonstrates that some, but not all, of these genes are contained in other H. pylori isolates (unpublished data). While each H. pylori strain is likely to contain a conserved core of genes, it is also likely that the remainder of the chromosomes in each H. pylori strain will carry a unique mixture of genes. This has already been shown in some strains using a subtractive hybridization technique (2). Whether these genes are clustered into a plasticity zone or are located in the coincident loci identified between J99 and 26695 will provide important information as to the evolution of the genomic structure of H. pylori.
A hypervariable plasticity zone was also detected when the two Chlamydia trachomatis genomes were compared (82). This locus also represented the major difference between these two genomes, although many of the genes contained in the island had a predicted function, including three tandemly arranged copies of a potential toxin molecule similar to an Escherichia coli O157 toxin, and did not display a skewed (G + C)% DNA content (82). It was suggested that the marked differences in the plasticity zone, compared with the high overall similarity of the remainder of the genome (see below), may represent the difference between the mouse and human trophic C. trachomatis genomes (82). In a similar fashion, the content of the H. pylori plasticity zones may have an influence on the type or severity of disease that is manifested by the host upon infection.
Genome of H. pylori Compared with Other Sequenced Microbial Genomes
Given the rapid rate at which diverse microbial genomes are being completed, there is a wealth of information to which the H. pylori sequence can be compared. The recent completion of the C. jejuni NCTC11168 genome (76) has now enabled the first comparison between two such closely related genera. There were several significant differences between the two organisms, including the ability to synthesize some amino acids and cofactors as well as differences in sulfur and energy metabolism (21). Further, the components of the flagellar systems were remarkably similar to each other but contained significant differences to that of the Enterobacteriaceae (38). However, there were significant differences between the chemotaxis systems of H. pylori and C. jejuni, including the presence of six additional putative methyl-accepting chemotaxis proteins in C. jejuni (38). Significantly, approximately 17% of the total number of predicted genes in H. pylori remained H. pylori specific with an unknown function after the comparison with C. jejuni. Whether these genes are truly specific to H. pylori, possibly resulting from adaptation to the gastric environment, or whether orthologs will be identified in other microorganisms should be answered as the field of genome sequencing becomes fully developed. Importantly, only representatives from 21 bacterial genera have been sequenced, a fraction of the 905 genera (containing 4,473 species) that have been validly described (74), suggesting that the understanding of bacterial metabolism is still in its infancy.
Whereas H. pylori was the first bacterial species to have two completed genome sequences available for comparison, since that time there have been several other reports of prokaryotic genome comparisons. These data enable one to view the H. pylori J99 and 26695 comparison in a different light, and the observed level of allelic or nucleotide diversity and genomic rearrangements can be placed in better perspective.
An initial report of the comparison of two Mycobacterium tuberculosis strains, one a recent clinical isolate and the other a well-passaged laboratory strain isolated in 1905, suggests that a single nucleotide polymorphism occurs approximately every 5 kb (0.02%) (31), which is approximately 2 orders of magnitude lower than that observed in H. pylori. One of the most significant changes between these two M. tuberculosis strains is the number of insertion elements. Indeed, most of the DNA-based epidemiological analysis of M. tuberculosis strains is based on mobile insertion elements (100, 105). The recent human isolate, CDC1551, has 4 copies of IS6110 whereas the heavily passaged H37Rv has 16 copies (31). The presence of an increased number of IS elements in the in vitro passaged strain parallels what is seen in H. pylori, with 26695 having seven complete IS elements compared to one in the recent clinical isolate J99. There are other minor differences such as copy number of tandem repeats (approximately every 90 kb) and gene duplications (approximately every 170 kb) in M. tuberculosis (31), but the physical genome structure and gene order appear far more conserved than observed for the two H. pylori strains.
