Sequence divergence and conservation in genomes of Helicobacter cetorum strains from a dolphin and a whale.

BACKGROUND AND OBJECTIVES: Strains of Helicobacter cetorum have been cultured from several marine mammals and have been found to be closely related in 16 S rDNA sequence to the human gastric pathogen H. pylori, but their genomes were not characterized further.
METHODS: The genomes of H. cetorum strains from a dolphin and a whale were sequenced completely using 454 technology and PCR and capillary sequencing.
RESULTS: These genomes are 1.8 and 1.95 mb in size, some 7-26% larger than H. pylori genomes, and differ markedly from one another in gene content, and sequences and arrangements of shared genes. However, each strain is more related overall to H. pylori and its descendant H. acinonychis than to other known species. These H. cetorum strains lack cag pathogenicity islands, but contain novel alleles of the virulence-associated vacuolating cytotoxin (vacA) gene. Of particular note are (i) an extra triplet of vacA genes with ≤50% protein-level identity to each other in the 5' two-thirds of the gene needed for host factor interaction; (ii) divergent sets of outer membrane protein genes; (iii) several metabolic genes distinct from those of H. pylori; (iv) genes for an iron-cofactored urease related to those of Helicobacter species from terrestrial carnivores, in addition to genes for a nickel co-factored urease; and (v) members of the slr multigene family, some of which modulate host responses to infection and improve Helicobacter growth with mammalian cells.
CONCLUSIONS: Our genome sequence data provide a glimpse into the novelty and great genetic diversity of marine helicobacters. These data should aid further analyses of microbial genome diversity and evolution and infection and disease mechanisms in vast and often fragile ocean ecosystems.

Introduction

The genus Helicobacter consists of Gram-negative bacterial species that live in the gastrointestinal tracts of diverse animal hosts [1]–[3]. H. pylori, the best known of these species, chronically infects the gastric (stomach) mucosa of billions of people worldwide, is a major cause of peptic ulcer disease and gastric cancer, and is very diverse genetically. It is transmitted preferentially within families and local communities, apparently without major environmental reservoirs or alternate hosts [4]–[7].

Much less is understood about transmission and infection mechanisms, virulence, and population biology and evolution of other Helicobacter species. Although most of these species are known from land animals, a few also have been discovered in marine mammals. Of particular note is H. cetorum from marine mammals, defined to date primarily by its 16 S rDNA sequences [8]–[13], which are more closely related to those of H. pylori and the big cat pathogen H. acinonychis [14] than to those of other known species. PCR and 16 S rDNA sequence data indicate that H. cetorum is present in oceans worldwide [8]–[13], and suggest that it or close relatives also caused gastric infections in some urban Venezuelans [15] and lymph node infections in mule deer in Montana [16]. Interestingly, the genus Helicobacter belongs to the Epsilonproteobacteria, some of whose other members are associated variously with coral and sponge disease, and gastropods and biofilms of deep-sea hydrothermal vents [17]–[21]. Here, we sequenced the genomes of H. cetorum strains from a whale and a dolphin to help define this species' gene content and diversity, with long-range goals of better understanding pathogen transmission and infection mechanisms in marine ecosystems, genome evolution, and possible impacts of non-pylori Helicobacter species on animal and human health.

Methods

H. cetorum Culture and Genome Sequencing

The two H. cetorum strains that we sequenced had been cultured by Harper et al [8] from the main (glandular) stomach of a beached Atlantic white sided dolphin (MIT 99–5656, here called “dolphin strain”), and the feces of a captive (Mystic Aquarium) Beluga whale with esophageal and stomach ulcers (MIT 00-7128, here called “whale strain”), and had been deposited as ATCC BAA-540 and ATCC BAA-429 (or CCUG 52418 T), respectively [8]. The whale strain, although cultured from feces, was inferred to have lived in its host's stomach because its 16 S rDNA sequence was identical to that obtained by PCR from the animal's gastric tissue [8]. We grew these strains from single colonies using standard H. pylori culture conditions (BHI blood agar plates at 37°C, in 5% CO2, 10% O2 and 85% N2) and extracted genomic DNA as described [22], [23]. Genomic DNAs were sequenced using 454 FLX Titanium paired-end shotgun sequencing (>40-fold coverage), and reads were assembled using 454 Corporation Newbler software (164 and 88 contigs, dolphin and whale strains, respectively) by MOGene Corporation (St Louis, MO). We determined relative positions of contigs by PCR and filled all gaps between contigs by capillary sequencing of PCR products. The genome sequences were deposited in GenBank as accessions CP003481.1 (chromosome) and CP003482.1 (plasmid) of the dolphin strain, and NC_017737.1 (chromosome) and NC_017738.1 (plasmid) of the whale strain, and were annotated by the NCBI Prokaryotic Genome Automatic Annotation Pipeline staff, as described [23].

Comparative Genomics and Phylogenetic Analysis

Complete, fully-annotated chromosome and plasmid sequences of the Helicobacter strains and species listed in Table 1 were downloaded from the NCBI ftp server; a database containing all predicted protein sequences was assembled and low-quality protein sequences were removed automatically. Reciprocal all-versus-all BLASTP was performed and results were processed by OrthoMCL using default parameters [24]. The OrthoMCL output was filtered using a perl script to produce different lists of ortholog groups (e.g. ortholog groups present in H. cetorum but not in H. pylori). Using the OrthoMCL output, we selected 126 genes in the core genome of gastric Helicobacter species with orthologs in a non-gastric outgroup species, H. hepaticus (Table S1). Alignments for each of these one-to-one rooted core genes were generated at the amino acid level using MAFFT-FFT-NS-i v.7 [25]; the proteins were back-translated to nucleotide sequence using Translatorx perl script [26]; aligned DNA sequences were concatenated using a perl script, and the phylogenetic tree was inferred using PhyML [27] by applying the following parameters: -b 2, -m GTR, -o tlr –a e, -c 6. A distance matrix of the concatenated aligned core genes was calculated using DISTMAT implemented in jEMBOSS using Kimura-2 [28].

Table 1

Strains and species used in this study.

	NCBI accession number
Species and strain designation	Chromosomes	Plasmid
Helicobacter cetorum
MIT-00-7128	NC_017737	NC_017738
Helicobacter cetorum
MIT-99-5656	NC_017735	NC_017736
Helicobacter pylori
2017	NC_017374
2018	NC_017381
26695	NC_000915
35A	NC_017360
51	NC_017382
83	NC_017375
908	NC_017357
Aklavik117	NC_019560	NC_019561
Aklavik86	NC_019563	NC_019564
B38	NC_012973
B8	NC_014256	NC_014257
Cuz20	NC_017358
ELS37	NC_017063	NC_017064
F16	NC_017368
F30	NC_017365	NC_017369
F32	NC_017366	NC_017370
F57	NC_017367
G27	NC_011333	NC_011334
Gambia94 24	NC_017371	NC_017364
HPAG1	NC_008086	NC_008087
HUP B14	NC_017733	NC_017734
India7	NC_017372
J99	NC_000921
Lithuania75	NC_017362	NC_017363
OK113	NC_020508
OK310	NC_020509	NC_020556
P12	NC_011498	NC_011499
PeCan18	NC_017742
PeCan4	NC_014555	NC_014556
Puno120	NC_017378	NC_017377
Puno135	NC_017379
Rif1	NC_018937
Rif2	NC_018938
SJM180	NC_014560
SNT49	NC_017376	NC_017380
Sat464	NC_017359	NC_017356
Shi112	NC_017741
Shi169	NC_017740
Shi417	NC_017739
Shi470	NC_010698
SouthAfrica7	NC_017361	NC_017373
UM032	NC_021215
UM037	NC_021217
UM066	NC_021218
UM299	NC_021216
XZ274	NC_017926	NC_017919
52	NC_017354
v225d	NC_017355	NC_017383
Helicobacter acinonychis
Sheeba	NC_008229	NC_008230
Helicobacter bizzozeronii
CIII-1	NC_015674	NC_015670
Helicobacter heilmannii
ASB1.4	NC_019674
Helicobacter felis
CS1	NC_014810
Helicobacter mustelae
12198	NC_013949
Helicobacter hepaticus
ATCC 51449	NC_004917

The two H. cetorum genome sequences were submitted to GGDC 2.0 [29], available at http://ggdc.dsmz.de, to calculate whole-genome distance and infer the degree of DNA-DNA hybridization between them.

