Improved method for electroporation of<i>Staphylococcus aureus</i>

Schenk, Steve; Laddaga, Richard A.

doi:10.1111/j.1574-6968.1992.tb05302.x

Cited by 370 publications

(42 citation statements)

References 16 publications

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“…Hyaluronic acid sodium salt purified from Streptococcus equi was purchased from Sigma-Aldrich. Plasmid DNA was purified from Escherichia coli and electroporated into S. aureus RN4220 as previously described (30). Plasmids and transposon insertions were moved from S. aureus RN4220 or S. aureus JE2 with bacteriophage ⌽11 to other S. aureus strains by transduction.…”

Section: Methodsmentioning

confidence: 99%

Staphylococcus aureus Hyaluronidase Is a CodY-Regulated Virulence Factor

et al. 2014

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Staphylococcus aureus is a Gram-positive pathogen that causes a diverse range of bacterial infections. Invasive S. aureus strains secrete an extensive arsenal of hemolysins, immunomodulators, and exoenzymes to cause disease. Our studies have focused on the secreted enzyme hyaluronidase (HysA), which cleaves the hyaluronic acid polymer at the ␤-1,4 glycosidic bond. In the study described in this report, we have investigated the regulation and contribution of this enzyme to S. aureus pathogenesis. Using the Nebraska Transposon Mutant Library (NTML), we identified eight insertions that modulate extracellular levels of HysA activity. Insertions in the sigB operon, as well as in genes encoding the global regulators SarA and CodY, significantly increased HysA protein levels and activity. By altering the availability of branched-chain amino acids, we further demonstrated CodY-dependent repression of HysA activity. Additionally, through mutation of the CodY binding box upstream of hysA, the repression of HysA production was lost, suggesting that CodY is a direct repressor of hysA expression. To determine whether HysA is a virulence factor, a ⌬hysA mutant of a community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 strain was constructed and found to be attenuated in a neutropenic, murine model of pulmonary infection. Mice infected with this mutant strain exhibited a 4-log-unit reduction in bacterial burden in their lungs, as well as reduced lung pathology and increased levels of pulmonary hyaluronic acid, compared to mice infected with the wild-type, parent strain. Taken together, these results indicate that S. aureus hyaluronidase is a CodY-regulated virulence factor.

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Section: Methodsmentioning

confidence: 99%

Staphylococcus aureus Hyaluronidase Is a CodY-Regulated Virulence Factor

et al. 2014

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“…Sequences of the chimeras were confirmed by dye-terminator DNA sequencing chemistry (Skirball DNA Sequencing Core Facility). pRN7128 derivatives were electroporated into S. aureus RN4220 (25) and moved by phage transduction into an agr-null strain, RN7206, to eliminate the effects of endogenous AIPs on function. The AgrC receptor histidine kinases (HKs) are referred to in the text as AgrC-N (N corresponding to the agr specificity group) and the chimeras are referred to as AgrC-N::C (designating AgrC group N for the N terminus of the receptor domain and AgrC group C for the C terminus).…”

Section: Methodsmentioning

confidence: 99%

Hydrophobic interactions drive ligand-receptor recognition for activation and inhibition of staphylococcal quorum sensing

