Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth

Eleaume, Heïdy; Jabbouri, Saı̈d

doi:10.1016/j.mimet.2004.07.015

Cited by 96 publications

(55 citation statements)

References 17 publications

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“…We believe this general approach represents the most reliable normalization protocol available to quantify gene expression in situ. Alternatively, quantification of mRNA from functional genes might be normalized to transcripts of a so-called 'housekeeping gene' that is constitutively and evenly transcribed during all growth phases and states of metabolic activity (Eleaume and Jabbouri, 2004;Johnson et al, 2005). However, to our knowledge, no such gene has been suggested across all of prokaryotic diversity and such normalization would require extensive a priori knowledge of the suites of genes present in natural soil microorganisms, which is currently not available.…”

Section: Discussionmentioning

confidence: 99%

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil

et al. 2008

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Expression of the functional gene tfdA involved in degradation of phenoxyacetic acids such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) was investigated during degradation scenarios in natural unseeded soil samples. The results illustrate how messenger RNA (mRNA)-based analysis is well suited to quantitatively study the activity of specific microbial populations in soil using phenoxyacetic acid biodegradation as a model system. Via quantitative real-time PCR, a clear response to the presence of phenoxy acids was shown during degradation in soil amended with 20 mg 2,4-D or MCPA per kg soil. Further, we found a relatively high degree of correlation between expression of the functional gene and the rates of mineralization. Melting curve analyses of real-time PCR products, supported by tfdA-denaturing gradient gel electrophoresis analysis showed that, although only class I tfdA genes were apparent in the indigenous microbial population, class III tfdA genes became predominant during incubation, and were the only genes expressed during degradation of MCPA in soil. In contrast, both classes were expressed during degradation of the structurally similar compound 2,4-D. The ability to quantify microbial transcripts directly in environmental samples will have a profound impact on our understanding of microbial processes in the environment in future studies.

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Section: Discussionmentioning

confidence: 99%

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil

et al. 2008

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“…Absolute quantification of transcript copy number was calculated by interpolating threshold cycle values against standard curves, as described previously for Staphylococcus aureus (38). To prepare the standard curves, the gelE and ahrC genes were PCR amplified in standard PCRs using Taq polymerase with primer pairs gelE ORF-F/gelE ORF-R and EF0983-F/EF0983-R, respectively.…”

Section: Methodsmentioning

confidence: 99%

AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

et al. 2013

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bEnterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species.

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“…No contaminating DNA was detected by PCR. DNase-treated RNA was reverse transcribed using an iScript kit (Bio-Rad), and real-time PCR was performed with an iCycler (Bio-Rad), using an iQ SYBR green kit (Bio-Rad) according to the manufacturer's recommended protocol and an initial denaturing step of 95°C for 3 min followed by 40 cycles of 95°C for 10 s and 55°C for 30 s. Expression of 16S rRNA (14) was monitored to allow for sample normalization. Results and all primers used are shown at http://www.scripps.edu/chem/romesberg/PublicationsMain.htm.…”

Section: Methodsmentioning

confidence: 99%

Complete and SOS-Mediated Response of Staphylococcus aureus to the Antibiotic Ciprofloxacin

Jones²,

et al. 2007

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Staphylococcus aureus infections can be difficult to treat due to both multidrug resistance and the organism's remarkable ability to persist in the host. Persistence and the evolution of resistance may be related to several complex regulatory networks, such as the SOS response, which modifies transcription in response to environmental stress. To understand how S. aureus persists during antibiotic therapy and eventually emerges resistant, we characterized its global transcriptional response to ciprofloxacin. We found that ciprofloxacin induces prophage mobilization as well as significant alterations in metabolism, most notably the up-regulation of the tricarboxylic acid cycle. In addition, we found that ciprofloxacin induces the SOS response, which we show, by comparison of a wild-type strain and a non-SOS-inducible lexA mutant strain, includes the derepression of 16 genes. While the SOS response of S. aureus is much more limited than those of Escherichia coli and Bacillus subtilis, it is similar to that of Pseudomonas aeruginosa and includes RecA, LexA, several hypothetical proteins, and a likely error-prone Y family polymerase whose homologs in other bacteria are required for induced mutation. We also examined induced mutation and found that either the inability to derepress the SOS response or the lack of the LexA-regulated polymerase renders S. aureus unable to evolve antibiotic resistance in vitro in response to UV damage. The data suggest that up-regulation of the tricarboxylic acid cycle and induced mutation facilitate S. aureus persistence and evolution of resistance during antibiotic therapy.

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Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth

Cited by 96 publications

References 17 publications

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil

AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

Complete and SOS-Mediated Response of Staphylococcus aureus to the Antibiotic Ciprofloxacin

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