To identify orthologs common to the two H. cetorum strains, the complete set of predicted proteins of one strain was compared with that of the other by reciprocal BLASTP. A BLAST score ratio cut-off of 0.4 was used to define two proteins as homologs.

Proteins identified by OrthoMCL as belonging to groups of orthologs that occur only in H. cetorum strains were then used as queries for BLASTP homology searches against the total NCBI database available in August 2013 to find related sequences, especially in H. pylori, and to better understand patterns of sequence conservation and divergence among related proteins.

Results

Phylogenetic Relationships of H. cetorum Strains

The chromosomes of the H. cetorum whale and dolphin and strains are 1.95 and 1.83 Mb Mb in size, respectively — a few hundred kb larger than is typical of H. pylori (1.55–1.71 Mb). Each strain also contains a plasmid, 12.5 and 14.1 kb in size, respectively (Table 2). The complete 16 S and 23 S rDNA sequences of these two strains differ by only 5 bp and 10 bp, respectively, and each is more closely related to the rDNAs of H. pylori and H. acinonychis than to those of other known species [8 and present results]. Whole genome BLASTN (http://blast.ncbi.nlm.nih.gov/) analyses confirmed and extended inferences from rDNA data — showing that these two strains are more closely related to various H. pylori strains or H. acinonychis than to any other known bacterial species. That said, only ∼64% of whale and ∼74% of dolphin strain genomes are found by BLASTN criteria in H. pylori genomes, and reciprocally, only ∼75–80% of representative H. pylori strain genome sequences are found in these H. cetorum genomes.

Table 2

General features of H. cetorum genomes.

Feature	MIT 00–7128, whale strain	MIT 99–5656, dolphin strain
Chromosome
Size bp	1 947 646	1 833 666
G+C content (%)	34,5	35,8
% Coding	88	88,4
Number genes	1 775	1 731
Protein coding	1 731	1 689
Structural rRNAs	38	36
16 S,23 S,5 S rRNAs	2,2,2	2,2,2
vacA	one next to cysS	one next to cysS, plus three divergent between ruvA, ruvB
cag pathogenicity island	Absent	absent
Urease	two: nickel & iron co-factored	two: nickel & iron co-factored
mobile DNAs	two TnPZ transposons; one near complete prophage with numerous rearrangements and insertions of probably non-phage DNAs	one IS605- and twenty IS606-like insertion sequences; one fragmented TnPZ transposon; multiple and duplicated prophage fragments
Plasmid	one, pHCW	one, pHCD
size (bp)	12 465	14 124
G+C content (%)	34,5	32,7
Number genes (orfs)	13	15
Other features	putative replication and transfer genes also present in dolphin strain plasmid	putative replication and transfer genes also present in whale strain plasmid; two IS606, nearly identical to chromosomal IS606

The phylogenetic positions of these strains (Figure 1) were also inferred by Maximum Likelihood using 126 concatenated core genes (Table S1). All nodes in this tree are well supported with Chi2-based parameter branch values of over 99%. The two strains clustered together in the sister clade of H. pylori/H. acinonychis, but are separated by relatively long branches. The kimura-2 corrected distance value between these two strains, calculated based on these 126 core genes, is 16.15 substitutions per 100 bp (16%). Using these same core genes, the average distance between H. pylori or H. acinonychis and H. cetorum is approximately 20%, whereas that among sequenced H. pylori genomes is only 4.1%. Thus, at 16% substitution, these two H. cetorum strains differ from each other far more than would have been expected based on the near identity of their 16 S rRNAs (1489/1494 bp).

Figure 1

Phylogram representing maximum-likelihood tree of gastric Helicobacter species based on 126 aligned and concatenated core genes.

The tree was inferred using PhyML applying General Time Reversible (GTR) model, estimating the gamma shape parameter by setting the number of substitution rate categories at 6. Statistical tests for branch support were conducted via a Chi2-based parametric approximate likelihood-ratio test (aLRT). All nodes are supported with aLRT values > 99%. The topology, branch lengths and rate parameters of the starting tree were optimized. The enteric (non-gastric) species H. hepaticus was used as outgroup. The core genes used for this figure are listed in Table S1.

Four additional tests were used to further characterize relationships of the H. cetorum strains to each other and to H. pylori, genome-wide. First, Mega BLAST analysis indicated that only 66% of dolphin strain DNA sequences are present in the larger whale strain genome. Similarly, BLASTN analysis of 1 kb chromosomal segments taken sequentially from the dolphin strain without regard to gene content indicated that some 30% of them have no significant homology to whale strain sequences. In contrast, pairs of H. pylori strains typically share >90% of chromosomal DNA sequences. The H. cetorum strain-specific DNAs are widely dispersed about their genomes, not concentrated in just one or a few sites (e.g., as chromosomal islands). Second, only 11% of sequential 1 kb chromosomal segments from the dolphin strain were at least 95% identical to whale strain sequences for at least 500 bp. In contrast, with even the least related pairs of H. pylori strains, ≥95% identities for >500 bp are found in more than 40% of such 1 kb segments. Third, chromosome alignment using MAUVE software revealed 204 differences in location and orientation of shared DNA segments between the H. cetorum strains (Figure 2A). In addition, the dolphin and whale strain chromosomes exhibited 135 and 203 differences, respectively, in DNA arrangement when aligned with that of a representative H. pylori strain (G27 [30]), whereas less than 10–15 DNA arrangement differences are found when comparing chromosomes of most other H. pylori strains with one another, as illustrated with strains G27 and Shi470 in Figure 2B [see also reference 23]. Fourth, DNA-DNA hybridization (DDH) parameters, estimated in silico by calculating whole-genome distance using the GGDC website, yielded a DDH estimate 29.1%±2.44 for these two strains. Based on conventional criteria [29], this indicates a probability via logistic regression of only 0.07% that they belong to the same species. A fifth test of relatedness and divergence emerged from our in silico proteome analyses, below.

Figure 2

MAUVE alignment of representative Helicobacter chromosomes.

For MAUVE software see http://gel.ahabs.wisc.edu/mauve/. A. Two H. cetorum genomes. B. Two representative H. pylori genomes. For further illustration of the higher conservation of gene order and orientation in H. pylori relative to H. cetorum, see [23].

