BACKGROUND
h. pylori has accompanied humans throughout evolution  <cit> , and as humans diverged, so did h. pylori.
based on multilocus sequences , h. pylori strains can be divided into populations that are specific for the geographic origin of their human hosts  <cit> .
strains from present-day africans include the most ancestral population hpafrica <dig> from southern africa, hpneafrica from northeastern africa and hpafrica <dig> from western  and southern africa .
h. pylori from europe, the middle east, western asia and india belong to the hpeurope population, and strains from asians include hpasia <dig> and hpeastasia.
the latter is subdivided into hspeasia , hspamerind , and hspmaori .
about 80% of the h. pylori strains isolated from mestizo hosts in latin america were assigned to hpeurope and almost 20% to hspwafrica, but no strains were assigned to hspamerind  <cit> .
conversely, h. pylori strains isolated from latin america amerindian hosts showed multi-locus haplotypes of the hspamerind and hpeurope populations in relatively equal proportions  <cit> .
geographic clustering also has been shown in virulence-associated genes, such as vaca <cit> .
all h. pylori strains recovered to date from mestizo hosts have carried european-types  of vaca, while the ones recovered from amerindian hosts exhibited similar amounts of vaca subtype s1c -clustering with east asia-pacific isolates- and european vaca subtype s1a and s1b <cit> .
we have also shown that the hpeurope strains isolated from mestizos and amerindians in latin america hosts exhibit a mosaic genetic structure; they are of predominantly european ancestry, containing some introgressions from african or asian strains  <cit> .
thus, this mosaicism suggests europeanization of the dna from the original latin america amerindian strains through several events of recombination  <cit> , including transformation  <cit>  or conjugation-like mechanisms  <cit> .
h. pylori population dynamics is known to be shaped by dna transformation and recombination, and the recombination rate in this bacterium is extraordinarily high  <cit> .
since several genetically distinct h. pylori strains can co-colonize a single stomach  <cit>  and since h. pylori are highly competent  <cit> , the net direction of transformation determines which genome would be invaded by foreign dna  <cit> .
instead of replacement of less fit strains, allelic competition via recombination among strains seems to dominate h. pylori evolution  <cit> .
recombination, as evidenced by the mosaic genetic structure of strains recovered from mestizo and european hosts, suggests the co-existence of at least two different haplotype-strains in a single host  <cit>  that allows recombination and provides a mechanism of competition, in this case, allelic competition rather than strain competition.
bacterial restriction-modification systems  confer protection against invasion by foreign dna, for example that from bacteriophages  <cit> , or from other bacteria  <cit> , by cleavage of this foreign dna.
in general, rms consist of a restriction endonuclease  that recognizes and cleaves specific dna sequences , and a counterpart methylase that catalyses the addition of a methyl group to adenine or cytosine residues in the same cognate recognition sites, protecting it from restriction by the cognate enzyme  <cit> .
according to their subunit composition, cofactor requirements, such as atp, adomet, or/and mg+ <dig> and mode of action, rms can be divided into types i, ii, iis, and iii.
type ii rmss are the simplest and most widely distributed among h. pylori strains  <cit> , in which methylases and restriction enzymes act independently.
type ii cognate recognition sites are often palindromic, 4– <dig> nt in length, with continuous  or interrupted  palindromes  <cit> .
similarly, type iis rmss, also found in h. pylori, have independent restriction and methylation enzymes, but the endonucleases act as monomers, restriction sites are uninterrupted , and dna cleavage occurs at specific distances from the recognition sites.
when cognate recognition sites are frequent, genomic or plasmid dna can be extensively cut, impairing recombination  <cit> .
however, cognate recognition sites also play a role in recombination, since they provide the locus for double stranded cuts suitable as substrate for recombination.
therefore, depending on the relative frequency of the cognate recognition sites, dna restriction and methylation systems modulate the capability of dna to recombine.
as such, we hypothesized that the dominance of hpeurope strains in latin america might be due to differences in the cognate restriction sites and active methylases between amerindian and european strains.
to test this hypothesis, we studied the frequencies of cognate recognition sites for  <dig> restriction enzymes in h. pylori strains that were assigned to different populations.
in addition, we estimated the number of active methylases and compared transformation rates in hpeurope and hspamerind h. pylori strains.
thus, we provide evidence of specific recombination events and mechanisms that indicate preferential receptor and donor status, respectively, in amerindian and european strains.
RESULTS
observed and expected number of cognate recognition sites
we examined the published multi-locus sequences  of  <dig> h. pylori strains   <cit> .
the previously assigned mls-based haplotypes were consistent with the geographic origin of their hosts: all of the h. pylori sequences from strains from european hosts were assigned to hpeurope  <cit> ; isolates from amerindians either belonged to hpeurope or hspamerind, and haplotypes from mestizos were mostly hpeurope with a few hpafrica <dig>  we also included  <dig> hpafrica <dig> strains from western africa to reflect the african genetic influx to the americas in colonial times, and  <dig> korean strains  to reflect the east asian origins of amerindians.
in addition, we extracted the mls sequences from  <dig> whole genomes available at the time of the analysis, including  <dig> from european hosts that were hpeurope , one from a north american host that was hpafrica <dig> , and two from south american native hosts that were hspamerind .
