Many of the adaptive mechanisms that allow Bacillus subtilis to adjust to changes in nutrient availability are controlled by CodY. Binding of CodY to its target genes is stimulated by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon nutrient limitation, intracellular pools of these effectors are depleted and CodY can no longer repress genes required for adaptation. In vitro studies reported here explored in more detail the interaction of CodY with GTP. DNase I footprinting experiments indicated that CodY has an affinity for GTP in the millimolar range. Further, CodY was shown to interact specifically with GTP and dGTP; no other naturally occurring nucleotides that were tested, including ppGpp and pppGpp, resulted in DNA protection. Two nonhydrolyzable analogs of GTP were fully able to activate CodY binding to target DNA, demonstrating that GTP hydrolysis is not necessary for CodY-dependent regulation. GTP and the BCAAs were shown to act additively to increase the affinity of CodY for DNA; increased protection was observed in DNase I footprinting experiments when both effectors were present, compared to either effector alone, and in in vitro transcription reactions, transcriptional repression by CodY was stronger in the presence of both GTP and BCAAs than of BCAAs alone. Thus, interaction of CodY with GTP is specific and results in increased affinity for its target genes. This increase in affinity is independent of GTP hydrolysis and is augmented in the presence of BCAAs.The ability to adapt to changes in nutrient availability is central to bacterial colonization of diverse habitats. When nutrients are limiting, the gram-positive bacterium Bacillus subtilis employs several adaptive strategies. It may migrate to a more favorable environment; secrete enzymes to degrade macromolecules; induce the expression of transport systems for amino acids, peptides, DNA, and other potential nutrients; and activate intracellular catabolic pathways. In addition, the cell may synthesize antibiotics to eliminate nearby competitors. If these adaptive strategies fail to restore conditions suitable for growth, B. subtilis can differentiate to form a dormant spore.CodY, a highly conserved regulatory protein in gram-positive bacteria (8,20,23,29,43,44), is one of the factors that control the expression of these adaptive mechanisms. CodY was initially identified in B. subtilis as a regulator of the dipeptide permease (dpp) operon (25, 34, 38-40), but we now know that the B. subtilis CodY regulon is large, encompassing nearly 200 genes (1,7,10,16,27,46). Many of these genes encode proteins that mediate the adaptive responses cited above. In addition, CodY has been proved to repress branched-chain amino acid biosynthesis (36) and activate the expression of the acetate kinase gene (35). CodY also participates in repression of the initiation of sporulation; unlike wild-type cells, those of B. subtilis codY mutant strains sporulate readily in a nutrientrich medium (12,15,31).CodY exists primarily as a dimer...

Background Group B streptococcus (GBS) causes serious diseases in newborn infants, often resulting in lifelong neurologic impairments or death. Prophylactic vaccination of pregnant women prior to delivery could provide comprehensive protection, as early onset and late-onset disease and maternal complications potentially could be addressed. Methods Capsular polysaccharide conjugate vaccine GBS6 was designed using surveillance data yielded by whole-genome sequencing of a global collection of recently recovered GBS isolates responsible for invasive neonatal GBS disease. Capsular polysaccharides were isolated, oxidized using sodium periodate, and conjugated to CRM 197 by reductive amination in dimethyl sulfoxide. Immune responses in mice and rhesus macaques were measured in a multiplex Luminex immunoglobulin G (IgG) assay and opsonophagocytic activity assays. Results The optimized conjugates were immunogenic, alone and in combination, in mice and rhesus macaques, inducing IgG antibodies that mediated opsonophagocytic killing. Active immunization of murine dams with GBS6 prior to mating resulted in serotype-specific protection of pups from a lethal challenge with GBS. Protection following passive administration of serotype-specific IgG monoclonal antibodies to dams demonstrated conclusively that anticapsular polysaccharide IgG alone is sufficient for protection. Conclusions The findings support the ongoing clinical evaluation of maternal GBS6 vaccination as a potential alternative method to prevent GBS disease in infants.

The production of polysaccharide intercellular adhesin (PIA) is an essential process in foreign body infections mediated by Staphylococcus epidermidis. Transcriptional regulation of the icaADBC operon, the genes responsible for production of enzymes that synthesize PIA, is multi-factorial and involves at least SarA and sigmaB. Transcriptional and promoter fusion studies revealed that the decreased transcription of the icaADBC operon observed in a S. epidermidis 1457 sigB mutant is not mediated through a direct interaction of sigmaB-RNA polymerase at the icaADBC promoter region but instead through the upregulation of IcaR, a known repressor of icaADBC transcription. Transcriptional analysis of a 1457 sigB-icaR double mutant confirmed that the decreased icaADBC transcript in 1457 sigB is IcaR dependent. Furthermore, primer extension studies suggest that the icaR promoter appears to be sigmaA dependent, suggesting that sigmaB indirectly controls icaR transcription through an unknown pathway. In addition, it was confirmed that the loss of SarA results in the loss of icaADBC transcription and PIA production in S. epidermidis. It was further demonstrated, through the over-production of SarA in 1457 sigB, that the loss of sarP1 promoter activity in 1457 sigB has little or no effect on the loss of PIA production in this mutant. Finally, it was demonstrated that PIA production could be restored in both 1457 sigB and 1457 sarA by complementing these mutants with a full-length icaADBC operon controlled by a cadmium-inducible noncognate promoter. It is concluded that sigmaB and SarA operate independently of each other to regulate PIA production and biofilm development in S. epidermidis.

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branchedchain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

Production of biofilm in Staphylococcus epidermidis is mediated through enzymes produced by the four-gene operon ica and is subject to phenotypic variation. The purpose of these experiments was to investigate the regulation of ica and icaR transcription in phenotypic variants produced by multiple unrelated isolates of S. epidermidis. Ten isolates were chosen for the study, four of which contained IS256. IS256 mediates a reversible inactivation of ica in approximately 30 % of phenotypic variants. All ten strains produced at least two types of phenotypic variant (intermediate and smooth) in which biofilm formation was significantly impaired. Reversion studies indicated that all phenotypic variants were stable after overnight growth, but began to revert to other phenotypic forms after 5 days of incubation at 37 8C. ica transcriptional analysis was performed on phenotypic variants from three IS256-negative isolates; 1457, SE5 and 14765. This analysis demonstrated that ica transcription was significantly reduced in the majority of phenotypic variants, although two variants from SE5 and 1457 produced wild-type quantities of ica transcript. Analysis of seven additional phenotypic variants from SE5 revealed that ica expression was only reduced in three. Expression of icaR transcript was unaffected in all smooth phenotypic variants. Mutations within ica were identified in two SE5 variants with wild-type levels of ica transcription. It is concluded that mutation and transcriptional regulation of ica are the primary mechanisms that govern phenotypic variation of biofilm formation within IS256-negative S. epidermidis.