Genomes from two Chlamydia pneumoniae and two C. trachomatis strains have been published (49, 82, 89), and a third strain of each has recently been completed by GENSET. Initial comparisons demonstrated that the two species were highly related, with C. pneumoniae containing 214 genes (~20%) that were absent in C. trachomatis, and most lacked orthologs in other bacterial species, whereas 70 C. trachomatis genes lacked orthologs in C. pneumoniae (49). When the two epidemiologically distinct C. pneumoniae 1.23-Mb genomes (strains AR39 and CWL029) were compared, they were only 377 bp different in size and contained only 296 single nucleotide polymorphisms and 21 single base-pair frameshift mutations, with many occurring in intergenic regions. Comparison of the protein products demonstrates that 86% of the orthologs were identical. The few changes identified in the physical arrangement of the two genomes were a 23-bp inverted region of DNA, the loss of a 393-bp iterated segment in an outer membrane protein gene, and the presence of an extrachromosomal bacteriophage in the AR39 genome (82). The close similarity of C. pneumoniae isolates had been demonstrated using amplified fragment length polymorphism (AFLP), although this method had suggested that the two sequenced strains represented phylogenetically separated isolates and that strain AR39 possessed a sequence variation of 6% (68), perhaps due to the presence of the bacteriophage (82). In contrast to C. pneumoniae, the two C. trachomatis genomes (strains MoPn and serovar D) contained some significant differences. The mouse C. trachomatis strain (MoPn) was almost 27 kb larger and contained 30 additional genes when compared to the human serovar D strain. The average nucleotide difference between genes was 10%, although with no major rearrangements, the conservation of overall physical structure was high (82), consistent with distinct AFLP fingerprint patterns between C. trachomatis isolates from different host origins (68). The one exception to this overall conservation was a ~50-kb plasticity zone that appears to have undergone a higher degree of genetic reorganization. The difference in size of the plasticity zones is essentially wholly responsible for the size difference between the genomes (82).
The genome sequences from two distinct Neisseria meningitidis serotypes have also recently been completed (75, 98). Our preliminary analyses indicate that over 10% of the predicted genes in each strain were not clearly identifiable in the other serotype, a number significantly higher than seen in the H. pylori genomic comparison. However, between the orthologous genes and the corresponding proteins in N. meningitidis, the level of nucleotide and amino acid identity is approximately 97%. These levels of identity are higher than found between H. pylori J99 and 26695 (9). However, the relative order of orthologous genes appears significantly more disrupted between the two N. meningitidis strains than between the two H. pylori isolates, punctuated by a large number of loci that contain the strain (serotype)-specific genes. This is similar to preliminary reports of the large number of islands of unique genes observed when the genomes of E. coli K-12 and E. coli O157 are compared.
Comparative analysis of the increasing availability of multiple genomic sequences from a single species provides important information about the genome structure and evolution of different bacterial species. While M. tuberculosis and C. pneumoniae are highly conserved and clonal organisms, the diversity seen in the two C. trachomatis and two N. meningitidis genomes is more similar to that observed between H. pylori J99 and 26695. The reported level of nucleotide diversity between C. trachomatis orthologs is ~10% (82), which is slightly higher than the 7.4% average in H. pylori (9) and the approximately 3% difference in N. meningitidis. Many of the changes between the isolates of C. trachomatis and H. pylori lie in a hyper-variable plasticity zone, although only H. pylori appears to have undergone any genomic reorganization, albeit limited when compared to the two serotypes of N. meningitidis.
Functional Genomes and Understanding the Host-Pathogen Interaction
H. pylori infection results in a wide range of clinical outcomes, and attempts have been made to correlate these with genetic diversity. The presence or absence of certain genes (i.e., cag pathogenicity island) has been associated with an increased risk of severe gastroduodenal disease in some, but significantly not all, populations (3, 29, 46, 59, 73). It is becoming increasingly clear that host, pathogen, and environmental factors all contribute to the clinical outcome of H. pylori infections (8). Bacterial factors such as strain-specific genes (either chromosomally encoded or present on plasmids), differential gene expression (potentially mediated by methylation and pseudogene generation), phase variation (mediated by slipped-strand repair), or allelic variation (such as found in cagA, which plays a role in the level of phosphorylation of the CagA protein ) may play a critical role in disease development. The increasing availability of genomic tools should now enable a more complete investigation into the participatory roles of specific bacterial or human genes in the H. pylori-host interaction.