*whole genome sequence strain.
we determine the number of cognate recognition sites on the  <dig> mls and  <dig> whole genome sequences  for  <dig> restriction/methylase enzymes previously reported in h. pylori.
the number of cognate recognition sites per kb on the  <dig> mls and the  <dig> were highly consistent and comparable between the two types of sequences.
to further validate that mls are representative of the whole genome sequences, we performed a linear regression analysis.
this analysis indicates a strong correlation between the observed cognate rms sites frequencies in the  <dig> mls and the seven wgs for the  <dig> rms .
thus, mls is representative of the whole genome sequences in terms of cognate rms sites.
of the  <dig> known cognate recognition sequences there were a mean  per kb of  <dig>   in wgs and  <dig>   sites in mls.
in both wgs and mls, the observed cognate recognition site frequencies were highly variable, ranging from  <dig> to  <dig>  sites per kb .
although the distributions were relatively uniform  along the dna, there were several regions that showed coverage of < <dig>  sites per kb.
such sites often corresponded to "genomic islands" with g-c ratios  that deviate from the intrinsic h. pylori ratio of about 39%.
expected recognition sites were calculated performing simulations on model sequences with the same length for the mls and the wgs.
these model sequences were constructed based on the average proportion of nucleotides of the actual sequences analyzed .
to establish the expected frequencies of appearance of a specific recognition site by chance, we randomized the order of the nucleotides in the model sequences and enumerated the occurrence of that specific recognition site .
we estimated a range of  <dig>  to  <dig>  expected cognate recognition sites in both the mls and wgs .
overall, there were no significant differences in the observed or expected number of cognate restriction sites, among the haplotypes .
arestriction endonucleases with palindromic recognition sites are indicated in bold.
code of degenerate nucleotide letters: r = g or a; y = c or t; s = g or c; w = a or t; d = not c ; h = not g ; n = any nucleotide.
bo/e ratio indicates the observed/expected  ratio values.
o/e ratios that are significantly  different from unity are highlighted in bold and bigger font.
cexclusively underrepresented in hpeurope mls.
the observed/expected  ratio indicates deviation from the expectation based on g + c ratio.
o/e ratios were highly similar for the wgs and mls , without any differences by haplotype.
analysis of the hpeurope and hspamerind sequences showed that  <dig> of the  <dig> cognate restriction sites were underrepresented in mls and  <dig> of those sites were also underrepresented in wgs .
one exception, hpy166iii  was exclusively underrepresented in hpeurope mls, but not in the hspamerind nor in wgs.
the underrepresented sites varied in their c + g content from  <dig>  to 75%.
most  of those  <dig> underrepresented sites were palindromic  <cit>  .
conversely, only one cognate recognition site: hpy99iii , was strongly overrepresented  in both hpeurope/hspamerind mls and wgs .
overall, similar results were found when analyzing hspeasia and hspwafrica strains .
in summary, the h. pylori genome has mostly evolved to avoid rms cognate recognition sites.
the total numbers of cognate restriction sites were not different among bacterial populations, based on h. pylori haplotypes.
profiles of cognate rms recognition sites
the rms profiles delineate the specific pattern of enzymatic recognition for each sequence, and offer more detailed information than the analysis of the total number of cognate recognition sites described above.
two strains with the same total number of cognate recognition sites among the combined pool of studied enzymes usually vary in the distribution of the specific cognate recognition sites for individual restriction enzymes within that pool.
we found that the profile of rms recognition sites varied significantly in a population-dependent manner .
four rms sites  showed very strong directionality in the rms strain profile, as shown by principal coordinate analysis  of the  <dig> mls .
another  <dig> cognate recognition sites  also contributed significantly, explaining 47% of the haplotype-strain variation  amongst strains .
the other  <dig> recognition sites cumulatively explain only 9% of the total variation.
non-parametric multidimensional scaling , based on those  <dig> cognate recognition site profiles that explain most of the variation in the pca analyses also separated the h. pylori strains in a population-dependent way .
both for mls and wgs analyses, the amerindian and asian strains exhibit similar profiles, that are distant from european and african strains that cluster apart .
in contrast to the homogeneous african and amerindian strains, the hpeurope strains from mestizo or amerindian hosts showed high heterogeneity in their restriction patterns .
these results provide evidence for a phylogenetic signal in the profile of the frequencies of the cognate recognition sites in h. pylori.
a non-hierarchical analysis of the cognate recognition site profile for the same  <dig> rms, with bidirectional clustering by frequency of the sites and by strain haplotype grouped rms recognition sites , and strains .
"strain cluster a", with hspamerind, hspeasia and some hpeurope strains  has a high frequency of cognate sites for "rms cluster 1".
in contrast, the euro-african "strain cluster c" has a low frequency of cognate sites for rms in cluster  <dig>  but high for "rms cluster 2" .
the cognate sites for rms cluster  <dig> have a significantly lower g + c content compared to the cluster  <dig> cognate sites .
"strain cluster b" includes hspeasia as well as hpeurope and hpafrica <dig> from mestizo and african hosts and shows a mosaic profile of the cognate recognition sites, consistent with the mosaic genetic structure shown in their mls .
strain-specific methylase representation
differences in transformation rates might be due to differences in the frequency of cognate restriction sites, but also to variation in the protection conferred by active methylases belonging to the rms.
we tested the hypothesis that cognate restriction sites are more protected by active methylases in hpeurope than in hspamerind strains.
we selected  <dig> representative h. pylori strains;  <dig> were hpeurope recovered from european , mestizo , and amerindian  hosts, and  <dig> were hspamerind from amerindian hosts .
to determine methylase protection, genomic dna from each strain was subject to digestion by each of  <dig> restriction endonucleases .
susceptibility to digestion indicated lack of an active methylase.
the restriction results showed a range of 5– <dig> active methylases  per h. pylori strain of the  <dig> examined.
there were non-significant differences in the number  or variances  of active methylases between hpeurope and hspamerind strains.