In silico Proteome Analysis

Examination of annotated genomes identified 86,309 predicted protein sequences in the chromosomes of 48 H. pylori strains and seven other Helicobacter species and in 25 Helicobacter plasmids (Table 1). Based on MCL clustering, 96% of the proteins were divided into 2,934 groups of orthologs (GOs), of which 1,478 and 1,434 GOs were detected in the whale and dolphin strain proteomes, respectively. Approximately 10% (164) of whale and 7% (112) of dolphin strain proteins have no orthologs in other genome sequenced Helicobacter species, and thus might be unique to H. cetorum. Among the 2,934 GOs, 157 are represented in whale but not dolphin strain proteomes, and 113 are represented in dolphin but not whale strain proteomes. The two H. cetorum strain proteomes were compared further using a BLAST score ratio cut-off of 0.4, which is more stringent than OrthoMCL, and can separate distant proteins that cluster in the same group by MCL. BLAST analysis identified 411 whale strain proteins (24% of proteome), with no significant homology to any dolphin strain protein, and conversely, 346 dolphin strain proteins (22% of proteome) with no significant homology to any whale strain protein. Thus, these data indicate considerable differences in the proteomes of these two H. cetorum strains.

H. cetorum-specific Genes

Forty-six GOs were found in the two H. cetorum strains but not in any H. pylori strain (Tables 3 and 4) by initial OrthoMCL-based screening using the genome-sequenced strains listed in Table 1. Of particular interest are enzymes of central intermediary metabolism such as a rhodanese-related sulfurtransferase (HCW_07590, HCD_02790), which KEGG pathway analysis suggests could catalyze synthesis of pyruvate and thiosulfate from 3-mercaptopyruvate (Figure 3; blue arrows) or possibly other substrates. Homologous sulfurtransferases seem to be absent from nearly all other genome-sequenced Epsilonproteobacteria, including all other Helicobacter spp. and Campylobacter spp. A second example is that of the NADP-dependent malic enzyme (HCW_01140, HCD_04775), that could catalyze synthesis of L-malate from pyruvate (Figure 4, blue arrows). Related malic enzymes have been found in many extragastric Helicobacter spp. and in Campylobacter spp., but not in any H. pylori strain. Conversely, 22 GOs were detected in the H. pylori/H. acinonychis clade but not in H. cetorum, as illustrated in Table 5. We note, in particular, enzymes that could mediate synthesis of L-homocysteine, conversion of L-cysteine to thiocysteine or pyruvate (Figures 3, red arrows); and syntheses of acetoacetyl-CoA and acetate from acetyl-CoA, and of acetoacetate from acetoacetyl-CoA (Figure 4, red arrows). Finally, a phosphoenolpyruvate carboxylase that could catalyze oxaloacetate synthesis from phosphoenolpyruvate (Figure 4; light green arrow) is encoded in the genomes of the whale strain and of several other Helicobacter species, but not in the dolphin strain genome, nor in any H. pylori or Campylobacter strain genome sequenced to date.

Table 3

H. cetorum whale strain proteins distinct from those in H. pylori strains.

Locus Tag	GO	# amino acids (aa)	Annotation	Matches in H. cetorum, aa identity (blastp)	Matches in H. pylori aa identity (blastp)
HCW_00105	HEL3581	246	Hypothetical	HCD_03325, 54% in aa 68–139	None
HCW_00115	HEL3059	122	Hypothetical	HCD_03315, 89%	None
HCW_00125	HEL3852	246	HcpA	HCD_03275, 95%	Most strains, ≤32% in aa ∼97–241,
HCW_00130	HEL3853	421	Hypothetical	HCD_03280, 94%	None
HCW_00595	HEL3854	208	hypothetical, COG0500, SAM-dependent methyltransferase	HCD_03930, 98%	None
HCW_01140	HEL3270	420	Malate dehydrogenase	HCD_04775, 95%	None (1)
HCW_01270	HEL3858	290	COG0338, DNA adenine methylase	HCD_08595	Two strains, 65%, 72% (2)
HCW_01595	HEL3859	1437	COG3468 anticodon nuclease masking agent;	None	None (3)
HCW_01740	HEL3860	390	COG0477, drug transport transmembrane	HCD_00760, 98%	None
HCW_02225	HEL3057	752	OMP; pfam01856	HCD_02935, 54%; HCD_05585, 50%; HCW_07955, 32%; HCD_00325, 33%; HCD_08430, 31%; HCD_05575, 30% (4)	Many strains, ≤28%
HCW_02500	HEL3861	488 aa	Hypothetical	HCD_03555, 38% in aa 1–281, 61% in aa 285–488	None
HCW_03170	HEL3864	891	OMP, HomB, pfam01856	HCD_05580, 66%	Many strains, ≤31%
HCW_03370	HEL3867	73	copper binding, chaperone	HCD_06365, 60%	Many strains, ≤42% (5)
HCW_03525	HEL3071	211	Hypothetical	HCD_07120, 50%; HCD_07510, 51%; HCD_00640, 42%; HCD_01920,42%	Two strains, ≤46%
HCW_04205	HEL3869	341	Hypothetical	HCD_08400, 92%; HCD_02210, 75% in aa 182–301	None
HCW_04215	HEL3870	146	Hypothetical	HCD_08395, 97%	None
HCW_04220	HEL3871	155	Hypothetical	HCD_08385, 74%; HCW_05395, 89% in aa 1–64	Several strains, ≤79% in aa 1–62
HCW_04245	HEL3872	74	Hypothetical	HCD_08370, 93%	None
HCW_04250	HEL3873	113	Hypothetical	HCD_08365, 95%	None
HCW_04255	HEL3874	332	COG0582 integrase (6)	HCD_08360, 92%	Many strains, ≤39%
HCW_04280	HEL3061	291	Hypothetical	HCD_07575, 51% in aa 1–167	None
HCW_04310	HEL3062	72	Hypothetical	HCD_07600, 88%	None
HCW_04320	HEL3625	603	Hypothetical	HCD_07610, 79% in aa 1–100, 80% in aa 336–603	Many strains, ≤28% in N terminal, C sub-terminal domains
HCW_04375	HEL3875	69	Hypothetical	HCD_07645, 94%	None
HCW_04395	HEL3876	191	Hypothetical	HCD_07665, 83%	None
HCW_04410	HEL3877	111	Hypothetical	HCD_07680, 80%	Many strains, ≤27%
HCW_04415	HEL3878	237	Hypothetical	HCD_07685, 96%	Many strains, ≤29%
HCW_04530	HEL2846	870	phosphoenolpyruvate carboxylase	None	None (7)
HCW_04560	HEL3620	385	COG0286 type I restriction-modification HsdM	HCD_02110, 93%	Many strains, ≥38% in aa 65–383
HCW_04565	HEL3879	194	COG0732 type I restriction-modification HsdS	HCD_02105, 97%	None
HCW_04635	HEL3880	470	OMP_2, pfam02521	HCD_05480, 78%; HCW_06475, 40%	Many strains, ≤38%
HCW_04920	HEL2809	799	OMP, HopG, pfam01856	HCD_06965, 65% in aa 64–799; HCD_06965, 65%; HCW_06795, 99%; HCW_07665, 76%; HCW_06910, 52%; HCW_07970, in 482–799, 79% (4)	Many strains, ≤40% in aa 627–799
HCW_05300	HEL2800	446	OMP, pfam01856	HCD_06515, 51%; HCD_02735, 45%; HCD_00320, 44% (4)	None
HCW_06445	HEL3881	720	Type I restriction-modification HsdM	HCD_02745, 72%	None
HCW_06450	HEL3882	481	SSF116734, Type I restriction-modification HsdS	HCD_02740, 40%	None
HCW_06795	HEL2809	799	OMP, HopG, pfam01856	HCD_06965, 65% in aa 64–799; HCD_06965, 65%; HCW_04920, 99%; HCW_07665, 76%; HCW_06910, 52%; HCW_07970, in 482–799, 79% (4)	None
HCW_06910	HEL2809	718	OMP, HopG, pfam01856	HCW _04920 & _06795, 52%; HCD_06965, 52%; HCW_07970, 74% in aa 509–718 (4)	None
HCW_07065	HEL3884	419	OMP3	HCD_07105, 45%; HCD_08025, 61%; HCW_07110, 52% in aa 154–419; HCW_07075, 52% in aa 152–419	Many strains, ≤48% in aa 161–419
HCW_07120	HEL3885	128	hypothetical; CRISPR/Cas system associated	HCD_08225, 83%in aa 69–128	None
HCW_07125	HEL3886	274	CRISPR/Cas system-associated RAMP superfamily protein Cas6	HCD_08220, 91% in aa 1–192	None
HCW_07130	HEL3887	550	CRISPR/Cas system-associated protein Cas10	HCD_08210, 46% in aa 16–132; HCD_08205, 54% in aa 350–421	None
HCW_07495	HEL3889	1054	COG1002 type II restriction-modification. N-6 adenine methylase	HCD_01155, 90%	One strain, 77%
HCW_07510	HEL7510	210	Hypothetical	HCD_00640, 50%; HCD_07210, 43%; HCD_02790, 64%; HCW_03525, 51%; HCW_01920, 50%	Two strains, ≤53%
HCW_07590	HEL3071	413	COG2897 rhodanese-related sulfur transferase	HCD_02790, 64%;	None (8)
HCW_07625	HEL3891	180	Hypothetical	HCD_0820, 67%; HCD_08215, 25%
HCW_07630	HEL3073	97	hypothetical; COG0790 FOG: Sel1-like repeat family	HCD_08525, 69%; HCW_07635, 75%	One strain; 50% in aa 59–88
HCW_07635	HEL3073	89	hypothetical; COG0790 FOG: Sel1-like repeat family	HCD_08525, 74%; HCW_07630, 75%	None
HCW_07665	HEL2809	810	OMP, HopG, pfam01856	HCD_06965, 66%; HCW_04920 & HCW_06795, 76%; HCW_06910, 52% in aa 97–810; HCW_07970, 43%(4)	Many strains; <38% in C terminal 120 aa
HCW_07955	HEL3892	731	OMP HomB	HCD_00325, 53%; HCD_02935, 33%; HCD_05585, 31%; HCD_03000, 30% in aa 13–503, 79% in aa 550–731; HCD_01075, 34% in aa 177–831; HCD_01075, 34% in aa 208–731; HCW_08600, 37%; HCW_02225, 32%	Many strains, ≤32% ident in aa 177–831
HCW_08150	HEL3894	117	Hypothetical	HCD_07625, 86%	None
HCW_08195	HEL3895	108	Hypothetical	HCD_08390, 83%; HCW_04200, 83%	None
HCW_08600	HEL3058	795	OMP; pfam01856	HCD_08430, 54%; HCD_03000, 54%; HCD_00325, 42%; HCD_05585, 32%; HCD_02935 in aa 184–795; HCD_01285, 30%; HCW_02225, 32% in aa 195–795	Many strains, ≤32% in aa 210–795