Regulation of gene expression in H. pylori is not particularly well understood. As mentioned above, only three sigma factors have been identified to date, and there are only four orthologs of histidine kinase sensor proteins and seven orthologs of DNA-binding response regulators. Some clear examples of gene expression regulation have been demonstrated for individual genes or pathways. In H. pylori flagellar biosynthesis there are two structural genes, flaA and flaB, each of which is under the control of a different sigma factor. Primer extension experiments have shown that while flaA is expressed by RNA polymerase in conjunction with σ28 (56), the flaB gene requires RNA polymerase plus σ54 for expression (93). Recently, transcription of the ferric citrate receptor homolog gene, fecA2, and the riboflavin synthesis gene ribBA were demonstrated to be iron-repressed in H. pylori using a modified Fur titration assay in an E. coli strain expressing the H. pylori Fur repressor (27).
Acid-related control of expression has been experimentally demonstrated for H. pylori wbcJ, the gene product of which is orthologous to bacterial O-antigen biosynthesis proteins, and for cagA, picB, and ureA (51, 67). Adherence of H. pylori to gastric epithelial cells in vitro has been shown to induce expression of iceA1 and the hpyIM gene that encodes an adenine methyltransferase (78), suggesting that regulation by DNA methylation may be important in H. pylori. The relative abundance of DNA methylation orthologs in the H. pylori genome, especially those with no apparent cognate restriction partners, supports this notion. The hspR gene product of H. pylori has been demonstrated to function as a transcriptional repressor of genes that encode the major chaperones, including groESL, hrcA-grpE-dnaK, and cbpA-hspR-orf (88).
The publication of the entire set of ORFs present in H. pylori has presented scientists with the opportunity to study gene expression and regulation on a whole-cell level. High-density filters containing fragments of the entire set of 26695 ORFs are already commercially available. Such tools are going to generate vast quantities of data in the coming years that will enable a greater understanding of the response of H. pylori to various environmental and host factors. One particular area of interest will be the potential for array and filter technologies to help identify the functions of unknown ORFs. For example, genes of unknown function that co-express with known pathways in response to certain stimuli can be identified and subsequently studied by classical molecular biology and biochemistry methods to elucidate their precise functions. Another potentially powerful use for high-density array technologies would be to compare expression profiles of wild-type strains versus defined mutants to identify regulatory circuits and knock-on effects that a mutation in one metabolic pathway may have on other cellular activities. High-density arrays would also allow the rapid identification of genes present in the arrayed genome (for example, J99 or 26695) that are absent in another H. pylori strain being queried. Other techniques such as subtractive hybridization would be required to identify the reverse situation, genes exclusively present in the "query" isolate but absent in the arrayed genome.
One of the greatest challenges facing the high-density array technologies is to be capable of providing data on gene expression from bacterial mRNA isolated from cells cultured in vivo. A number of techniques for monitoring in vivo expression or essentiality have been described in recent years (e.g., IVET, signature-tagged mutagenesis, reverse transcription-PCR, and others; for a review, see reference 16). The advantage held by the high-density array technologies is that expression data can be theoretically collected on every gene in H. pylori from cells harvested from human biopsies.
Diagnostic and Therapeutic Potential
As more bacterial genomes are completed, the ability of researchers to dissect bacterial pathogenesis will be considerably enhanced. Two important medical areas that will undoubtedly benefit from this are diagnostics and therapeutics. In the case of H. pylori, the urea breath test or serum antibody diagnostic tests could readily be supplemented by PCR-based diagnostic tests. Having the complete DNA sequence available of two independent isolates, as well as the ability to compare it with that of other species, allows the design of specific PCR primers, which would readily determine the presence or absence of H. pylori in patients suspected of harboring an infection. Perhaps even more useful may be the application of diagnostic primer sets that could discriminate between particular strains of H. pylori. For example, primers designed to amplify regions of the 23S rRNA sequence could identify whether a strain is resistant or susceptible to the antibiotic clarithromycin (90) prior to treatment and may decrease treatment failures due to resistance.
