Regulated gene expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action

Zhang, Ling; Fan, Frank; Palmer, Leslie M.; Lonetto, Michael A.; Petit, Chantal; Voelker, LeRoy L.; John, Annemarie St.; Bankosky, Brent; Rosenberg, Martin; McDevitt, Damien

doi:10.1016/s0378-1119(00)00325-5

Cited by 91 publications

(101 citation statements)

References 26 publications

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“…The need to identify such untapped potential antibiotic targets in bacterial pathogens has fostered innovative approaches for essential gene identification. These include gene disruption or deletion (Joyce and Grindley, 1984;Link et al, 1997;Xia et al, 1999), promoter replacement (Jana et al, 2000;Zhang et al, 2000), saturation transposon mutagenesis in Mycoplasma genomes to deduce essential genes (Hutchison et al, 1999), transposon delivery of conditionally expressed promoters (Chow and Berg, 1988), comparative genomics combined with conditional expression (Arigoni et al, 1998;Freiberg et al, 2001), and approaches that use in vitro transposition in combination with genetic footprinting or with polymerase chain reaction (PCR) and Southern analysis (Akerley et al, 1998;Reich et al, 1999).…”

mentioning

confidence: 99%

A genome‐wide strategy for the identification of essential genes in Staphylococcus aureus

Forsyth¹,

Haselbeck²,

Ohlsen³

et al. 2002

Molecular Microbiology

454

360

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SummaryTo address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.

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mentioning

confidence: 99%

A genome‐wide strategy for the identification of essential genes in Staphylococcus aureus

Forsyth¹,

Haselbeck²,

Ohlsen³

et al. 2002

Molecular Microbiology

454

360

View full text Add to dashboard Cite

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“…Variants of P xyl/tet promoters provide a wide dynamic range of tet regulation Many previous studies have shown that the P xyl/tet promoter is highly active when induced in Bacillales such as B. subtilis, S. aureus or Streptococcus mutans (Geissendörfer & Hillen, 1990;Wang & Kuramitsu, 2005;Zhang et al, 2000). In fact, the activity of P xyl/tet in the TetR unbound state exceeds that of native S. aureus promoters of low and intermediate strength, such as P folA , P zwf or P fabI and rather reflects the transcriptional level of the highly active rpsF promoter (Stary et al, 2010;Xu et al, 2010).…”

Section: Quantification Of Regulatory Capacities Of Prmc2 and Prab11mentioning

confidence: 99%

Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus

et al. 2011

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A set of vectors for improved tetracycline-dependent gene regulation in Staphylococcus aureus is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second tet operator within the TetR-regulated promoter P xyl/tet . Pronounced repression was observed in the absence of anhydrotetracycline (ATc) combined with high induction in the presence of the drug, as demonstrated for pRAB11 bearing staphylococcal nuclease nuc1, lacZ or gfp. Also, in plasmid pCG261, the pRAB11 tetR-P xyl/tet regulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target gene rny (putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed P xyl/tet -like fragments, mutated at up to six defined positions, were constructed. Sixteen mutant sequences with single to quadruple exchanges were analysed for transcriptional strength and ATc-dependent inducibility. A set of promoters with gradually decreased activities and improved repression is presented. Finally, the implementation of reverse TetR revtetR-r2, which exhibits three amino acid exchanges and binds to tetO in the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly, revtetR was found to contain a fourth mutation only after propagation in S. aureus. We predict that the described vectors constitute valuable tools for staphylococcal genetics.

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“…In order to normalise the data the 16S rRNA gene was chosen. This gene has been previously used as the endogenous control gene in qPCR experiments (Hardwick et al, 2008;Zhang et al, 2000). As the genomes of all bacteria have not as yet been sequenced the exact number of copies of 16S rRNA genes per cell is currently unknown.…”

Section: Introductionmentioning

confidence: 99%

Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

Walsh

Ingenfeld

Zampicolli

et al. 2011

Journal of Microbiological Methods

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Antibiotic application in plant agriculture is primarily used to control fire blight caused by Erwinia amylovora in pome fruit orchards. In order to facilitate environmental impact assessment for antibiotic applications, we developed and validated culture-independent quantitative real-time PCR multiplex assays for streptomycin (strA, strB, aadA and insertion sequence IS1133) and tetracycline (tetB, tetM and tetW) resistance elements in plant and soil samples. The qPCR were reproducible and consistent whether the DNA was extracted directly from bacteria, plant and soil samples inoculated with bacteria or soil samples prior to and after manure slurry treatment. The genes most frequently identified in soils pre-and post-slurry treatment were strB, aadA, tetB and tetM. All genes tested were detected in soils pre-slurry treatment, and a decrease in relative concentrations of tetB and the streptomycin resistance genes was observed in samples taken post-slurry treatment. These multiplex qPCR assays offer a cost-effective, reliable method for simultaneous quantification of antibiotic resistance genes in complex, environmental sample matrices.

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Regulated gene expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action

Cited by 91 publications

References 26 publications

A genome‐wide strategy for the identification of essential genes in Staphylococcus aureus

A genome‐wide strategy for the identification of essential genes in Staphylococcus aureus

Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus

Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

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