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      Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

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          ABSTRACT

          Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of S. aureus. Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages.

          IMPORTANCE

          The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus.

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          Most cited references36

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          N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.

          N(6)-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction-modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA-protein interactions. In gamma-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In alpha-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.
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            sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4.

            The accessory sigma factor sigmaB controls a general stress response that is thought to be important for Staphylococcus aureus survival and may contribute to virulence. The strain of choice for genetic studies, 8325-4, carries a small deletion in rsbU, which encodes a positive regulator of sigmaB activity. Consequently, to enable the role of sigmaB in virulence to be addressed, we constructed an rsbU(+) derivative, SH1000, using a method that does not leave behind an antibiotic resistance marker. The phenotypic properties of SH1000 (8325-4 rsbU(+)) were characterized and compared to those of 8325-4, the rsbU mutant, parent strain. A recognition site for sigmaB was located in the promoter region of katA, the gene encoding the sole catalase of S. aureus, by primer extension analysis. However, catalase expression and activity were similar in SH1000 (8325-4 rsbU(+)), suggesting that this promoter may have a minor role in catalase expression under normal conditions. Restoration of sigmaB activity in SH1000 (8325-4 rsbU(+)) resulted in a marked decrease in the levels of the exoproteins SspA and Hla, and this is likely to be mediated by reduced expression of agr in this strain. By using Western blotting and a sarA-lacZ reporter assay, the levels of SarA were found to be similar in strains 8325-4 and SH1000 (8325-4 rsbU(+)) and sigB mutant derivatives of these strains. This finding contrasts with previous reports that suggested that SarA expression levels are altered when they are measured transcriptionally. Inactivation of sarA in each of these strains resulted in an expected decrease in agr expression; however, the relative level of agr in SH1000 (8325-4 rsbU(+)) remained less than the relative levels in 8325-4 and the sigB mutant derivatives. We suggest that SarA is not likely to be the effector in the overall sigmaB-mediated effect on agr expression.
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              The methylomes of six bacteria.

              Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and C. jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N(6)-methyladenine ((m6)A) and N(4)-methylcytosine ((m4)C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases, it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTase genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. Two of these proved active. No attempt was made to detect 5-methylcytosine ((m5)C) recognition motifs from the SMRT® sequencing data because this modification produces weaker signals using current methods. However, all predicted (m6)A and (m4)C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active but also revealing their recognition sequences.
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                Author and article information

                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                26 May 2015
                May-Jun 2015
                : 6
                : 3
                : e00308-15
                Affiliations
                [ a ]Moyne Institute of Preventative Medicine, Department of Microbiology, School of Genetics and Microbiology, Trinity College Dublin, Dublin, Ireland
                [ b ]Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Australia
                [ c ]Austin Centre for Infection Research (ACIR), Infectious Diseases Department, Austin Health, Melbourne, Australia
                [ d ]Microbiological Diagnostics Unit, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Australia
                Author notes
                Address correspondence to Ian R. Monk, imonk@ 123456unimelb.edu.au .
                [*]

                Present address: Ian R. Monk, Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia.

                T.P.S. and T.J.F. are joint senior authors.

                Editor Steven J. Projan, MedImmune

                Article
                mBio00308-15
                10.1128/mBio.00308-15
                4447248
                26015493
                8f2b860d-2f63-48b0-b0e2-0677fc51b27b
                Copyright © 2015 Monk et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 23 February 2015
                : 21 April 2015
                Page count
                supplementary-material: 0, Figures: 8, Tables: 3, Equations: 0, References: 45, Pages: 12, Words: 9242
                Categories
                Research Article
                Custom metadata
                May/June 2015

                Life sciences
                Life sciences

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