As is the case with most pathogens with available genome sequences, there has been significant interest in the potential use of these data for the generation of novel therapeutics and vaccines. The recent publication of the N. meningitidis genome sequence (98) was accompanied by a report of the utilization of these data to identify, clone, and express 350 potential vaccine antigens in E. coli (79). A number of companies and institutions have been investigating the genomes of H. pylori in attempts to identify and evaluate vaccine candidates that would not only offer prophylactic but also therapeutic efficacy. Vaccination has been successfully demonstrated in animal models with recombinant H. pylori antigens (23, 28, 35, 41, 54, 61, 80), although an attempt using recombinant urease in humans was less convincing. The availability of the complete genome has also allowed other approaches, such as proteomics, to be employed to identify novel vaccine candidates (65, 66, 69).
Currently the only approved anti-H. pylori therapies involve a combination of antimicrobial and acid suppression compounds. Although often successful in the majority of patients, problems with this therapy include patient noncompliance with the regimen, due to its complexity and accompanying side effects, as well as the occurrence of increasing resistance to the commonly used antibiotics such as metronidazole and clarithromycin (6, 37). Future antibiotics targeted at H. pylori would ideally be specific for this bacterium in order to decrease the overall use of broad-spectrum antibiotics (overuse is a major cause of increased rates of resistance in other bacteria) as well as to decrease the side effects commonly seen when nonselective antibiotics interfere with the patient's commensal flora. Using the genomic information of H. pylori, a metabolic blueprint of the organism has been predicted and then experimentally confirmed. This information can then be used to predict biological pathways or functions that are essential for the viability of the organism. Target proteins or pathways subsequently proven to be essential in vitro or in vivo can then be used in screening approaches to identify chemical compounds that can inhibit their respective biochemical activities. Subsequent optimization of these compounds to obtain suitable microbiological and pharmacological characteristics will result in novel, specific anti-H. pylori agents. An alternative approach is to identify compounds from direct antibacterial screening programs that already possess anti-H. pylori activity, but whose targets are unknown. Genomic data coupled with technologies such as high-density microarrays can be used to determine the mechanism of action of such compounds.
The future of Helicobacter basic and applied research has been irrevocably changed by the advent of the genomic era. With two complete H. pylori genomes available, the next few years will see an increasing body of knowledge arising from the application of the genomic sequence data to all aspects of H. pylori biology, from basic information about metabolism and mechanisms of pathogenesis, to novel vaccines and antibiotics. It is likely that the genomes of more Helicobacter species will be sequenced and the genes that are involved in host specificity and disease severity will be identified.
Perhaps the greatest challenge will be to determine the functions of the unknown genes in H. pylori. It seems likely that these orphan genes encode many of the phenotypic characteristics that allow H. pylori to function and thrive in the hostile gastric environment. Classical knockout approaches can be used to determine if these genes encode proteins essential for growth or pathogenesis but will not provide much information on the biochemical role of the encoded proteins. Certainly, new and more sophisticated computer algorithms are needed to pave the way for a rapid determination of predicted function from primary sequence data. Structural genomics applied to the solution of X-ray crystal structures of unknown proteins may prove useful in helping identify function and, once a sufficiently large X-ray structure database is available, could lead to virtual predictions of high-resolution structures directly from sequence data.
A major challenge will be to gain a better understanding of the role of host factors in H. pylori infections. All the evidence to date points to the human–H. pylori interaction as being complex and multifaceted. The application of techniques such as expression profiling with both human and bacterial microarrays should help answer many of the questions relating to this unique host-pathogen interaction. Knowledge about the intricacies of this interaction will greatly increase our chances of finding novel, effective treatments for this medically significant pandemic.
We thank our colleagues at AstraZeneca R&D Boston for their willingness to participate in many stimulating discussions. We give special thanks to Dave Schultz for his bioinformatics help and to Rishi Sanyal for performing many of the comparisons between H. pylori and C. jejuni.
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Richard A. Alm and Brian Noonan.
ASM Press, Washington (DC)
Alm RA, Noonan B. The Genome. In: Mobley HLT, Mendz GL, Hazell SL, editors. Helicobacter pylori: Physiology and Genetics. Washington (DC): ASM Press; 2001. Chapter 27.