High-frequency transformation ofStaphylococcus aureus by electroporation

Kraemer, G. R.; Iandolo, John J.

doi:10.1007/bf02199440

Cited by 161 publications

(110 citation statements)

References 10 publications

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“…Most promoters in the array were obtained after transfer of individual clones of a partial Sau3AI digest library from E. coli into S. aureus because direct cloning into the latter was inefficient and did not result in a sufficiently large number of luminescent clones. 21,42 Francis et al (2000) 43 recovered 73 clones in a different lux vector with at least moderate luminescence out of 20 000 S. aureus transformants, and selected only the six brightest for use in in vivo tracking. In our study, 53 luminescent clones were generated.…”

Section: Discussionmentioning

confidence: 99%

Staphylococcus aureus promoter-lux reporters for drug discovery

Mesak

Villanueva

et al. 2010

J Antibiot

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We describe a collection of antibiotic-activated Staphylococcus aureus promoter-lux reporter strains that can be used to discriminate among antibiotic classes on the basis of their light production response profile. We screened over 400 culture supernatants from previously uncharacterized actinomycetes from soil for the production of aminocoumarin-type compounds and DNA-damaging agents. Novobiocin production was determined in three isolates of Streptomyces, and streptonigrin, a DNA-damaging agent, together with several other bioactive compounds (oxopropaline D and G), was identified from a novel Kitasatospora isolate. This array provides an effective and specific whole-cell approach to search for classes of antimicrobial compounds in unfractionated culture broths.

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Section: Discussionmentioning

confidence: 99%

Staphylococcus aureus promoter-lux reporters for drug discovery

Mesak

Villanueva

et al. 2010

J Antibiot

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“…S. aureus strains were cultivated on tryptic soy agar (Difco). Electroporation in S. aureus was carried out by the procedure of Kraemer & Iandolo (1990). Nitrocefin was purchased from Becton Dickinson Microbiology Section.…”

Section: Methodsmentioning

confidence: 99%

The arl locus positively regulates Staphylococcus aureus type 5 capsule via an mgrA-dependent pathway

Luong

Lee

2006

Microbiology

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Most clinical Staphylococcus aureus strains produce either type 5 or type 8 capsular polysaccharides. The production of these capsules is influenced by various environmental factors. To study the regulation of capsule, Tn551 transposon mutagenesis and transcriptional reporter gene fusion were employed to identify several putative regulatory loci that influenced capsule gene expression. One of these, the arl locus, was chosen for further analysis. Tn551 was found to insert within the coding region (near the translational start site of the arlR gene). ArlR, along with ArlS, forms a two-component system that has been previously shown to affect autolysis and production of several secreted proteins. Phenotypic analyses of the arlR-specific mutant and gene fusion analyses showed that arlR activated capsule production at the transcriptional level. However, gel mobility shift assays did not support activation of the capsule genes by direct ArlR binding to the primary cap5 promoter region upstream of the operon. In contrast, it was found that arl activated mgrA, an activator for capsule production, whereas mgrA did not have a significant effect on arlR. Genetic studies supported the notion that arlR functions upstream of mgrA with respect to the regulation of capsule production, although gene fusion studies indicated that arl could also regulate capsule independently from mgrA. Collectively, the results suggest that arl positively regulates capsule production at the transcriptional level primarily through an mgrA-dependent pathway.

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“…Preparation and transformation of E. coli DH5␣ were accomplished as described by Inoue et al (14). Electroporation of DNA into S. aureus was carried out using the procedure of Kraemer and Iandolo (17). S. aureus 11-mediated transduction was performed using the method of Shafer and Iandolo (30).…”

Section: Methodsmentioning

confidence: 99%

Analysis of Genetic Elements ControllingStaphylococcus aureus lrgABExpression: Potential Role of DNA Topology in SarA Regulation

2000

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Penicillin-induced killing and murein hydrolase activity in Staphylococcus aureus are dependent on a variety of regulatory elements, including the LytSR two-component regulatory system and the virulence factor regulators Agr and Sar. The LytSR effects on these processes can be explained, in part, by the recent finding that a LytSR-regulated operon, designated lrgAB, affects murein hydrolase activity and penicillin tolerance. To examine the regulation of lrgAB expression in greater detail, we performed Northern blot and promoter fusion analyses. Both methods revealed that Agr and Sar, like LytSR, positively regulate lrgAB expression. A mutation in the agr locus reduced lrgAB expression approximately sixfold, while the sar mutation reduced lrgAB expression to undetectable levels. cis-acting regulatory elements involved in lrgAB expression were identified by fusing various fragments of the lrgAB promoter region to the xylE reporter gene and integrating these constructs into the chromosome. Catechol 2,3-dioxygenase assays identified DNA sequences, including an inverted repeat and intrinsic bend sites, that contribute to maximal lrgAB expression. Confirmation of the importance of the inverted repeat was achieved by demonstrating that multiple copies of the inverted repeat reduced lrgAB promoter activity, presumably by titrating out a positive regulatory factor. The results of this study demonstrate that lrgAB expression responds to a variety of positive regulatory factors and suggest that specific DNA topology requirements are important for optimal expression.Peptidoglycan or murein hydrolases are a family of enzymes that catalyze the cleavage of specific structural components of the bacterial cell wall. These enzymes are involved in several important physiological processes, including peptidoglycan turnover and recycling, cell wall expansion during bacterial growth, septation, and daughter cell separation (13, 32). Due to the potential of these enzymes to compromise cell wall integrity, leading to cell lysis (autolysis), murein hydrolase activity must be carefully regulated. This regulation includes transcriptional control, blocked access to the specific substrate, inhibition by choline or teichoic acid, and substrate modification (13, 32).The Staphylococcus aureus lytS and lytR genes, whose products are members of the two-component regulator family of proteins, are involved in the control of murein hydrolase activity (3). A lytS mutant phenotypically displays altered murein hydrolase activity and an increased rate of penicillin-and Triton X-100-induced lysis (3, 11). Immediately downstream of lytS and lytR are two genes, lrgA and lrgB, whose transcription is dependent upon lytS and lytR. LrgA and LrgB show no sequence similarity to known murein hydrolases. Instead, LrgA has structural characteristics in common with the bacteriophage-encoded holin proteins involved in murein hydrolase export (4, 11). Recent data generated in our laboratory indicate that the LrgA and LrgB gene products inhibit extracellular murein hy...

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High-frequency transformation ofStaphylococcus aureus by electroporation

Cited by 161 publications

References 10 publications

Staphylococcus aureus promoter-lux reporters for drug discovery

Staphylococcus aureus promoter-lux reporters for drug discovery

The arl locus positively regulates Staphylococcus aureus type 5 capsule via an mgrA-dependent pathway

Analysis of Genetic Elements ControllingStaphylococcus aureus lrgABExpression: Potential Role of DNA Topology in SarA Regulation

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