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Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations.

Roberts GA, Houston PJ, White JH, Chen K, Stephanou AS, Cooper LP, Dryden DT, Lindsay JA - Nucleic Acids Res. (2013)

Bottom Line: A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants.We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently.This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium.

View Article: PubMed Central - PubMed

Affiliation: EaStCHEM School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh EH9 3JJ, UK and Division of Clinical Sciences, St. George's, University of London, Cranmer Terrace, London, SW17 0RE, UK.

ABSTRACT
A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA.

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Electroporation of plasmid pCN36 is dependent on Sau1 Modification and Restriction. (A) Plasmid methylation profiles of pCN36 when grown in different donor backgrounds. There are two TRS for the CC5-1 enzyme (recognized by both CC8 and CC5 isolates), one target site for CC1-2 (recognized by CC8), and no target sites for CC5-2 (recognized by CC5). CC5-1− and CC1-2− refer to S. aureus JE2 (CC8) isolates with mutations in sau1hsdSCC5-1 and sau1hsdSCC1-2, respectively. (B) Transformation efficiency of pCN36 (tetracycline resistant colonies per 1 μg DNA) into S. aureus JE2 (CC8) is dependent on modification with both CC5-1 and CC1-2 and restriction by sau1hsdR (5,6), but not with restriction by the Type IV restriction system (35,36). pCN36 prepared from S. aureus N315 (CC5) is not readily accepted by S. aureus JE2 (CC8). (C) Transformation efficiency of pCN36 from S. aureus JE2 (CC8) to S. aureus N315 (CC5) is dependent on CC5-1 modification, and not CC5-2. S. aureus N315 (CC5) accepts plasmid at high rates from S. aureus JE2 (CC8), as pCN36 does not contain a TRS for CC5-2. Data presented represent average transformation efficiency of three experiments ± SD. Asterisk denotes significant difference P < 0.001.
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gkt535-F5: Electroporation of plasmid pCN36 is dependent on Sau1 Modification and Restriction. (A) Plasmid methylation profiles of pCN36 when grown in different donor backgrounds. There are two TRS for the CC5-1 enzyme (recognized by both CC8 and CC5 isolates), one target site for CC1-2 (recognized by CC8), and no target sites for CC5-2 (recognized by CC5). CC5-1− and CC1-2− refer to S. aureus JE2 (CC8) isolates with mutations in sau1hsdSCC5-1 and sau1hsdSCC1-2, respectively. (B) Transformation efficiency of pCN36 (tetracycline resistant colonies per 1 μg DNA) into S. aureus JE2 (CC8) is dependent on modification with both CC5-1 and CC1-2 and restriction by sau1hsdR (5,6), but not with restriction by the Type IV restriction system (35,36). pCN36 prepared from S. aureus N315 (CC5) is not readily accepted by S. aureus JE2 (CC8). (C) Transformation efficiency of pCN36 from S. aureus JE2 (CC8) to S. aureus N315 (CC5) is dependent on CC5-1 modification, and not CC5-2. S. aureus N315 (CC5) accepts plasmid at high rates from S. aureus JE2 (CC8), as pCN36 does not contain a TRS for CC5-2. Data presented represent average transformation efficiency of three experiments ± SD. Asterisk denotes significant difference P < 0.001.

Mentions: Electroporation of plasmids into S. aureus JE2, a CC8 SCCmecIV USA300 isolate typical of MRSA circulating in the community in the USA, was controlled by the Type I RM system (Figure 5). Plasmids grown in S. aureus JE2 donors deficient in each of the two hsdS genes recognizing the CC5-1 and CC1-2 TRS were not modified, and when transferred to parental JE2 recipients, these plasmids were recognised as foreign and restricted (Figure 5A and B). Restriction was due to the Type I RM system and hsdR dependent, as when this gene was deleted the unmodified plasmid was transferred at high frequency. Transfer was not restored by deleting the Type IV restriction system, showing no role for this system in transfer of plasmids between the MRSA isolates (although it does prevent transformation of cytosine-methylated plasmids prepared from E.coli containing the dcm MTase) (35,36). Similarly, plasmids grown in S. aureus N315, a clinical MRSA from lineage CC5, were recognized as foreign and digested by the Type I and not the Type IV restriction system (Figure 5B).Figure 5.


Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations.

