Differential fluorescence induction reveals Streptococcus pneumoniae loci regulated by competence stimulatory peptide

Abstract

SummaryDifferential fluorescence induction (DFI) in Streptococcus pneumoniae was used as a method for the discovery of genes activated in specific growth environments. Competence stimulatory peptide (CSP) was used as the model inducing system to identify differentially expressed genes. To identify CSP-induced promoters, a plasmid library was constructed by inserting random pieces of S. pneumoniae chromosomal DNA upstream of the promoterless gfpmut2 gene in an Escherichia coli/S. pneumoniae shuttle vector. S. pneumoniae carrying the library were induced with CSP and enriched for green fluorescent protein (GFP)-expressing bacteria using fluorescenceactivated cell sorting. A total of 886 fluorescent clones was screened, and 12 differentially activated promoter elements were identified. Sequence analysis of these clones revealed that three were associated with novel competence loci, one of which we show is essential for DNA uptake, and six are known CSP-inducible promoters. We also explored whether competence proteins have a role in virulence and found that mutations in three CSP-inducible genes resulted in attenuated virulence phenotypes in either of two murine infection models. These results demonstrate the utility of DFI as a method for identifying differentially expressed genes in S. pneumoniae and the potential utility of applying DFI to other Gram-positive bacteria.

In vivo characterization of the psa genes from Streptococcus pneumoniae in multiple models of infection

“…The S. pneumoniae promoter-trap library in vector pNE1gfp has been described previously (Bartilson et al, 2000). Briefly, the library consists of 200-500 bp DNase I fragments of S. pneumoniae D39 DNA cloned upstream of the promoterless gfp gene in pNE1gfp.…”

“…Briefly, the library consists of 200-500 bp DNase I fragments of S. pneumoniae D39 DNA cloned upstream of the promoterless gfp gene in pNE1gfp. Following ligation, the library was electroporated into E. coli RR1, and plasmid DNA was prepared and used to transform S. pneumoniae D39 as described (Bartilson et al, 2000). The S. pneumoniae D39 promoter-trap library was used to infect groups of three mice via intranasal instillation ; as a negative control for sorting, D39 carrying the promoterless gfp fusion plasmid pNE1gfp was used to infect another set of mice.…”

“…We have previously used DFI to identify S. pneumoniae promoters induced under certain in vitro laboratory conditions (Bartilson et al, 2000 ;Marra et al, 2002) with the intention of mimicking aspects of the in vivo environment to which a pathogen may respond ; subsequent experiments screened for promoters induced during infection. It was hoped that these identified promoters expressed genes whose functions were necessary for causing infection, and thus interruption of their expression would decrease virulence.…”

“…shuttle plasmid (Bartilson et al, 2000). The resulting library was transformed into S. pneumoniae and grown under inducing and non-inducing conditions, and pools of clones grown under the inducing conditions were analysed and sorted by fluorescence-activated cell sorting (FACS).…”

The HtrA Protease from Streptococcus pneumoniae Digests Both Denatured Proteins and the Competence-stimulating Peptide

“…The ability to develop competence is critical for the virulence of this organism (1)(2)(3)(4) and has promoted the development of antibiotic resistance (5,6) that now restricts the options available for treating pneumococcal infections. A central component of this process (reviewed in Ref.…”