Virulence Gene Identification by Differential Fluorescence Induction Analysis of Staphylococcus aureus Gene Expression during Infection-Simulating Culture

Abstract

We have employed a strategy utilizing differential fluorescence induction (DFI) in an effort to identify Staphylococcus aureus genes whose products can be targeted for antimicrobial drug development. DFI allows identification of promoters preferentially active under given growth conditions on the basis of their ability to drive expression of a promoterless green fluorescent protein gene (gfp). A plasmid-based promoter trap library was constructed of 200-to 1,000-bp fragments of S. aureus genomic DNA fused to gfp, and clones with active promoters were isolated under seven different in vitro growth conditions simulating infection. Six thousand two hundred sixty-seven clones with active promoters were screened to identify those that exhibited differential promoter activity. Bioinformatic analysis allowed the identification of 42 unique operons, containing a total of 61 genes, immediately downstream of the differentially active putative promoters. Replacement mutations were generated for most of these operons, and the abilities of the resulting mutants to cause infection were assessed in two different murine infection models. Approximately 40% of the mutants were attenuated in at least one infection model.

Genomic Profiling of Iron-Responsive Genes in Salmonella enterica Serovar Typhimurium by High-Throughput Screening of a Random Promoter Library

“…The procedure for cloning random DNA fragments into promoterless vectors to identify promoters displaying condition-dependent gene regulation is a well-established method (29,32,43,(48)(49)(50). We adapted this approach and incorporated the luxCDABE reporter to develop sensitive methods FIG.…”

“…Complications of microarray analysis include its reproducibility and standardization (9,10,21,24,27,44). Differential fluorescence induction uses flow cytometry to select bacteria containing transcriptionally active regulatory regions cloned upstream of a promoterless green fluorescence protein (GFP) reporter gene (29,43,(48)(49)(50); however, there are technical challenges associated with sorting bacteria. The LuxArray system of genome-wide transcriptional analysis uses specific bioluminescent reporter strains arrayed on solid-phase media (51).…”

Structure of Coenzyme A−Disulfide Reductase from Staphylococcus aureus at 1.54 Å Resolution,

“…Reinforcing the concept that CoASH assumes the intracellular redox function of GSH in S. aureus, a novel NADPHdependent flavoprotein disulfide reductase, coenzyme A-disulfide reductase (CoADR 1 ), was identified (6,7). Consistent with the important functional role attributed to CoADR, staphylococcal virulence in mice was shown to be attenuated for CoADR-deficient strains (8,9).…”

Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

“…This technique was subsequently adapted to study induction of Staphylococcus aureus and Streptococcus pneumoniae gene expression by in vitro stimuli that mimic the host environment, such as temperature shift, increased osmolarity, iron limitation, increased acidity, presence of competence stimulatory peptide, and cation starvation (13,160,237).…”