Roberts GA, Houston PJ, White JH, Chen K, Stephanou AS, Cooper LP, Dryden DT, Lindsay JA - Nucleic Acids Res. (2013)

Electroporation of plasmid pCN36 is dependent on Sau1 Modification and Restriction. (A) Plasmid methylation profiles of pCN36 when grown in different donor backgrounds. There are two TRS for the CC5-1 enzyme (recognized by both CC8 and CC5 isolates), one target site for CC1-2 (recognized by CC8), and no target sites for CC5-2 (recognized by CC5). CC5-1− and CC1-2− refer to S. aureus JE2 (CC8) isolates with mutations in sau1hsdSCC5-1 and sau1hsdSCC1-2, respectively. (B) Transformation efficiency of pCN36 (tetracycline resistant colonies per 1 μg DNA) into S. aureus JE2 (CC8) is dependent on modification with both CC5-1 and CC1-2 and restriction by sau1hsdR (5,6), but not with restriction by the Type IV restriction system (35,36). pCN36 prepared from S. aureus N315 (CC5) is not readily accepted by S. aureus JE2 (CC8). (C) Transformation efficiency of pCN36 from S. aureus JE2 (CC8) to S. aureus N315 (CC5) is dependent on CC5-1 modification, and not CC5-2. S. aureus N315 (CC5) accepts plasmid at high rates from S. aureus JE2 (CC8), as pCN36 does not contain a TRS for CC5-2. Data presented represent average transformation efficiency of three experiments ± SD. Asterisk denotes significant difference P < 0.001.
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gkt535-F5: Electroporation of plasmid pCN36 is dependent on Sau1 Modification and Restriction. (A) Plasmid methylation profiles of pCN36 when grown in different donor backgrounds. There are two TRS for the CC5-1 enzyme (recognized by both CC8 and CC5 isolates), one target site for CC1-2 (recognized by CC8), and no target sites for CC5-2 (recognized by CC5). CC5-1− and CC1-2− refer to S. aureus JE2 (CC8) isolates with mutations in sau1hsdSCC5-1 and sau1hsdSCC1-2, respectively. (B) Transformation efficiency of pCN36 (tetracycline resistant colonies per 1 μg DNA) into S. aureus JE2 (CC8) is dependent on modification with both CC5-1 and CC1-2 and restriction by sau1hsdR (5,6), but not with restriction by the Type IV restriction system (35,36). pCN36 prepared from S. aureus N315 (CC5) is not readily accepted by S. aureus JE2 (CC8). (C) Transformation efficiency of pCN36 from S. aureus JE2 (CC8) to S. aureus N315 (CC5) is dependent on CC5-1 modification, and not CC5-2. S. aureus N315 (CC5) accepts plasmid at high rates from S. aureus JE2 (CC8), as pCN36 does not contain a TRS for CC5-2. Data presented represent average transformation efficiency of three experiments ± SD. Asterisk denotes significant difference P < 0.001.
Mentions: Electroporation of plasmids into S. aureus JE2, a CC8 SCCmecIV USA300 isolate typical of MRSA circulating in the community in the USA, was controlled by the Type I RM system (Figure 5). Plasmids grown in S. aureus JE2 donors deficient in each of the two hsdS genes recognizing the CC5-1 and CC1-2 TRS were not modified, and when transferred to parental JE2 recipients, these plasmids were recognised as foreign and restricted (Figure 5A and B). Restriction was due to the Type I RM system and hsdR dependent, as when this gene was deleted the unmodified plasmid was transferred at high frequency. Transfer was not restored by deleting the Type IV restriction system, showing no role for this system in transfer of plasmids between the MRSA isolates (although it does prevent transformation of cytosine-methylated plasmids prepared from E.coli containing the dcm MTase) (35,36). Similarly, plasmids grown in S. aureus N315, a clinical MRSA from lineage CC5, were recognized as foreign and digested by the Type I and not the Type IV restriction system (Figure 5B).Figure 5.

Bottom Line: A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants.We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently.This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium.

View Article: PubMed Central - PubMed

Affiliation: EaStCHEM School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh EH9 3JJ, UK and Division of Clinical Sciences, St. George's, University of London, Cranmer Terrace, London, SW17 0RE, UK.

ABSTRACT
A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA.

Show MeSH
Related in: MedlinePlus