the only exception was the enzyme hpaii, to which dna from the hspamerind strains was significantly more resistant  than dna from the hpeurope strains .
overall, the results confirm that h. pylori strains conserve similar active methylase protection, regardless of their population assignment.
genetic transformation rates
to assess differences in natural competence, five h. pylori hspamerind strains isolated from amerindians and five hpeurope strains recovered from european  or mestizo  hosts each were transformed with two plasmids: i) p801r, a plasmid with an  <dig> bp insertion that introduces a single-base mutation of the gene rpsl, conferring resistance to streptomycin ; or ii) pctb <dig>  a plasmid with a  <dig>  kb insertion with an exogenous apha cassette that produces kanamycin-resistant  strains  <cit> .
hspamerind strains exhibited a significantly higher number of strr transformants than did hpeurope strains .
introduction of pctb <dig> showed much lower rates of transformation: very few kanr colonies  were recovered, which did not allow comparison of the transformation frequency with this plasmid between the different h. pylori populations .
we have hypothesized that the replacement of hspamerind strains by hpeurope strains in latin america was mainly facilitated by the introgression of dna from hpeurope strains into hspamerind strains  <cit> .
to test this hypothesis, we reproduced the encounter of hspamerind and hpeurope h. pylori strains by co-culturing and evaluating the directionality of the dna horizontal transfers among strains in vitro.
we produced double plasmid/resistant hspamerind and hpeurope strains by transforming the single plasmid trains described above with an additional suicide plasmid, pad1-cat that includes an exogenous  <dig>  kb cat cassette that elicits chloramphenicol resistance .
thus, we obtained double resistant strains exhibiting: strr/cmr or kmr/cmr.
to evaluate the direction of the dna transformation, we co-cultured a single plasmid strain  with the double plasmid/resistant strain .
we first assessed the ability of h. pylori hspamerind or hpeurope strains to acquire a plasmid with a single-base mutation  from each other, co-culturing strr strains  and cmr/kmr strains .
transformants acquiring the single-base mutation from strr strains  will exhibit a triple antibiotic resistant phenotype: strr/cmr/kmr.
the frequency of hspamerind strains acquiring this single-base mutation from hpeurope strains was slightly higher  than hpeurope strains acquiring it from hspamerind strains .
to extend our observation, we also co-cultured strr/cmr and kmr strains.
we expected that during co-culturing, transformants acquiring the single-base mutation  from a strr/cmr strain will be strr/kmr but cms, while transformants acquiring the  <dig>  kb apha cassette from a kmr strain will be triple antibiotic-resistant .
we observed that the frequency of transformation  with a single-base mutation , was higher than the frequency of transformants that had acquired the large dna fragment  for both the hspamerind and hpeurope strains.
control  inoculations were included in all the transformation and co-culture experiments  to control for spontaneous mutation events.
the frequency of transformation of hspamerind strains with the single-base mutation  from hpeurope  strains was significantly higher  than that of hpeurope strains from hspamerind strains .
for transformation events in which the  <dig>  kb apha cassette is acquired from a kmr strain , we observed that this cassette is not a suitable genetic marker to evaluate transformation between h. pylori strains because of the low frequency of transformation ; however, the few transform colonies  were predominantly hspamerind strains acquiring the cassette from hpeurope strains.
in total, these observations support that amerindian strains are more receptive to acquiring european dna than vice versa.
discussion
phylogenetic signal of h. pylori rms cognate sites and its correlation with human evolution
our results confirm h. pylori genomic avoidance of many cognate restriction sites  <cit>  in some bacteria, bacteriophages mimic the avoidance pattern of cognate recognition sites of their hosts  <cit>  and exert selective pressure on the pattern of bacterial restriction sites  <cit> .
since bacteriophages do not appear important in h. pylori, presumably most of the pressure came from the rmss themselves .
although we did not find significant haplotype differences in the frequencies of cognate recognition sites, we found population-specific differences in the profiles of the cognate recognition sites.
the relatively more recent asian and amerindian h. pylori strains have lower frequencies of palindromic restriction sites rich in g + c than the african strains and also than the european strains which have been shown to be hybrids between an ancestral h. pylori population  from central and western asia and another ancestral population  from northeast africa  <cit> .
the genetic bottlenecks experienced by humans as they migrated from africa  <cit> , might also have influenced changes in the profile of frequency of restriction words in h. pylori strains.
indeed, the more homogeneous profile of restriction word frequencies in amerindian h. pylori strains in relation to those from african and european strains , is consistent with the lower genetic diversity of both amerindian hosts and their h. pylori strains  <cit> .
what are the implications of this phylogenetic signature for the pattern of restriction site frequency in h. pylori?
that g + c-rich restriction sites were both underrepresented and overrepresented, indicates a lack of selection for total g + c-content.
given that genetic drift is expected to be functionally neutral  <cit> , we cannot discard that differences in the frequency of cognate restriction sites might be functionally relevant in h. pylori.
this is consistent with the idea that rms cognate recognition sites are important for recombination, an important force that drives the evolution of h. pylori.
if modulation of natural competence occurs preferentially in one direction, this leads to genetic subversion of one of the transformed strains in a pair  <cit> .
the results of this work suggest that the specific rms cognate restriction site profile might lead to a recombination dynamic that favors "europeanization" of amerindian strains, explaining at least in part the replacement of amerindian strains by european strains in latin america.
in the context of human evolution, the human divergence within africa and the worldwide divergence after the out-of-africa migrations, were followed by genetic convergence by mixing in modern times.
h. pylori strains differing in the use of cognate recognition words might have optimized fitness in the specific environment in which they evolved, but not in new host environments with different competitors.