Campylobacter species.(1) Homologs of HCW_01140 in many

% aa identity in many other H. pylori strains.(2) Distant homologs of HCW_01270 with up to 29

Helicobacter species such as H. bilis, H. winghamensis, and H. fennelliae.(3) Distant homologs of HCW_01595 in other

% and 50% identity overall to HCD_02935 and HCD_05585, but >85% identity to these proteins starting at aa position ∼590 of the 752 aa long protein.(4) For most OMPs in this table, distribution of identities throughout protein is distinctly non-random, with highest sequence conservation in carboxy terminal, and in some cases also amino terminal domains. For example, HCW_02225 exhibits 54

% aa identity in other Helicobacter species including H. cinaedi, H. bizzozeronii, and H. hepaticus.(5) Homologs of HCW_03370 with up to 39

(6) HCW_04255 is just one of four "integrases" annotated in the whale strain proteome.

–54% in H. bizzozeronii, H. felis, H. bilis, H. fennelliae, H. mustelae, H. hepaticus and Wolinella.(7) Homologs of HCW_04530 with identities of 47

–38% in multiple strains of Leptotrichia, Actinobacillus, Providencia, Haemophilus, Morganella, etc.(8) Homologs of HCW_07590 with aa identities of 35

Table 4

H. cetorum dolphin strain proteins distinct from those in H. pylori strains.

Locus Tag	GO	# amino acids (aa)	protein annotation	Matches in H. cetorum, aa identity (blastp)	Matches in H. pylori, aa identity (blastp)
HCD_00320	HEL2800	487	OMP, HopK, pfam01856	HCW_05300, 50% in aa 182–487; HCD_08540, 37% in aa 23–367; HCD_02735, 55%; HCD_06515, 51%;	Many strains, ≤37% in aa 330–465
HCD_00325	HEL3892	741	hypothetical	HCD_03000 & _08430, 47%; HCD_05585, 31%; HCW_07955, 53%; HCW_08600, 42%	Many strains, ≤31%
HCD_00760	HEL3860	396	COG0477, sugar/drug transport membrane	HCW_01740, 98%;	None
HCD_01155	HEL3889	1054	Type II restriction-modification, N-6 adenine methylase	HCW_07495, 90%	One strain, 78%
HCD_02105	HEL3879	194	COG0732 Type I restriction-modification. HsdS	HCW_04565, 97%;	Many strains, ≤25%
HCD_02110	HEL3620	385	COG0286 Type I restriction-modification. HsdM	HCW_04560, 93%	Many strains, ≤37%
HCD_02735	HEL2800	506	OMP, HopK	HCD_08540, 100% in aa 58–336; HCD_06515, 58%; HCD_00320, 55%	Many strains, ≤37% in aa 306–506
HCD_02740	HEL3882	498	SSF116734: Type I restriction modification. DNA specificity domain superfamily HsdS	HCD_06450, 40%	Many strains, ≤30% ident for <∼200 aa from many parts of protein
HCD_02745	HEL3881	720	COG0286 Type I restriction-modification. N-6 adening methylase HsdM	HCD_06445, 72%	Many strains, ≤24% identity, C terminal ∼half of protein
HCD_02790	HEL3890	403	COG2897 rhodanese-related sulfur transferase	HCW_07590, 64%	None (1)
HCD_02935	HEL3057	746	OMP, pfam01856	HCW_02225, 55%; HCW_07955, 34%; HCW_08600, 34% in aa 135–746; HCW_03765, 29% in aa 197–746; HCW_02225, 55%; HCD_05585, 55%; HCD_00325, 34%; HCD_05575, 32%	Many strains, ≤28%
HCD_03000	HEL3058	806	OMP, HomB, pfam01856	HCW_08600, 54%;HCW_07955, 39%; HCD_08430, 78%; HCD_00325, 46%; HCD_05585, 31%; HCD_01285, 30%	Many strains, ≤31% in aa 213–806
HCD_03265	HEL3059	122	hypothetical	HCW_00115, 89%; HCD_03315, 100%.	None (2)
HCD_03275	HEL3852	248	HcpA, cysteine rich protein	HCW_00125, 95%	All strains, 32% in aa 93–241
HCD_03280	HEL3853	274	hypothetical	HCW_00130, 94%	None
HCD_03315	HEL3059	122	hypothetical	HCW_00115, 89%; HCD_03265, 100%	None (2)
HCD_03325	HEL3851	72	hypothetical	HCW_00105, 54%	None
HCD_03555	HEL3861	575	hypothetical	HCW_02500, 40% in aa 1–308 & 61% in aa 410–575 (deletion, codons 309–409)	None
HCD_03930	HEL3854	208	hypothetical, COG0500 SAM-dependent methyltransferase	HCW_00595, 98%	None
HCD_04775	HEL3270	422	NADP-dependent malic enzyme	HCW_01140, 95%	None (3)
HCD_04915	HEL3859	63	anti-codon nuclease masking agent (fragment of >1400 aa protein)	HCW_01595, 68% (match to internal segment)	None
HCD_05580	HEL3864	675	OMP, HomB, pfam01856	HCW_03170, 66%; HCW_01770, 31%; HCW_06190, 32%; HCW_03165, 32%; HCD_01070, 31%; HCD_00840, 32%; HCD_05575	Many strains, ≤32%
