Membrane Composition Changes and Physiological Adaptation byStreptococcus mutansSignal Recognition Particle Pathway Mutants

Abstract

Previously, we presented evidence that the oral cariogenic species Streptococcus mutans remains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (⌬ffh, ⌬scRNA, and ⌬ftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription-and translation-related proteins, the ␤ subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression in S. mutans UA159 (parent strain) and AH333 (⌬ffh mutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant.

The 4.5S RNA component of the signal recognition particle is required for group A Streptococcus virulence

“…In the absence of stress, growth yield was similar between the parental and SRP mutant S. mutans strains, although the doubling time increased. Consistent with the function of the SRP pathway, the concentration of membraneassociated proteins differed between parental and SRP mutant strains (Hasona et al, 2007). The abundance of 116 gene transcripts also differed between parental and ffh mutant strains, highlighting the extensive transcriptional remodelling required to overcome disruption of the SRP pathway (Hasona et al, 2007).…”

Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation

“…All primers designed for qRT-PCR analysis are listed in Supplementary Table S1. The expression levels of target genes were normalized using the gyrA gene of S. mutans as an internal standard, since it produced little variation of expression (Ajdic & Pham, 2007;Hasona et al, 2007). A standard curve was plotted with cycle threshold (C T ) values obtained from amplification of known quantities of cDNAs and also used to determine the efficiency of amplification.…”

“…The aciduricity of this organism has been attributed largely to the proton-extruding F 1 F 0 -ATPase that functions well at pH 5.0 and below, allowing the organism to maintain adequate DpH when the external pH falls to 4.0 and lower (Bender et al, 1986;Cotter & Hill, 2003). Other mechanisms of acid resistance in S. mutans include induction of stress proteins (Hamilton & Svensäter, 1998;Len et al, 2004;Svensäter et al, 1997;Wilkins et al, 2002), changes in membrane-associated proteins and fatty acid composition (Fozo et al, 2007;Hasona et al, 2007), DNA repair enzymes (Hahn et al, 1999;Hanna et al, 2001) and increase in alkali production through several metabolic pathways (Burne et al, 1999;Griswold et al, 2006). Moreover, the ComCDE quorum-sensing system has been found to play a role in cell-density-dependent acid tolerance by S. mutans (Li et al, 2001b).…”

YidC1 and YidC2 are functionally distinct proteins involved in protein secretion, biofilm formation and cariogenicity of Streptococcus mutans

“…Formation of wild-type and YidC mutant strain biofilms was assessed as described previously (Hasona et al, 2007). Statistical significance relative to the wild-type value at each time point and growth condition was determined using Student's t-test.…”