there may have been an ancestral h. pylori rms pool, before out-of-africa  followed by apparent differential selection for and avoidance of particular rms, as h. pylori evolved with different isolated human groups.
selection against certain cognate recognition sites, particularly palindromes  <cit> , has been shown in several bacteria and bacteriophages  <cit> , which we again observe in h. pylori.
the avoidance of specific palindromes may reflect selection pressure exerted by restriction enzymes with incomplete methylation  <cit> , and their effects on genetic regulatory control  <cit> .
when methylation protection fails, strains that avoid specific cognate restriction sites have a fitness advantage over those with more frequent cognate sites  <cit> .
consistent with this hypothesis is that life forms lacking rms, such as some dna viruses, mitochondria, and chloroplasts, do not show palindrome avoidance  <cit> .
differences in rms profiles in the isolated sub-populations of h. pylori that derived from the worldwide spread of humans could reflect rms competition, founder effects, and locale-specific selection.
the biological significance of overrepresentation of palindromic sites is harder to explain in the light of the defensive role of rms.
however, the frequent occurrence of small dna fragments might increase recombination frequency, which may improve fitness  <cit> .
similarly, methylation of dna promoters and origins of replication might provide benefits for the regulation of gene expression  <cit>  and replication  <cit> .
this study confirms prior observations that the mean numbers of active methylases are conserved in h. pylori strains recovered from hosts of different geographical origins  <cit> , suggesting selection for an optimal rms number across the universe of h. pylori cells  <cit> .
such selection might be achieved by horizontal gene transfer of rms genes among h. pylori strains, with a consequent equilibrium in the number of active methylases.
rmss have been postulated to behave as "selfish" mobile genetic elements  <cit> .
selection favors the maintenance of the system of restriction endonuclease and methylase, because loss of methylase function is lethal.
however, intact methylase genes with apparently truncated restriction genes have been observed in completed h. pylori genomes, suggesting that active methylases are involved in the regulation of essential physiological processes that are independent of rms  <cit> .
however, the process of restriction and methylation might be a dynamic mechanism that can vary in vivo.
for example, hpyi methylase  expression varied dramatically within h. pylori cells colonizing the gastric tissue  <cit> .
dominance of european over amerindian strains
despite a similar number of active methylases, hspamerind strains exhibited higher rates of transformation than hpeurope strains.
dna incorporation into the chromosome during transformation can be divided into three general steps: i) dna uptake or binding to the cell; ii) degradation of one strand of the invading dna, and iii) recombination of the remnant dna fragments into the genome  <cit> .
for the first step, extensive evidence supports the fact that h. pylori is highly competent in uptake of "non-self" dna.
h. pylori is genetically diverse within a single stomach niche and is subject to a very high rate of intraspecific recombination  <cit> .
proteins such as comb <dig>  comb7–comb <dig> of the type iv secretion system encoded by the comb genes,  <cit>  are homologs to virb proteins  of a. tumefaciens and resemble their conjugation-like function in h. pylori dna transformation  <cit> .
mutations of comb in h. pylori strains abrogate transformation  <cit> .
whether haplotype differences in the proteins involved in dna uptake and access to foreign dna can affect the efficiency of dna uptake and incorporation, remains to be tested.
step  involves the degradation of one dna strand and processing of the foreign dna.
although h. pylori isolates from different bacterial populations exhibit a similar number of methylases, the differences in the cognate recognition sites can explain differences in the "dna availability" as a substrate for recombination.
for example, four-base cognate recognition sites are  more frequent that six-base recognition sites.
step , homologous recombination, requires at least a single stranded break; dna differences in the location of the homologous sites may favor higher transformation in amerindian strains.
when two h. pylori strains meet in a host’s stomach, they can recombine in an asymmetric fashion, leading to subversion of one strain by the other.
an additional explanation of european dominance might rely on host selection that seems to favor european strains, for example, host mixing with europeans.
host selection is evidenced by the h. pylori adhesin phenotypes in relation to human blood groups.
up to 95% of "generalist" european h. pylori strains can bind a, b or o antigens whereas 60% of amerindian strains bind only o antigens  <cit> .
this binding-specialization of h. pylori strains coincides with the unique predominance of blood group o antigens in amerindian hosts.
our results provide evidence that asymmetric recombination rates lead to dominance of one strain over another by means of genetic subversion.
if amerindian strains recombine at higher rates, they are more likely to become mosaic strains integrating european loci and gradually become "europeanized".
CONCLUSIONS
in conclusion, geographical variations in the pattern of cognate recognition sites provide evidence for ancestral differences in rms representation and possibly also in function.
the higher transformation rates in amerindian strains support the hypothesis of europeanization of latin american strains via recombination.
a potential scenario, supported by our results is that during colonial times when spanish conquers, african slaves, and native amerindians mix also did their h. pylori haplotypes, thus a new generation of h. pylori strains arise, exhibiting mosaic genetic structure result of several events of recombination among strains with different rms profile.
in this mixing, hpeurope alleles succeed dominating their incorporation into dna from amerindian strains .
future studies are needed to evaluate differences by haplotype in competence-related function driven by comb, dpra and comh genes  <cit> .