HCD_05585	HEL3057	784	OMP, pfam01856	HCW_02225, 50%; HCW_07955, 33%; HCW_08600, 32%; HCD_02935, 55%; HCD_00325, 33%; HCD_08430, 32%; HCD_03000, 32%; HCD_01075, 31%	Many strains, ≤28%
HCD_05840	HEL3880	483	OMP-2, pfam02521	HCW_04635, 80%; HCW_06475, 39%; HCW_04640, 36%; HCW_06805, 39%; HCW_05840, 38%; HCW_05835, 34%; HCW_03775, 32%; HCW_04625, 31%; HCD_05570, 39%; HCD_06420, 39%; HCD_05545, 37%; HCD_05845, 34%; HCD_07420, 32%; HCD_05850, 32%; HCD_08485, 31%; HCD_05830, 30%	Many strains, ≤39%
HCD_06365	HEL3867	73	COG2608, copper (metal) binding, chaperone	HCW_03370, 60%; HCW_03375, 42%; HCD_06360, 43%	Many strains, ≤45%
HCD_06515	HEL2800	473	OMP, HopK, pfam01856	HCW_05300, 52% in aa 151–473; HCD_08540, 45% in aa 47–353; HCD_02735, 58%; HCD_00320, 51%	Many strains, ≤35% in aa 281–473
HCD_06965	HEL2809	757	OMP, HopF, pfam01856	HCD_07970 54% in aa 279–834; HCD_02585, 52% in aa 584–757; HCW_07665, 66%; HCW_04920, 65%; HCW_06795, 65%; HCW_06910, 52%	Many strains, ≤42% in aa 564–757
HCD_07210	HEL3071	207	hypothetical	HCW_03525, 50%; HCW_01920, 40%; HCW_07510, 43%; HCD_00640, 41%	Two strains, 42%; and 45% in aa 88–207
HCD_07600	HEL3062	72	hypothetical	HCW_04310, 88%	None
HCD_07610	HEL3625	434	hypothetical	HCW_04320, 80%, but 603 aa (has internal replacement of 67 by 235 aa)	Many strains, ≤35%, most from aa 36 or 97 to aa 315
HCD_07625	HEL3894	108	hypothetical	HCW_08150, 80%	None
HCD_07645	HEL3875	69	hypothetical, type III restriction	HCW_04375, 94%	Many, ≤43% in aa 19–58
HCD_07680	HEL3876	110	hypothetical, COG0841,cation efflux, TrBC2/VirB2 family	HCW_04410, 80%
HCD_07685	HEL3878	237	hypothetical	HCW_04415, 96%	None
HCD_08025	HEL3884	375	OMP-3	HCW_07065, 61%; HCW_07110, 45% in aa 125–375; HCW_07075, 50% in aa 127–375; HCW_07105, 33%; HCW_07115, 36% in aa 83–328; HCW_04520, 31%; HCD_02500, 41% in aa 127–375;	Many strains, ≤45% in aa 127–375
HCD_08210	HEL3887	133	CRISPR/Cas system protein Cas10	HCW_07130, 46%	None
HCD_08220	HEL3886	195	CRISPR/Cas system RAMP superfamily protein Cas6	HCW_07125, 91%	None (4)
HCD_08225	HEL3885	60	CRISPR/Cas system protein	HCW_07120, 83% (aa 69–128 of 128 aa long protein)	None (5)
HCD_08310	HEL3062	72	hypothetical	HCW_04310, 88%	None
HCD_08345	HEL3061	242	hypothetical	HCD_07575, 100%; HCW_04280, 51% in aa 1–154 (167 aa long protein)	None
HCD_08360	HEL3874	332	integrase	HCW_04255, 92%	Many strains, ≤38%
HCD_08365	HEL3873	113	hypothetical	HCW_04250, 95%	None
HCD_08370	HEL3872	74	hypothetical	HCW_04245, 93%	None
HCD_08385	HEL3871	153	hypothetical	HCW_04220, 74%; HCW_05395, 78% in aa 11–65	Two strains, 80% in aa 5–63
HCD_08390	HEL3872	108	hypothetical	HCW_08195, 83%; HCW_04200, 82%	None
HCD_08395	HEL3870	147	hypothetical	HCW_04215, 97%	None
HCD_08400	HEL3869	340	hypothetical	HCW_04205, 92%; HCW_02210, 75% in aa 182–299 (127 aa protein)	None
HCD_08430	HEL3058	812	OMP, HomB, pfam01856	HCW_08600, 54%; HCW_07955, 39%; HCD_03000, 79%; HCD_00325, 47%; HCD_01285, 31%	Many, ≤33% in aa 216–812
HCD_08520	HEL3891	179	hypothetical	HCW_07625, 67%; HCD_03555, 31% in aa 70–178	None
HCD_08525	HEL3073	58	COG0790 FOG Sel1 repeat c102723	HCW_07635, 74% and HCW_07630, 69% in aa 4–58. Homologs have 17 and 34 aa N-terminal extensions	None
HCD_08540	HEL2800	331	membrane, protein export, secD	No close HCW homolog. HCD_02735, 100% in aa 2–300; HCD_06515, 47% in aa 2–330; HCD_00320, 37% in aa 1–330	None
HCD_08595	HEL3858	291	COG0338 DNA adenine methylase	HCW_01270, 89%;	Several strains with aa identities of 31%–71%

–40% in multiple strains of Actinobacillus, Leptotrichia, Haemophilus, Morganella, Providencia, etc.(1) Homologs of HCD_02790 with aa identities of 35

H. felis, H. bizzozeronii, and H. fennelliae.(2) Distant homologs of HCD_03265 and HCD_03315 in

Campylobacter strains.(3) Homologs of HCD_04775 in many

H. pullorum, H. cinaedi and Campylobacter gracilis.(4) Homologs of HCD_08220 in several species including

Campylobacter and Helicobacter species.(5) Homologs of HCD_08225 in several

Figure 3

Schematic representation of cysteine and methionine metabolism (based on KEGG pathway 00270).

