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High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor
1Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University, New York, New York 10021.
Abstract:
Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.
Insights
Neisseria gonorrhoeae uses restriction systems to block foreign plasmid DNA. Methylation of plasmids by specific enzymes overcomes these barriers, increasing antibiotic resistance gene transfer from E. coli to gonococci.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Antibiotic resistance in Neisseria gonorrhoeae is a growing public health concern.
- The spread of R plasmids between bacterial species contributes to antibiotic resistance.
- Broad-host-range plasmids, such as IncP and IncQ, are implicated in this genetic exchange.
Purpose of the Study:
- To investigate the role of gonococcal restriction systems in limiting plasmid mobilization from Escherichia coli.
- To determine if plasmid modification, specifically methylation, can overcome these restriction barriers.
Main Methods:
- Utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) plasmids for conjugation experiments.
- Assessed plasmid mobilization from E. coli into Neisseria gonorrhoeae strains F62 and PGH 3-2.
- Investigated the effect of coresident methylase-producing plasmids (pCAL7 and pFnuDI) on plasmid protection from gonococcal restriction enzymes in vitro and in vivo.
Main Results:
- Gonococcal restriction systems significantly impede plasmid mobilization from E. coli.
- Partially purified gonococcal restriction enzymes digest RSF1010 plasmid DNA in vitro.
- Coresident methylase plasmids (pCAL7, pFnuDI) in E. coli protect RSF1010 from gonococcal restriction.
- Plasmid mobilization into Neisseria gonorrhoeae increased by four orders of magnitude when protective methylases were present.
Conclusions:
- Restriction systems in Neisseria gonorrhoeae act as a major barrier to efficient conjugal transfer of plasmids from heterologous hosts like E. coli.
- Plasmid modification via methylation can overcome these barriers, facilitating the transfer of genetic material, potentially including antibiotic resistance genes.