 h. pylori has accompanied humans throughout evolution, and as humans diverged, so did h. pylori. based on multilocus sequences, h. pylori strains can be divided into populations that are specific for the geographic origin of their human hosts. strains from present-day africans include the most ancestral population hpafrica from southern africa, hpneafrica from northeastern africa and hpafrica from western and southern africa. h. pylori from europe, the middle east, western asia and india belong to the hpeurope population, and strains from asians include hpasia and hpeastasia. the latter is subdivided into hspeasia, hspamerind, and hspmaori. about 80% of the h. pylori strains isolated from mestizo hosts in latin america were assigned to hpeurope and almost 20% to hspwafrica, but no strains were assigned to hspamerind. conversely, h. pylori strains isolated from latin america amerindian hosts showed multi-locus haplotypes of the hspamerind and hpeurope populations in relatively equal proportions. geographic clustering also has been shown in virulence-associated genes, such as vaca. all h. pylori strains recovered to date from mestizo hosts have carried european-types of vaca, while the ones recovered from amerindian hosts exhibited similar amounts of vaca subtype s1c -clustering with east asia-pacific isolates- and european vaca subtype s1a and s1b. we have also shown that the hpeurope strains isolated from mestizos and amerindians in latin america hosts exhibit a mosaic genetic structure; they are of predominantly european ancestry, containing some introgressions from african or asian strains. thus, this mosaicism suggests europeanization of the dna from the original latin america amerindian strains through several events of recombination, including transformation or conjugation-like mechanisms. h. pylori population dynamics is known to be shaped by dna transformation and recombination, and the recombination rate in this bacterium is extraordinarily high. since several genetically distinct h. pylori strains can co-colonize a single stomach and since h. pylori are highly competent, the net direction of transformation determines which genome would be invaded by foreign dna. instead of replacement of less fit strains, allelic competition via recombination among strains seems to dominate h. pylori evolution. recombination, as evidenced by the mosaic genetic structure of strains recovered from mestizo and european hosts, suggests the co-existence of at least two different haplotype-strains in a single host that allows recombination and provides a mechanism of competition, in this case, allelic competition rather than strain competition. bacterial restriction-modification systems confer protection against invasion by foreign dna, for example that from bacteriophages, or from other bacteria, by cleavage of this foreign dna. in general, rms consist of a restriction endonuclease that recognizes and cleaves specific dna sequences, and a counterpart methylase that catalyses the addition of a methyl group to adenine or cytosine residues in the same cognate recognition sites, protecting it from restriction by the cognate enzyme. according to their subunit composition, cofactor requirements, such as atp, adomet, or/and mg+ and mode of action, rms can be divided into types i, ii, iis, and iii. type ii rmss are the simplest and most widely distributed among h. pylori strains, in which methylases and restriction enzymes act independently. type ii cognate recognition sites are often palindromic, 4– nt in length, with continuous or interrupted palindromes. similarly, type iis rmss, also found in h. pylori, have independent restriction and methylation enzymes, but the endonucleases act as monomers, restriction sites are uninterrupted, and dna cleavage occurs at specific distances from the recognition sites. when cognate recognition sites are frequent, genomic or plasmid dna can be extensively cut, impairing recombination. however, cognate recognition sites also play a role in recombination, since they provide the locus for double stranded cuts suitable as substrate for recombination. therefore, depending on the relative frequency of the cognate recognition sites, dna restriction and methylation systems modulate the capability of dna to recombine. as such, we hypothesized that the dominance of hpeurope strains in latin america might be due to differences in the cognate restriction sites and active methylases between amerindian and european strains. to test this hypothesis, we studied the frequencies of cognate recognition sites for restriction enzymes in h. pylori strains that were assigned to different populations. in addition, we estimated the number of active methylases and compared transformation rates in hpeurope and hspamerind h. pylori strains. thus, we provide evidence of specific recombination events and mechanisms that indicate preferential receptor and donor status, respectively, in amerindian and european strains.  observed and expected number of cognate recognition sites we examined the published multi-locus sequences of h. pylori strains. the previously assigned mls-based haplotypes were consistent with the geographic origin of their hosts: all of the h. pylori sequences from strains from european hosts were assigned to hpeurope; isolates from amerindians either belonged to hpeurope or hspamerind, and haplotypes from mestizos were mostly hpeurope with a few hpafrica we also included hpafrica strains from western africa to reflect the african genetic influx to the americas in colonial times, and korean strains to reflect the east asian origins of amerindians. in addition, we extracted the mls sequences from whole genomes available at the time of the analysis, including from european hosts that were hpeurope, one from a north american host that was hpafrica, and two from south american native hosts that were hspamerind. *whole genome sequence strain. we determine the number of cognate recognition sites on the mls and whole genome sequences for restriction/methylase enzymes previously reported in h. pylori. the number of cognate recognition sites per kb on the mls and the were highly consistent and comparable between the two types of sequences. to further validate that mls are representative of the whole genome sequences, we performed a linear regression analysis. this analysis indicates a strong correlation between the observed cognate rms sites frequencies in the mls and the seven wgs for the rms. thus, mls is representative of the whole genome sequences in terms of cognate rms sites. of the known cognate recognition sequences there were a mean per kb of in wgs and   sites in mls. in both wgs and mls, the observed cognate recognition site frequencies were highly variable, ranging from to sites per kb. although the distributions were relatively uniform along the dna, there were several regions that showed coverage of < sites per kb. such sites often corresponded to "genomic islands" with g-c ratios that deviate from the intrinsic h. pylori ratio of about 39%. expected recognition sites were calculated performing simulations on model sequences with the same length for the mls and the wgs. these model sequences were constructed based on the average proportion of nucleotides of the actual sequences analyzed. to