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      Expression of a Cryptic Secondary Sigma Factor Gene Unveils Natural Competence for DNA Transformation in Staphylococcus aureus

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          Abstract

          It has long been a question whether Staphylococcus aureus, a major human pathogen, is able to develop natural competence for transformation by DNA. We previously showed that a novel staphylococcal secondary sigma factor, SigH, was a likely key component for competence development, but the corresponding gene appeared to be cryptic as its expression could not be detected during growth under standard laboratory conditions. Here, we have uncovered two distinct mechanisms allowing activation of SigH production in a minor fraction of the bacterial cell population. The first is a chromosomal gene duplication rearrangement occurring spontaneously at a low frequency [≤10 −5], generating expression of a new chimeric sigH gene. The second involves post-transcriptional regulation through an upstream inverted repeat sequence, effectively suppressing expression of the sigH gene. Importantly, we have demonstrated for the first time that S. aureus cells producing active SigH become competent for transformation by plasmid or chromosomal DNA, which requires the expression of SigH-controlled competence genes. Additionally, using DNA from the N315 MRSA strain, we successfully transferred the full length SCC mecII element through natural transformation to a methicillin-sensitive strain, conferring methicillin resistance to the resulting S. aureus transformants. Taken together, we propose a unique model for staphylococcal competence regulation by SigH that could help explain the acquisition of antibiotic resistance genes through horizontal gene transfer in this important pathogen.

          Author Summary

          Staphylococcus aureus is a major human pathogen responsible for a broad spectrum of infections, emphasized by the emergence of multiple antibiotic-resistant strains with up to 60% of strains worldwide resistant to methicillin ( Methicillin Resistant Staphylococcus aureus or MRSA). Indeed, MRSA-related infections are now one of the leading causes of death in the USA, highlighting the growing threat this bacterium poses to human health. Many bacteria have the ability to acquire novel genetic characteristics, including antibiotic resistance, through the uptake of extracellular DNA, a phenomenon known as natural genetic transformation or competence. We have shown that the SigH staphylococcal sigma factor is a likely key component for competence development, but that its gene is not expressed under standard laboratory conditions. Here, we have uncovered two distinct mechanisms allowing activation of SigH production in S. aureus: a chromosomal gene duplication rearrangement and post-transcriptional regulation through an upstream inverted repeat sequence. Importantly, we have demonstrated for the first time that S. aureus cells producing active SigH become competent for natural transformation by plasmid or chromosomal DNA, and we were able to confer methicillin resistance to a methicillin-sensitive strain by transformation with chromosomal DNA. SigH-dependent competence development in S. aureus could help explain the acquisition of antibiotic resistance genes and the rise of the so-called “superbug."

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          Most cited references74

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          Waves of resistance: Staphylococcus aureus in the antibiotic era.

          Staphylococcus aureus is notorious for its ability to become resistant to antibiotics. Infections that are caused by antibiotic-resistant strains often occur in epidemic waves that are initiated by one or a few successful clones. Methicillin-resistant S. aureus (MRSA) features prominently in these epidemics. Historically associated with hospitals and other health care settings, MRSA has now emerged as a widespread cause of community infections. Community or community-associated MRSA (CA-MRSA) can spread rapidly among healthy individuals. Outbreaks of CA-MRSA infections have been reported worldwide, and CA-MRSA strains are now epidemic in the United States. Here, we review the molecular epidemiology of the epidemic waves of penicillin- and methicillin-resistant strains of S. aureus that have occurred since 1940, with a focus on the clinical and molecular epidemiology of CA-MRSA.
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            Bistability, epigenetics, and bet-hedging in bacteria.

            Clonal populations of microbial cells often show a high degree of phenotypic variability under homogeneous conditions. Stochastic fluctuations in the cellular components that determine cellular states can cause two distinct subpopulations, a property called bistability. Phenotypic heterogeneity can be readily obtained by interlinking multiple gene regulatory pathways, effectively resulting in a genetic logic-AND gate. Although switching between states can occur within the cells' lifetime, cells can also pass their cellular state over to the next generation by a mechanism known as epigenetic inheritance and thus perpetuate the phenotypic state. Importantly, heterogeneous populations can demonstrate increased fitness compared with homogeneous populations. This suggests that microbial cells employ bet-hedging strategies to maximize survival. Here, we discuss the possible roles of interlinked bistable networks, epigenetic inheritance, and bet-hedging in bacteria.
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              New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria.

              A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                November 2012
                November 2012
                1 November 2012
                : 8
                : 11
                : e1003003
                Affiliations
                [1 ]University of Tsukuba, Division of Biomedical Science, Faculty of Medicine, Tsukuba, Japan
                [2 ]Institut Pasteur, Biology of Gram Positive Pathogens, Department of Microbiology, Paris, France
                [3 ]CNRS ERL 3526, Paris, France
                University of Tubingen, Germany
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: KM TM. Performed the experiments: KM AJT MT LTNT. Analyzed the data: KM YI TO TM. Wrote the paper: KM TM.

                Article
                PPATHOGENS-D-12-00430
                10.1371/journal.ppat.1003003
                3486894
                23133387
                04026a41-90e7-40b4-ac5f-0ed1003fad69
                Copyright @ 2012

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

                History
                : 16 February 2012
                : 17 September 2012
                Page count
                Pages: 20
                Funding
                This work was supported by research funds from the Institut Pasteur, European Commission (StaphDynamics [LHSM-CT-2006-019064] grant) and the Centre National de la Recherche Scientifique (CNRS URA 2172) (to TM), Grant-in-Aid for Young Scientists B of the Japan Society for the Promotion of Science (18790301), the Ichiro Kanehara Foundation (09KI202), the Asahi Glass Foundation (to KM), and FY2006 Researcher Exchange Program between JSPS and CNRS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Microbiology
                Bacterial Pathogens
                Staphylococci
                Bacteriology
                Bacterial Physiology
                Microbial Pathogens
                Microbial Physiology

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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