Wright

Lyon

George

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Two-component systems represent the most widely used signaling paradigm in living organisms. Encoding the prototypical twocomponent system in Gram-positive bacteria, the staphylococcal agr (accessory gene regulator) operon uses a polytopic receptor, AgrC, activated by an autoinducing peptide (AIP), to coordinate quorum sensing with the global synthesis of virulence factors. The agr locus has undergone evolutionary divergence, resulting in the formation of several distinct inter-and intraspecies specificity groups, such that most cross-group AIP-receptor interactions are mutually inhibitory. We have exploited this natural diversity by constructing and analyzing AgrC chimeras generated by exchange of intradomain segments between receptors of different agr groups. Functional chimeras fell into three general classes: receptors with broadened specificity, receptors with tightened specificity, and receptors that lack activation specificity. Testing of these chimeric receptors against a battery of AIP analogs localized the primary ligand recognition site to the receptor distal subdomain and revealed that the AIPs bind primarily to a putative hydrophobic pocket in the receptor. This binding is mediated by a highly conserved hydrophobic patch on the AIPs and is an absolute requirement for interactions in self-activation and cross-inhibition of the receptors. It is suggested that this recognition scheme provides the fundamental basis for agr activation and interference.staphylococci ͉ agr ͉ intradomain chimera ͉ autoinducing peptide T wo-component signaling systems function in the sensing of the cell's external environment and are probably the most widely used signaling paradigm in living organisms (1). Many twocomponent signaling systems in Gram-positive bacteria use polytopic transmembrane receptors that are activated by autoinducing peptides (AIPs) (2, 3). Although these systems regulate virulence in staphylococci (agr) (4) and enterococci ( fsr) (5), competence in bacilli (6) and pneumococci (com) (7), and bacteriocin production in lactic acid bacteria (pin and ssp) (8, 9), constituting a major component of the regulatory biology of these organisms, the mechanism(s) by which peptides bind to and activate the respective receptors are unknown.We have found the staphylococcal agr (accessory gene regulator) system to be particularly amenable to mechanistic investigation in this context for two reasons: first, because it is conserved throughout the staphylococci but has undergone a highly significant evolutionary divergence, resulting in four (or more) different specificity variants in Staphylococcus aureus and at least 20 others in the non-aureus species (10-12); and second, because the 7-to 10-aa AIPs of different staphylococcal species form a close family with a conserved structure consisting of a 5-aa thiolactone ring (a lactone ring in one case) and a linear 2-to 5-aa ''tail'' (13-16). Because the AIP from any one specificity group generally inhibits agr activation in the other groups (10,11,15,17,18), one is provided...

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“…The sequences of all PCR products used above were confirmed by DNA sequencing. Transformation of plasmid DNA were done by either the protoplast method (26) or electroporation (28).…”

Section: Methodsmentioning

confidence: 99%

Transmembrane Topology of AgrB, the Protein Involved in the Post-translational Modification of AgrD in Staphylococcus aureus

Zhang

Gray

Novick

et al. 2002

Journal of Biological Chemistry

119

140

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The accessory gene regulator (agr) of Staphylococcus aureus is the central regulatory system that controls the gene expression for a large set of virulence factors. This global regulatory locus consists of two transcripts: RNAII and RNAIII. RNAII encodes four genes (agrA, B, C, and D) whose gene products assemble a quorum sensing system. RNAIII is the effector of the Agr response. Both the agrB and agrD genes are essential for the production of the autoinducing peptide, which functions as a signal for the quorum sensing system. In this study, we demonstrated the transmembrane nature of AgrB protein in S. aureus. A transmembrane topology model of AgrB was proposed based on AgrB-PhoA fusion analyses in Escherichia coli. Two hydrophilic regions with several highly conserved positively charged amino acid residues among various AgrBs were found to be located in the cytoplasmic membrane as suggested by PhoA-AgrB fusion studies. However, this finding is inconsistent with the putative transmembrane profile of AgrB by computer analysis. Furthermore, we detected an intermediate peptide of processed AgrD from S. aureus cells expressing AgrB and a 6 histidine-tagged AgrD. These results provide direct evidence that AgrB is involved in the proteolytic processing of AgrD. We speculate that AgrB is a novel protein with proteolytic enzyme activity and a transporter facilitating the export of the processed AgrD peptide.

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Improved method for electroporation ofStaphylococcus aureus

Cited by 370 publications

References 16 publications

Staphylococcus aureus Hyaluronidase Is a CodY-Regulated Virulence Factor

Staphylococcus aureus Hyaluronidase Is a CodY-Regulated Virulence Factor

Hydrophobic interactions drive ligand-receptor recognition for activation and inhibition of staphylococcal quorum sensing

Transmembrane Topology of AgrB, the Protein Involved in the Post-translational Modification of AgrD in Staphylococcus aureus

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