establish the expected frequencies of appearance of a specific recognition site by chance, we randomized the order of the nucleotides in the model sequences and enumerated the occurrence of that specific recognition site. we estimated a range of to expected cognate recognition sites in both the mls and wgs. overall, there were no significant differences in the observed or expected number of cognate restriction sites, among the haplotypes. arestriction endonucleases with palindromic recognition sites are indicated in bold. code of degenerate nucleotide letters: r = g or a; y = c or t; s = g or c; w = a or t; d = not c; h = not g; n = any nucleotide. bo/e ratio indicates the observed/expected ratio values. o/e ratios that are significantly different from unity are highlighted in bold and bigger font. cexclusively underrepresented in hpeurope mls. the observed/expected ratio indicates deviation from the expectation based on g + c ratio. o/e ratios were highly similar for the wgs and mls, without any differences by haplotype. analysis of the hpeurope and hspamerind sequences showed that of the cognate restriction sites were underrepresented in mls and of those sites were also underrepresented in wgs. one exception, hpy166iii was exclusively underrepresented in hpeurope mls, but not in the hspamerind nor in wgs. the underrepresented sites varied in their c + g content from to 75%. most of those underrepresented sites were palindromic. conversely, only one cognate recognition site: hpy99iii, was strongly overrepresented in both hpeurope/hspamerind mls and wgs. overall, similar results were found when analyzing hspeasia and hspwafrica strains. in summary, the h. pylori genome has mostly evolved to avoid rms cognate recognition sites. the total numbers of cognate restriction sites were not different among bacterial populations, based on h. pylori haplotypes. profiles of cognate rms recognition sites the rms profiles delineate the specific pattern of enzymatic recognition for each sequence, and offer more detailed information than the analysis of the total number of cognate recognition sites described above. two strains with the same total number of cognate recognition sites among the combined pool of studied enzymes usually vary in the distribution of the specific cognate recognition sites for individual restriction enzymes within that pool. we found that the profile of rms recognition sites varied significantly in a population-dependent manner. four rms sites showed very strong directionality in the rms strain profile, as shown by principal coordinate analysis of the mls. another cognate recognition sites also contributed significantly, explaining 47% of the haplotype-strain variation amongst strains. the other recognition sites cumulatively explain only 9% of the total variation. non-parametric multidimensional scaling, based on those cognate recognition site profiles that explain most of the variation in the pca analyses also separated the h. pylori strains in a population-dependent way. both for mls and wgs analyses, the amerindian and asian strains exhibit similar profiles, that are distant from european and african strains that cluster apart. in contrast to the homogeneous african and amerindian strains, the hpeurope strains from mestizo or amerindian hosts showed high heterogeneity in their restriction patterns. these results provide evidence for a phylogenetic signal in the profile of the frequencies of the cognate recognition sites in h. pylori. a non-hierarchical analysis of the cognate recognition site profile for the same rms, with bidirectional clustering by frequency of the sites and by strain haplotype grouped rms recognition sites, and strains. "strain cluster a", with hspamerind, hspeasia and some hpeurope strains has a high frequency of cognate sites for "rms cluster 1". in contrast, the euro-african "strain cluster c" has a low frequency of cognate sites for rms in cluster but high for "rms cluster 2". the cognate sites for rms cluster have a significantly lower g + c content compared to the cluster cognate sites. "strain cluster b" includes hspeasia as well as hpeurope and hpafrica from mestizo and african hosts and shows a mosaic profile of the cognate recognition sites, consistent with the mosaic genetic structure shown in their mls. strain-specific methylase representation differences in transformation rates might be due to differences in the frequency of cognate restriction sites, but also to variation in the protection conferred by active methylases belonging to the rms. we tested the hypothesis that cognate restriction sites are more protected by active methylases in hpeurope than in hspamerind strains. we selected representative h. pylori strains; were hpeurope recovered from european, mestizo, and amerindian hosts, and were hspamerind from amerindian hosts. to determine methylase protection, genomic dna from each strain was subject to digestion by each of restriction endonucleases. susceptibility to digestion indicated lack of an active methylase. the restriction results showed a range of 5– active methylases per h. pylori strain of the examined. there were non-significant differences in the number or variances of active methylases between hpeurope and hspamerind strains. the only exception was the enzyme hpaii, to which dna from the hspamerind strains was significantly more resistant than dna from the hpeurope strains. overall, the results confirm that h. pylori strains conserve similar active methylase protection, regardless of their population assignment. genetic transformation rates to assess differences in natural competence, five h. pylori hspamerind strains isolated from amerindians and five hpeurope strains recovered from european or mestizo hosts each were transformed with two plasmids: i) p801r, a plasmid with an bp insertion that introduces a single-base mutation of the gene rpsl, conferring resistance to streptomycin; or ii) pctb a plasmid with a kb insertion with an exogenous apha cassette that produces kanamycin-resistant strains. hspamerind strains exhibited a significantly higher number of strr transformants than did hpeurope strains. introduction of pctb showed much lower rates of transformation: very few kanr colonies were recovered, which did not allow comparison of the transformation frequency with this plasmid between the different h. pylori populations. we have hypothesized that the replacement of hspamerind strains by hpeurope strains in latin america was mainly facilitated by the introgression of dna from hpeurope strains into hspamerind strains. to test this hypothesis, we reproduced the encounter of hspamerind and hpeurope h. pylori strains by co-culturing and evaluating the directionality of the dna horizontal transfers among strains in vitro. we produced double plasmid/resistant hspamerind and hpeurope strains by transforming the single plasmid trains described above with an additional suicide plasmid, pad1-cat that includes an exogenous kb cat cassette that elicits chloramphenicol resistance. thus, we obtained double resistant strains exhibiting: strr/cmr or kmr/cmr. to evaluate the direction of the dna transformation, we co-cultured a single plasmid strain with the double plasmid/resistant strain. we first assessed the ability of h. pylori