Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains

Aras, Rahul; Small, Aaron J.; Ando, Takafumi; Blaser, Martin J.

doi:10.1093/nar/gkf686

Cited by 60 publications

(58 citation statements)

References 32 publications

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“…Transformation studies in other bacteria have clearly shown that endogenously modified plasmid DNA can more efficiently transform strains possessing the same R-M system (2,3,15,37,39). The data provided by this study, as well as previous work by other investigators, indicate that a primary limitation to transformation of B. burgdorferi with shuttle vector DNA from E. coli is the barrier presented by the endogenous plasmid-borne R-M systems of B. burgdorferi.…”

Section: Discussionsupporting

confidence: 63%

Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

2011

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The restriction-modification (R-M) systems of many bacteria present a barrier to the stable introduction of foreign DNA. The Lyme disease spirochete Borrelia burgdorferi has two plasmid-borne putative R-M genes, bbe02 and bbq67, whose presence limits transformation by shuttle vector DNA from Escherichia coli. We show that both the bbe02 and bbq67 loci in recipient B. burgdorferi limit transformation with shuttle vector DNA from E. coli, irrespective of its dam, dcm, or hsd methylation status. However, plasmid DNA purified from B. burgdorferi transformed naïve B. burgdorferi much more efficiently than plasmid DNA from E. coli, particularly when the bbe02 and bbq67 genotypes of the B. burgdorferi DNA source matched those of the recipient. We detected adenine methylation of plasmid DNA prepared from B. burgdorferi that carried bbe02 and bbq67. These results indicate that the bbe02 and bbq67 loci of B. burgdorferi encode distinct R-M enzymes that methylate endogenous DNA and cleave foreign DNA lacking the same sequence-specific modification. Our findings have basic implications for horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. Further characterization and identification of the nucleotide sequences recognized by BBE02 and BBQ67 will facilitate efficient genetic manipulation of this pathogenic spirochete.Borrelia burgdorferi sensu lato is a zoonotic pathogen whose natural infectious cycle alternates between a tick vector and rodent or bird reservoir hosts (1,7,8,14,32,33,36). Transmission of B. burgdorferi to humans occurs through the bite of an infected tick and can lead to Lyme disease, which is a major public health concern in areas of North America and Europe where B. burgdorferi is endemic (8, 53).The genomic structure of the spirochete B. burgdorferi is unique, consisting of a linear chromosome of approximately 900 kb and more than 20 linear (lp) and circular (cp) plasmids, ranging in size from ϳ5 kb to 56 kb, in the type strain B31 (9,10,11,19,42). The plasmids of B. burgdorferi are present at unit copy number relative to the chromosome (22), and some are relatively unstable during in vitro propagation (52, 57). The loss of linear plasmids lp25, lp28-1, and lp36 by strain B31 was found to correlate with the loss of infectivity in mice (20,31,45,56), leading to the identification of genes carried on these plasmids that are dispensable in vitro but required in vivo during an experimental infectious cycle (21,26,35,44,47). The loss of two linear plasmids, lp25 and lp56, was shown to correlate with enhanced shuttle vector transformation, suggesting that specific lp25 and lp56 gene products present a barrier to stable introduction of foreign DNA (34). Further studies linked the transformation phenotype of B. burgdorferi strain B31 with the bbe02 and bbq67 genes on lp25 and lp56, respectively, and the putative restriction-modification (R-M) enzymes that they encode (11,27,29,34). The recent demonstration by Chen and colleagues of enhanced transformation of B. burgdorferi follow...

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Section: Discussionsupporting

confidence: 63%

Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

2011

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“…3B). These results indicated that REs of the recipient strains inhibit transformation by this exogenous 1127-bp cat cassette (synthesized in E. coli), which is consistent with functional restriction barriers in the wild-type strain (4,48).…”

Section: Figuresupporting

confidence: 66%

Natural Transformation of an Engineered Helicobacter pylori Strain Deficient in Type II Restriction Endonucleases

Zhang

2012

J Bacteriol

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Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counterselection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log 10 for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.

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“…Helicobacter pylori) can prevent genome rearrangements by chromosomal DNA from competing strains which are non-isogenic (Aras et al, 2002). Lastly, R-M systems may have evolved as molecular parasites, a notion which has only recently received much attention (Naito et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems

2003

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The gene encoding the LlaCI methyltransferase (M.LlaCI) from Lactococcus lactis subsp. cremoris W15 was overexpressed in Escherichia coli. The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein of M r 31 300±1000 under denaturing conditions. The exact position of the start codon AUG was determined by protein microsequencing. This enzyme recognizes the specific palindromic sequence 59-AAGCTT-39. Purified M.LlaCI was characterized. Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer. It modifies the first adenine residue at the 59 end of the specific sequence to N 6 -methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII (Haemophilus influenzae Rd) and EcoVIII (Escherichia coli E1585-68) restriction-modification systems. This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved among N 6 -adenine b-class methyltransferases. However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII. Restriction endonucleases require Mg 2+ for phosphodiester bond cleavage. Mg 2+ was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues. This observation suggests that sensitivity of the M.LlaCI to Mg 2+ may strengthen the restriction activity of the cognate endonuclease in the bacterial cell. Other biological implications of this finding are also discussed. INTRODUCTIONLactic acid bacteria (LAB) are probiotic micro-organisms that have been used for millennia in the processing of dairy products. It was shown recently that dairy establishments in Britain can be dated back to 5000 years BC (Copley et al., 2003). Manufacturing of food such as cheese and yogurt, but also sauerkraut and dill pickles, as well as the existence of some regional cuisine, is heavily dependent on lactic fermentation. LAB and food processed by them therefore have armies of admirers, but on the other side, these bacteria have also powerful enemies. Their enemies are not people who hate buttermilk, but virulent phages whose existence relies on propagation within bacterial cells. To combat phages, LAB have developed multiple molecular strategies.These measures can be divided into four groups: (i) inhibition of phage adsorption, (ii) blockage of phage DNA injection, (iii) abortive infection mechanisms, and (iv) restriction of phage DNA (Forde & Fitzgerald, 1999;Coffey & Ross, 2002). These defence mechanisms are often located on plasmids. That feature can facilitate their horizontal spread among LAB and when acquired affords them protection against phage invasion (Allison & Klaenhammer, 1998). Among the defence mechanisms listed above, restriction-modification (R-M) systems form the most diverse group. Based on their molecular structure and cofactor requireme...

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Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains

Cited by 60 publications

References 32 publications

Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

Natural Transformation of an Engineered Helicobacter pylori Strain Deficient in Type II Restriction Endonucleases

Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems

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