In blue, reactions predicted in H. cetorum but not H. pylori with locus tags of the unique H. cetorum rhodanese-related sulfurtransferase gene indicated. In red, reactions predicted in H. pylori but not H. cetorum. In black, reactions predicted in both H. cetorum and H. pylori. A reaction for which no predicted enzymes were found in Helicobacter genomes is indicated by the dotted line and arrowheads in gray. Of note, DNA sequences matching those of HP1045 (acetyl CoA synthetase) are missing by BLASTN criteria from each H. cetorum strain, and also from 14 of the 48 fully sequenced H. pylori genomes screened. HP1045 was not included in Table 5 because of its absence from a significant minority of H. pylori strains.

Figure 4

Schematic representation of pyruvate metabolism (based on KEGG pathway 00620).

In blue, reactions predicted in H. cetorum but not H. pylori, with locus tags of the unique H. cetorum malate dehydrogenase indicated. In red, reactions predicted in H. pylori but not H. cetorum. In black, reactions predicted in both H. cetorum and H. pylori. In green, a reaction predicted only in the H. cetorum whale strain, not in the dolphin strain, nor in H. pylori.

Table 5

H. pylori strain 26695 proteins(1) belonging to 22 GOs in H. pylori/H. acinonychis clade not in H. cetorum.

H. pylori 26695 Locus_tag(1)	GO	NCBI annotation (H. pylori 26695)
HP0085	HEL2215	Hypothetical protein
HP0092	HEL1980	Type II restriction enzyme M protein (HsdM)
HP0104	HEL2216	2′,3′-cyclic-nucleotide 2′-phosphodiesterase
HP0105	HEL2077	S-ribosylhomocysteinase (LuxS)
HP0106	HEL2078	Cystathionine gamma-synthase/cystathionine beta-lyase (MetB
HP0309	HEL2219	N-carbomoyl-D-amino acid amidohydrolase (2)
HP0311	HEL2220	Hypothetical protein
HP0312	HEL2221	ATP-binding protein
HP0338	HEL2222	Hypothetical protein
HP0614	HEL2224	Hypothetical protein
HP0630	HEL2096	NAD(P)H-quinone reductase (MdaB)
HP0690	HEL2098	Acetyl Co A acetyltransferase
HP0691	HEL2099	Succinyl-CoA-transferase subunit A
HP0692	HEL2191	Succinyl-CoA-transferase subunit B (3)
HP0696	HEL2100	Acetone carboxylase alpha subunit
HP0697	HEL2226	Acetone carboxylase gamma subunit
HP0730	HEL2227	membrane protein (2)
HP0851	HEL2107	Pap2-like membrane protein (2)
HP0871	HEL2229	CDP-diacylglycerol pyrophosphatase
HP0879	HEL2230	Putative nuclease (2)
HP0935	HEL2200	Putative N-acyltransferase (2)
HP1177	HEL1225	Outer membrane protein (HopQ)
HP1185	HEL2045	Sugar efflux transporter

(1) Gene names from original (1997) genome sequence deposition (NC_000915.1). The NCBI database also contains a recent deposition of a separately determined 26695 genome sequence with entirely different gene numbers (CP003904.1).

(2) Designated as hypothetical in original 1997 publication; the function indicated here was suggested by other groups analyzing corresponding sequences in other strains.

H. pylori genomes inspected (Table 1), although its protein product was not identifiied in annotations of Shi417 and XZ274 because of apparent frameshift or nonsense mutations, which we suspect may result from DNA sequencing errors.(3) The HP0692 gene sequence is present in all

Also of note are H. cetorum genes for an integrase, DNA restriction-modification, CRISPR/cas (anti-phage defense) systems, and metal (copper) binding, and numerous outer membrane proteins (OMPs; discussed further below) (Tables 3 and 4). For some of these, no homologs at all are found by BLASTP analyses in current H. pylori sequence databases. Many of the OMPs, however, are mosaic, with some segments well matched to those in H. pylori next to segments that are so divergent that we postulate functional differences, e.g., in their molecular or host cell targets or interaction partners. We suggest that many of the present strain-specific H. cetorum genes or gene fragments had been transferred from unrelated phyla, and that Helicobacter spp. adaptation to particular hosts can involve acquisition or loss of specific metabolic pathways, as was suggested during H. bizzozeronii genome analysis [31].

Genes Likely to be Involved In Bacterial-Host Interaction

Genes implicated in bacterial host interactions and that differ markedly between H. cetorum and H. pylori, that are absent from H. cetorum, or that are present in H. cetorum but not H. pylori merit special attention.

vacA

H. pylori strains encode a potent vacuolating cytotoxin (VacA) that contributes to bacterial fitness and can cause multiple structural and functional changes in host tissues — prominent among them, formation of anion-selective channels and cytoplasmic vacuoles, increased permeability of cell monolayers and mitochondrial membranes, and interference with antigen presentation, inflammatory responses and immune cell activation and proliferation [32]–[35]. To our knowledge, no intact vacA genes have been found in species other than H. pylori. vacA sequences are found in H. acinonychis, but only as fragmented pseudogenes in each of the several strains examined [14], [36]). In contrast, the two H. cetorum strains each contain intact vacA homologs next to cysS, the location also occupied in H. pylori (HCD_01900, 1342 codons, and HCW_04035, 1316 codons, in dolphin and whale strains, respectively). These H. cetorum vacA genes exhibit only 60%–68% protein-level identity to their most closely related H. pylori homologs, and only ∼66% identity to one another (Figure 5).

Figure 5

vacuolating cytotoxin (vacA) genes of H. cetorum.

A, Chromosomal region containing vacA genes from the H. cetorum whale and dolphin strains. Arrows indicate gene orientation. B, Sequence conservation and divergence among vacA genes of H. cetorum. Lighter and darker shades of same color indicate ≥60% identity by BLASTP criteria. Completely different colors (black, green, blue, red) indicate ≤51% identity. To illustrate, amino acids (aa) 130–881 of gene HCD_01900 (vacA at normal location next to cysS) exhibit 40%, 50% and 65% identity to corresponding regions of HCD_01865, HCD_01875 and HCW_05035, respectively, and also 34–46% identity to corresponding regions of HCD_01870 (which itself has an internal divergent duplication with aa 1–694, just 67% identical to aa 734–1428). In contrast, aa 920–1342 of HCD_01900 exhibit 99% identity to corresponding carboxy terminal regions of HCD_01865 and HCD_01875, although only 58% and 69% identity to corresponding regions of HCD_01870 and HCW_04035. Similarly, the amino terminal ∼720 aa of HCD_01865 and HCD_01875 are each ≤50% identical to corresponding regions of other VacA proteins, whether from H. cetorum or H. pylori.

The dolphin strain contains, in addition, an extraordinary extra triplet of contiguous but divergent vacA genes (HCD_01865, HCD_01870, HCD_01875) inserted 6.5 kb from the cysS-linked vacA gene (HCD_01900) between two DNA repair/recombination genes, ruvA and ruvC, which are adjacent to one another in the whale strain (Figure 5A) (and curiously, adjacent or very near to one another in six of 16 genome sequenced H. pylori strains screened, including four strains from Africa). The dolphin strain's four vacA genes exhibit only 40% to 51% protein level identity to one another in the first ∼700–800 codons, a region important for VacA protein's secretion and multiple host cell intoxication functions [32]–[35]. In contrast, the protein from the first and third triplet members and the cysS-linked gene are 99% identical to one another in the last ∼340 amino acids (which determine VacA's autotransporter activity), but these well matched sequences are only 70% identical to the corresponding segment from the second member of the triplet (HCD_01870). The second triplet member's protein also contains an unusual divergent duplication of nearly 700 amino acids whose two components are only 67% identical to one another (Figure 5). The vacA triplet members each seem to lack ≥80 codons corresponding to 5′-ends of typical toxigenic H. pylori homologs (Figure 5) and thus may not be functional. Nevertheless these extra genes may contribute novel sequences and functionalities to other vacA genes by intragenic recombination. Just how these various vacA alleles affect the transport, actions and interactions of their encoded proteins, and bacterial virulence, host range and host responses to infection all merit further study.