hspamerind or hpeurope strains to acquire a plasmid with a single-base mutation from each other, co-culturing strr strains and cmr/kmr strains. transformants acquiring the single-base mutation from strr strains will exhibit a triple antibiotic resistant phenotype: strr/cmr/kmr. the frequency of hspamerind strains acquiring this single-base mutation from hpeurope strains was slightly higher than hpeurope strains acquiring it from hspamerind strains. to extend our observation, we also co-cultured strr/cmr and kmr strains. we expected that during co-culturing, transformants acquiring the single-base mutation from a strr/cmr strain will be strr/kmr but cms, while transformants acquiring the kb apha cassette from a kmr strain will be triple antibiotic-resistant. we observed that the frequency of transformation with a single-base mutation, was higher than the frequency of transformants that had acquired the large dna fragment for both the hspamerind and hpeurope strains. control inoculations were included in all the transformation and co-culture experiments to control for spontaneous mutation events. the frequency of transformation of hspamerind strains with the single-base mutation from hpeurope strains was significantly higher than that of hpeurope strains from hspamerind strains. for transformation events in which the kb apha cassette is acquired from a kmr strain, we observed that this cassette is not a suitable genetic marker to evaluate transformation between h. pylori strains because of the low frequency of transformation; however, the few transform colonies were predominantly hspamerind strains acquiring the cassette from hpeurope strains. in total, these observations support that amerindian strains are more receptive to acquiring european dna than vice versa. discussion phylogenetic signal of h. pylori rms cognate sites and its correlation with human evolution our results confirm h. pylori genomic avoidance of many cognate restriction sites in some bacteria, bacteriophages mimic the avoidance pattern of cognate recognition sites of their hosts and exert selective pressure on the pattern of bacterial restriction sites. since bacteriophages do not appear important in h. pylori, presumably most of the pressure came from the rmss themselves. although we did not find significant haplotype differences in the frequencies of cognate recognition sites, we found population-specific differences in the profiles of the cognate recognition sites. the relatively more recent asian and amerindian h. pylori strains have lower frequencies of palindromic restriction sites rich in g + c than the african strains and also than the european strains which have been shown to be hybrids between an ancestral h. pylori population from central and western asia and another ancestral population from northeast africa. the genetic bottlenecks experienced by humans as they migrated from africa, might also have influenced changes in the profile of frequency of restriction words in h. pylori strains. indeed, the more homogeneous profile of restriction word frequencies in amerindian h. pylori strains in relation to those from african and european strains, is consistent with the lower genetic diversity of both amerindian hosts and their h. pylori strains. what are the implications of this phylogenetic signature for the pattern of restriction site frequency in h. pylori? that g + c-rich restriction sites were both underrepresented and overrepresented, indicates a lack of selection for total g + c-content. given that genetic drift is expected to be functionally neutral, we cannot discard that differences in the frequency of cognate restriction sites might be functionally relevant in h. pylori. this is consistent with the idea that rms cognate recognition sites are important for recombination, an important force that drives the evolution of h. pylori. if modulation of natural competence occurs preferentially in one direction, this leads to genetic subversion of one of the transformed strains in a pair. the results of this work suggest that the specific rms cognate restriction site profile might lead to a recombination dynamic that favors "europeanization" of amerindian strains, explaining at least in part the replacement of amerindian strains by european strains in latin america. in the context of human evolution, the human divergence within africa and the worldwide divergence after the out-of-africa migrations, were followed by genetic convergence by mixing in modern times. h. pylori strains differing in the use of cognate recognition words might have optimized fitness in the specific environment in which they evolved, but not in new host environments with different competitors. there may have been an ancestral h. pylori rms pool, before out-of-africa followed by apparent differential selection for and avoidance of particular rms, as h. pylori evolved with different isolated human groups. selection against certain cognate recognition sites, particularly palindromes, has been shown in several bacteria and bacteriophages, which we again observe in h. pylori. the avoidance of specific palindromes may reflect selection pressure exerted by restriction enzymes with incomplete methylation, and their effects on genetic regulatory control. when methylation protection fails, strains that avoid specific cognate restriction sites have a fitness advantage over those with more frequent cognate sites. consistent with this hypothesis is that life forms lacking rms, such as some dna viruses, mitochondria, and chloroplasts, do not show palindrome avoidance. differences in rms profiles in the isolated sub-populations of h. pylori that derived from the worldwide spread of humans could reflect rms competition, founder effects, and locale-specific selection. the biological significance of overrepresentation of palindromic sites is harder to explain in the light of the defensive role of rms. however, the frequent occurrence of small dna fragments might increase recombination frequency, which may improve fitness. similarly, methylation of dna promoters and origins of replication might provide benefits for the regulation of gene expression and replication. this study confirms prior observations that the mean numbers of active methylases are conserved in h. pylori strains recovered from hosts of different geographical origins, suggesting selection for an optimal rms number across the universe of h. pylori cells. such selection might be achieved by horizontal gene transfer of rms genes among h. pylori strains, with a consequent equilibrium in the number of active methylases. rmss have been postulated to behave as "selfish" mobile genetic elements. selection favors the maintenance of the system of restriction endonuclease and methylase, because loss of methylase function is lethal. however, intact methylase genes with apparently truncated restriction genes have been observed in completed h. pylori genomes, suggesting that active methylases are involved in the regulation of essential physiological processes that are independent of rms. however, the process of restriction and methylation might be a dynamic mechanism that can vary in vivo. for example, hpyi methylase expression varied dramatically within h. pylori cells colonizing the gastric tissue. dominance