has a panmictic population structure due to high genetic diversity promoted by both inter- as well as intra-strain transformation (37
). Intergenomic recombination is subject to strain-specific restriction in H. pylori
). Annotation of the genomes of H. pylori
strains 26695 and J99 shows the presence of nearly two dozen R–M systems out of which 16 were postulated to be Type II for J99 (40–42
). These R–M systems act as a barrier to transformation (43
). On the other hand, restriction barriers do not restrict all transformation, which could be due to some additional regulation of restriction systems. This balance between restriction and transformation in turn regulates the gene flow to equilibrate competition and cooperation between various H. pylori
RecA, DprA and DprB have been shown to be involved in the presynaptic pathway of recombination (44
). Our biochemical characterization of HpDprA revealed ability to bind ssDNA and dsDNA (). Binding of HpDprA to both ssDNA and dsDNA results in large nucleoprotein complexes that do not enter the native PAGE. However, DNA trapped in the wells could be released by the addition of excess of competitor DNA, illustrating that the complexes are reversible and do not represent dead-end reaction products (). A large DNA–protein complex that sits in the well has also been observed with other DNA-binding proteins such as RecA (45
). HpDprA interaction with ssDNA and dsDNA was stable under high salt condtion (200 mM NaCl) indicating that these interactions are specific (Supplementary Figure S2A
). The interaction of HpDprA with dsDNA is biologically important since dsDNA plays an important role in natural transformation of H. pylori
. The pathway of transformation by dsDNA is highly facilitated (nearly 1000-fold) when compared with ssDNA (15
). However, dsDNA is a preferred substrate for REases which are a barrier to horizontal gene transfer. This implies that the decision of ‘restriction’ or ‘facilitation for recombination’ of incoming DNA might be made before the conversion of dsDNA into ssDNA. The incoming DNA has been reported to be in the double-stranded form in periplasm and in single-stranded form in the cytoplasm (10
). Hence, the temporal and spatial events surrounding endonuclease cleavage remain to be understood. These studies suggest a very important role of dsDNA in natural transformation process in H. pylori
. Hence, the binding and protection of dsDNA by HpDprA is possibly of crucial importance in the success of the natural transformation process for this organism.
Since HpDprA binds dsDNA, one would expect that most of the protein will be bound to the chromosomal DNA of H. pylori
. DprA shows polar localization along with CoiA, RecA and SsbB (21
). These four processing proteins show co-localization with ComGA and/or ComFA. Thus, DprA is less abundant in cytoplasm and localized at cell poles thus interacting with incoming foreign DNA. It may be noted that HpDprA has a higher affinity for ssDNA than dsDNA () and therefore, HpDprA will bind preferentially to incoming ssDNA than to chromosomal DNA.
Both R–M systems and DprA have been shown to have a presynaptic role in the natural transformation process (16
). While DprA has a protective role, the R–M systems have an inhibitory role for incoming DNA. This indicates a functional interaction between both of them. Our results suggest that when HpDprA interacts with dsDNA, it prevents Type II restriction enzymes from acting on it and at the same time stimulates the activity of MTases thereby resulting in increased methylation of bound DNA (Figures and ). This observation is of significance as the only way a bacterial cell discriminates between self- and non-self-DNA is through the pattern of methylation. Binding of HpDprA to incoming DNA inhibits access to exonucleases, endonucleases and REases but not to MTases. Moreover, HpDprA may promote the methylation activity of the MTases on incoming dsDNA. As a result, the exogenous DNA will be methylated with the same pattern as that of the host cell and will no longer remain a substrate for restriction enzymes. Thus, HpDprA effectively alleviates the restriction barrier. However, it remains to be understood how DNA in complex with HpDprA, while not accessible to REases or other cellular nucleases, is accessible to a MTase? It has been shown that there is an overlap between DprA dimerization and RecA interaction interfaces and in presence of RecA, DprA–DprA homodimer is replaced with DprA–RecA heterodimer allowing RecA nucleation and polymerization on DNA followed by homology search and synapsis with the chromosome (33
). A similar scenario may be possible for the interaction of HpDprA with the MTase.
R–M systems play an important role in protection of genomic DNA from bacteriophage DNA. Hence, dampening the restriction enzymes activity by HpDprA may not be desirable by the host during entire life cycle. Therefore, the positive regulation of DprA expression by ComK, which happens only when competence is achieved, is noteworthy (21
). In H. pylori
, DNA damage induces a genetic exchange via natural competence (47
). Direct DNA damage leads to a significant increase in intergenomic recombination (48
). Taken together it can be proposed that when genetic competence is induced, R–M systems are down regulated to allow increased genetic exchange and thus, increasing adaptive capacity in a highly selective environment such as that of the gastric mucosa.
On the basis of the results from this investigation, we propose a model for the modulation of restriction enzymes activity by HpDprA. As describes, during inter-strain transformation, the incoming DNA is cleaved by restriction enzymes, due to recognition of a different pattern of methylation other than host DNA. However, in the presence of HpDprA, incoming DNA is coated by DprA and thus made inaccessible to restriction enzymes and other nucleases. Additionally, the MTase activity on DprA-coated DNA is stimulated and thus the incoming DNA is modified with the same pattern of methylation as that of the host DNA, thereby rendering it resistant to restriction activity.
Figure 8. Role of HpDprA in alleviating restriction barrier during natural transformation. Diagrammatic illustration of the proposed DprA involvement in alleviating restriction barrier. Coating of DNA by HpDprA occludes restriction enzymes but HpDprA stimulates (more ...)
There is an evolutionary arms race between bacterial genomes and invading DNA molecules. R–M system and anti-restriction systems have co-evolved to maintain an evolutionary balance between the prey and the predator. For example, phage and plasmid employ anti-restriction strategies to avoid restriction barrier by (a) DNA sequence alteration, (b) transient occlusion of restriction sites and (c) subversion of R–M activities (49
). The observations of MTase stimulation and site occlusion of restriction sites by HpDprA appear to be analogous to such anti-restriction strategies, otherwise employed by bacteriophages. Thus, HpDprA could be a unique bacterial anti-restriction protein used by the organism for downregulating its own R–M systems to maintain the balance between fidelity and diversity.
In conclusion, we have demonstrated a novel role for H. pylori DprA in the modulation of REase and MTase activity during transformation. HpDprA not only protects incoming DNA from REases but also interacts with MTases and promotes methylation of exogenous DNA to allow it to escape host self-/non-self-recognition. Thus, HpDprA alleviates the R–M barrier and promotes natural transformation in competence-induced conditions. It would be interesting to further study the effects of competence and stress-dependent regulation of DprA and R–M systems in vivo, to understand these mechanisms better.