H. pylori strains typically contain several genes annotated as toxin-like or vacA-like because the C-terminal autotransporter domains of their encoded proteins exhibit ∼30% identity to that of VacA. The H. cetorum strains also contain several such toxin-like genes, including one with ≥65% protein-level identity to H. pylori imaA (HP0289), found recently to help modulate host inflammatory responses to infection [37].

cag PAI and adjacent HP0159 gene

Each H. cetorum strain lacks a cag pathogenicity island (cag PAI), a ∼30 kb DNA segment present in more than half of H. pylori strains worldwide that is a major contributor to infection-associated inflammation and changes in epithelial structure and development, and that is disease-associated epidemiologically and a contributor to H. pylori fitness and virulence in cell culture and animal infection models [38]–[42]. Also absent is a close homolog of gene HP0519, which is next to one cag PAI end in cag-positive H. pylori, seems to have undergone intense selection for amino acid sequence change in certain populations, and is suspected of helping manage host responses to infection [23], [43]. Homologs of genes that flank the HP0519-cag PAI cluster in H. pylori are next to each other in both H. cetorum strains (e.g., HCD_05445 and HCD_05440; and HCW_05215 and HCW_05220); it is not known whether H. cetorum had never obtained a cag PAI or HP0519, vs. if this DNA segment was lost by deletion.

Extra urease genes

Stomach-colonizing Helicobacter species produce a urease that hydroylzes urea using nickel as a cofactor, and that is essential for gastric infection [44]. Remarkably several species from carnivore hosts each produce an additional urease, cofactored by iron rather than nickel [H. acinonychis (big cats), H. felis (domestic cats and dogs), and H. mustelae (ferrets)] [45], [46]. The two H. cetorum strains also contain genes for both iron- and nickel-cofactored ureases – for example, in the dolphin strain, genes HCD_02705 and HCD_02710, 94% and 97% protein level identity to H. acinonychis ureA2 and ureB2 (iron) and HCD_03580 and HCD_03585, ∼94% and ∼98% identity to H. pylori ureA and ureB (nickel). Equivalent homologs are found in the whale strain. Since nickel is limiting and iron is abundant in meat, an iron-cofactored urease is considered adaptive for carnivore infection [45], [46] (although H. heilmannii sensu stricto and H. bizzozeronii, which infect cats and dogs, respectively, have only a nickel-dependent urease).

Sel1-like repeat (slr) family genes

Seven and nine members of the divergent slr gene family, whose encoded products are secreted, and contain one or more copies of a motif characteristic of Sel1-type eukaryotic regulatory factors, were found in the dolphin and whale strain, respectively. The three best known H. pylori SLR proteins are: HcpA, which may modulate immune responses to infection by stimulating the release of cytokines IFN-γ, TNF-α, IL-6, IL-10 and IL-12, and differentiation of Thp1 monocytes to macrophages [47]; HcpC, which facilitates GroEL chaperone and urease translocation to the bacterial surface, and stimulates H. pylori growth in mammalian cell cultures [48] and also interacts with eukaryotic protein kinase Nek9 (implicated in eukaryotic cell cycle regulation) [49]; and HP0519, which, as noted above, has undergone intense selection for amino acid change in particular human populations [23], [43]. Of these, only genes closely related to hcpC were found in H. cetorum genomes (genes HCD_08435 and HCW_08325; 86% and 79% protein level identity, respectively, to closest H. pylori hcpC homologs), although the C terminal 150 codons of HCD_03275 and HCW_00125 exhibit ∼32% protein level identity to corresponding regions of H. pylori HcpA.

Virulence-associated Leptospira/Bartonella paralog gene family

A remarkable multigene family implicated in pathogenesis in species of Leptospira and Bartonella (PF07598; up to 12 divergent copies in the most virulent strains) [50] is represented by one distant homolog in each H. cetorum strain (HCW_01460 and HCD_04445). No member of this family is found in any of the many dozens of H. pylori strains genome sequenced to date. Just how this gene family can contribute to infection, virulence or other phenotypes that increase fitness is not yet known.

Outer membrane protein (OMP) genes

The H. cetorum strains each contain 78 or more putative OMP genes, whose various functions should include bacterial adherence to host tissues, uptake of ions, solutes and larger molecules; export of effectors and toxic metabolites, antimicrobial resistance, outer membrane assembly, etc. This gene number compares with the approximately 64 OMP genes found in annotations of H. pylori genomes [51, and unpublished]. A first-pass BLASTP comparison indicates that the most closely matched OMP pairs from the two H. cetorum strains tend to be very divergent from one another. For example, the median level of identity of whale strain OMPs to the most closely related dolphin strain homologs is only about 62%, with a range from 0% (no significant homolog) to >86% in the 35 representative proteins screened. This contrasts with the median ∼95% identity (>90% identity of some 84% of individual H. pylori OMPs) between unrelated H. pylori strains such as 26695 and J99 [51]. Superimposed on this diversity, many H. cetorum OMPs are more related to other OMPs in the same strain than to any homolog in the other strain; and many pairs of H. cetorum OMPs, although ≥80% identical in C terminal ∼200 amino acids, exhibit <30% sequence identity in their more central segments, which are likely to mediate interactions with other molecules or cells. In H. pylori such central region protein divergence patterns is typical of OMPs encoded by different genes, not products of strain-specific alleles of the same OMP gene. These divergences suggest OMP gene transfer from other bacterial phyla and/or different selective forces once these genes appeared in H. cetorum lineages, which, in turn, may have led to significantly different spectra of OMP functions in the two strains and affected cell type or host specificity.

Competence Genes

The three separate clusters of genes needed collectively for H. pylori DNA transformation (genes HP0014-HP0018  =  comB1-comB5; HP0036-HP0042  =  comB6-comB10; and dprA and dprB) are present in H. cetorum genomes. The comB-encoded type IV secretion system is used in recipient cells to facilitate DNA transfer by bacterial conjugation [52]. DprA protein binds DNA and can help protect it from restriction and stimulate its methylation [53]. The presence of these genes supports ideas of DNA exchange as a force in H. cetorum evolution.

Transposable Elements

Distributions of bacterial transposable elements reflect patterns of horizontal DNA transfer (genetic exchange) in populations. Three distinct classes are known in Helicobacter: 1) the IS605 family of IS elements, whose five known types are each ∼2 kb long and contain a transposase gene (orfA) and one or two auxiliary genes of unknown function [54]–[57]; 2) the ∼40 kb TnPZ “plasticity zone” transposons, which contain genes implicated epidemiologically in virulence in some human populations [22], and also genes for a type IV secretion system (tfs3) and for a novel putative integrase protein (xerT) [22], [58]; 3) inducible plaque-forming prophages, found in a few East Asian H. pylori strains [59], [60] and remnants of them found in some other strains [14, 61, and present analyses].