of european over amerindian strains despite a similar number of active methylases, hspamerind strains exhibited higher rates of transformation than hpeurope strains. dna incorporation into the chromosome during transformation can be divided into three general steps: i) dna uptake or binding to the cell; ii) degradation of one strand of the invading dna, and iii) recombination of the remnant dna fragments into the genome. for the first step, extensive evidence supports the fact that h. pylori is highly competent in uptake of "non-self" dna. h. pylori is genetically diverse within a single stomach niche and is subject to a very high rate of intraspecific recombination. proteins such as comb comb7–comb of the type iv secretion system encoded by the comb genes, are homologs to virb proteins of a. tumefaciens and resemble their conjugation-like function in h. pylori dna transformation. mutations of comb in h. pylori strains abrogate transformation. whether haplotype differences in the proteins involved in dna uptake and access to foreign dna can affect the efficiency of dna uptake and incorporation, remains to be tested. step involves the degradation of one dna strand and processing of the foreign dna. although h. pylori isolates from different bacterial populations exhibit a similar number of methylases, the differences in the cognate recognition sites can explain differences in the "dna availability" as a substrate for recombination. for example, four-base cognate recognition sites are more frequent that six-base recognition sites. step, homologous recombination, requires at least a single stranded break; dna differences in the location of the homologous sites may favor higher transformation in amerindian strains. when two h. pylori strains meet in a host’s stomach, they can recombine in an asymmetric fashion, leading to subversion of one strain by the other. an additional explanation of european dominance might rely on host selection that seems to favor european strains, for example, host mixing with europeans. host selection is evidenced by the h. pylori adhesin phenotypes in relation to human blood groups. up to 95% of "generalist" european h. pylori strains can bind a, b or o antigens whereas 60% of amerindian strains bind only o antigens. this binding-specialization of h. pylori strains coincides with the unique predominance of blood group o antigens in amerindian hosts. our results provide evidence that asymmetric recombination rates lead to dominance of one strain over another by means of genetic subversion. if amerindian strains recombine at higher rates, they are more likely to become mosaic strains integrating european loci and gradually become "europeanized".  in conclusion, geographical variations in the pattern of cognate recognition sites provide evidence for ancestral differences in rms representation and possibly also in function. the higher transformation rates in amerindian strains support the hypothesis of europeanization of latin american strains via recombination. a potential scenario, supported by our results is that during colonial times when spanish conquers, african slaves, and native amerindians mix also did their h. pylori haplotypes, thus a new generation of h. pylori strains arise, exhibiting mosaic genetic structure result of several events of recombination among strains with different rms profile. in this mixing, hpeurope alleles succeed dominating their incorporation into dna from amerindian strains. future studies are needed to evaluate differences by haplotype in competence-related function driven by comb, dpra and comh genes. 
this phenomenon might occur via dna recombination, modulated by restriction-modification systems, in which differences in cognate recognition sites and in active methylases will determine direction and frequency of gene flow. amerindians strains also exhibited higher transformation rates than european strains, and were more susceptible to be subverted by larger dna hpeurope-fragments than vice versa. we hypothesized that genomes from hspamerind strains that evolved from a small founder population have lost crs for rms and active methylases, promoting hpeurope’s dna invasion. we also measured the number of active methylases by resistance to in vitro digestion by restriction enzymes of genomic dna from hpeurope and hspamerind strains, and determined the direction of dna uptake in co-culture experiments of hspamerind and hpeurope strains. although neither the frequency of crs nor the number of active methylases differ among the bacterial populations, hspamerind strains had a restriction profile distinct from that in hpeurope strains, with recognition sites accounting for the differences. we determined the observed and expected frequencies of crs for rms in dna from h. pylori whole genomes and multilocus sequences. most of the crs were underrepresented with consistency between whole genomes and multilocus sequences. the geographical variation in the pattern of crs provides evidence for ancestral differences in rms representation and function, and the transformation findings support the hypothesis of europeanization of the amerindian strains in latin america via dna recombination. recent evidence suggests that in the current human mixing in latin america, european h. pylori are increasingly dominant at the expense of amerindian haplotypes.
 helicobacter pylori has diverged in parallel to its human host, leading to distinct phylogeographic populations. recent evidence suggests that in the current human mixing in latin america, european h. pylori are increasingly dominant at the expense of amerindian haplotypes. this phenomenon might occur via dna recombination, modulated by restriction-modification systems, in which differences in cognate recognition sites and in active methylases will determine direction and frequency of gene flow. we hypothesized that genomes from hspamerind strains that evolved from a small founder population have lost crs for rms and active methylases, promoting hpeurope’s dna invasion. we determined the observed and expected frequencies of crs for rms in dna from h. pylori whole genomes and multilocus sequences. we also measured the number of active methylases by resistance to in vitro digestion by restriction enzymes of genomic dna from hpeurope and hspamerind strains, and determined the direction of dna uptake in co-culture experiments of hspamerind and hpeurope strains.  most of the crs were underrepresented with consistency between whole genomes and multilocus sequences. although neither the frequency of crs nor the number of active methylases differ among the bacterial populations, hspamerind strains had a restriction profile distinct from that in hpeurope strains, with recognition sites accounting for the differences. amerindians strains also exhibited higher transformation rates than european strains, and were more susceptible to be subverted by larger dna hpeurope-fragments than vice versa.  the geographical variation in the pattern of crs provides evidence for ancestral differences in rms representation and function, and the transformation findings support the hypothesis of europeanization of the amerindian strains in latin america via dna recombination. h. pylorihaplotypesrestriction-modification systemrecombination 