The dolphin strain chromosome contains two IS605 family members — one copy of an element closely related to IS605 itself, plus 20 nearly identical copies of an IS606-type element (∼82% DNA identity to H. pylori IS606) [54]. Also present are multiple fragments of a TnPZ element plus more than 20 fragments with significant matches to 1961P-type H. pylori phages [59], [60]. Among these are three near perfect repeats of fragments with lengths of ∼631 bp, 908 bp and 1260 bp in four, two and three locations, respectively, in the dolphin strain chromosome.

The whale strain chromosome, in contrast, lacks IS605-family elements, and contains two apparently complete TnPZ elements, one classified as “type 2” based on gene order and 80–85% DNA identity to H. pylori type 2 TnPZs described in [22], and another that could be considered a type 1/type 2 hybrid or a third TnPZ transposon type [22]. Also present is a 39 kb sequence that contains most genes found in the 1961P phage group (from genes HCW_02700 through HCW_02905). The first 19 kb consists of a relatively uninterrupted set of homologs of phage 1961P genes gp1 to gp18 [59] (HCW_02700 to HCW_02770), whereas the remaining ∼20 kb contain homologs of known phage genes interspersed with other (probably bacterial) genes in an order that is scrambled relative to that in 1961P and related plaque forming phages.

Plasmids

The dolphin and whale H. cetorum strains contain partially related plasmids, 14.1 kb and 12.5 kb in length, respectively. Some 40% of the smaller whale strain plasmid exhibits 71%–92% DNA identity to the larger dolphin strain plasmid and contains genes implicated in plasmid DNA replication; the other 60% of this plasmid is absent by BLASTN criteria from the dolphin strain plasmid. Among features unique to the dolphin strain plasmid are (i) genes provisionally classified as encoding NTPase – DNA partitioning (HCD_08789), DNA nicking (nikB, HCD_08804) and DNA mobilization (mobC, HCD_08799) functions, which suggests that the plasmid might be readily transferred to other bacterial strains; and (ii) a direct non-tandem repeat of IS606 elements that are nearly identical to those in the chromosome.

The fragmentation of prophages in both strains suggests ancient phage infection and lysogenization event(s); in contrast, the number and homogeneity of the dolphin strain's IS606 elements suggests evolutionarily recent introduction and rapid copy number expansion by tranposition.

Discussion

We sequenced the genomes of two strains of H. cetorum, a taxonomic group that infects marine mammals worldwide and that, based on 16 S rDNA sequences, seemed most closely related to the human gastric pathogen H. pylori and its derivative from big cats, H. acinonychis. Our genome sequences and analyses of shared genes confirm this close relationship genome-wide. That said, less than three-fourths of whale and dolphin strain genome sequences are found by BLASTN default criteria in H. pylori genome sequences. In addition, these strains differ remarkably from one another in: (i) sequences of many shared genes, (ii) overall content of strain-specific DNAs, and (iii) chromosomal gene arrangement. These differences are far more pronounced than are seen with strains of H. pylori, which is generally considered one of the most genetically diverse of bacterial species. Further studies, especially using additional H cetorum strains from various hosts and geographic regions are needed to learn if the two strains studied here represent different discrete groups that perhaps should be designated as separate species, vs. simply points on a genetic continuum of one extraordinarily diverse species. In considering this issue, we note that the traditional species concept as developed for higher organisms is poorly suited to bacteria. This is because many bacterial phyla have rich histories of DNA transfer from unrelated groups, superimposed on reproduction by clonal growth without need for gene exchange [62].

Multiple features distinguish the genomes of these H. cetorum strains from those of H. pylori and H. acinonychis, most prominently: (i) their positions in a phylogenetic tree based on sequences of shared core genes (Figure 1); and (ii) the 36% of the whale strain and 26% of the smaller dolphin strain genomes not found in H. pylori genomes by Mega BLASTN criteria. Such features suggest H. cetorum genome evolution driven by horizontal DNA transfer from other phyla, in addition to in situ mutation, selection for adaptive change and genetic drift. Supporting this view are differences in metabolic enzymes illustrated in Figures 3 and 4; OMPs and other proteins likely to participate directly in bacterial host interaction; and contents of mobile DNAs (the IS605-family elements, TnPZ transposons and prophage remnants). We note, in particular the differences in ∼80 putative outer membrane proteins, many of which may participate in adherence and signaling to host tissues, uptake or export of ions and molecules, and membrane synthesis (Tables 3 and 4); and also the remarkably divergent alleles of the vacA (vacuolating cytotoxin) gene in the usual location next to cysS and in the dolphin strain's extra triplet of vacA genes inserted nearby (Figure 5). The most intense divergence among the various H. cetorum VacA proteins is in the first ∼700–800 amino acids, which in well characterized VacA proteins, contains a signal sequence needed for VacA secretion and determinants of the protein's multiple host cell intoxication activities [32]–[35]. Future studies may reveal novel functionalities of these various vacA alleles, how their divergent sequences affect the transport, actions and interactions of their encoded proteins, and the selective forces that drive their evolution.

Metabolic differences also merit particular attention: Prominent among them are H. cetorum's rhodonase sulfurtransferase, which may catalyze synthesis of pyruvate and thiosulfate from 3-mercaptopyruvate (Figure 3; blue arrows). These sulfurtransferases are related to enzymes found in diverse genera including Haemophilius and Actinobacillus, but in few if any other members of the Epsilonproteobacteria. A second example is provided by H. cetorum's distinctive NADP-dependent malic enzyme, which should catalyze production of L-malate from pyruvate (Figure 4, blue arrows), and whose homologs occur in multiple extragastric Helicobacter spp, but not in H. pylori. Also noteworthy are the metabolic enzymes found in H. pylori but not H. cetorum: in particular those for synthesis of L-homocysteine and conversion of L-cysteine to thiocysteine or pyruvate (Figures 3; red arrows); and those for syntheses of acetoacetyl-CoA and acetate from acetyl-CoA, and of acetoacetate from acetoacetyl-CoA (Figure 4; red arrows). Finally we note the phosphoenolpyruvate carboxylase (production of oxaloacetate from phosphoenolpyruvate) in the whale but not the dolphin strain (Figure 4; green arrow). Although direct experimental analyses are needed to fully understand these enzymes and their actions and importance in vivo, our findings fit with a suggestion, made while describing H. bizzozeronii [31], that Helicobacter adaptation to particular hosts could in part involve acquisition or loss of specific metabolic pathways,

Many additional features of interest to particular readers will be found in our two H. cetorum genome sequences, which should also aid further analyses of issues such as: (i) this species' great diversity and how these microbes have adapted for chronic infection of their various marine mammal hosts; (ii) how genetically interconnected or separate H. cetorum populations from different oceans or host species may be; (iii) mechanisms of H. cetorum transmission within and among host species; (iv) host ranges and factors that determine host specificity; (v) the relative importance for H. cetorum strain genetic divergence of mutation and horizontal gene transfer, and of selection for adaptive change and genetic drift (e.g., due to specialization for different host species or the vastness of the world's oceans); and (vi) finally the pathogenic vs. benign or beneficial interactions of H. cetorum strains with their various hosts, an issue of particular interest in today's fragile marine ecosystems.

*Annotation from H. pylori 26695 NCBI BioProject PRJNA178201.

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