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In bacterial two-component regulatory systems ( TCSs), dephosphorylation of phosphorylated response regulators is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a protein complex with the sensor kinase SaeS and activate the sensor kinase's phosphatase activity. Efficient activation of the phosphatase activity required the presence of both SaeP and SaeQ. When SaeP and SaeQ were ectopically expressed, the expression of coagulase, a sae target with low affinity for phosphorylated SaeR, was greatly reduced, while the expression of alpha-haemolysin, a sae target with high affinity for phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by the SaeRS TCS, these results suggest that the SaeRS TCS returns to the pre-activation state by a negative feedback mechanism. [ABSTRACT FROM AUTHOR]

Macrophages are central players in immune response, manifesting divergent phenotypes to control inflammation and innate immunity through release of cytokines and other signaling factors. Recently, the focus on metabolism has been reemphasized as critical signaling and regulatory pathways of human pathophysiology, ranging from cancer to aging, often converge on metabolic responses. Here, we used genome-scale modeling and multi-omics (transcriptomics, proteomics, and metabolomics) analysis to assess metabolic features that are critical for macrophage activation. We constructed a genome-scale metabolic network for the RAW 264.7 cell line to determine metabolic modulators of activation. Metabolites well-known to be associated with immunoactivation (glucose and arginine) and immunosuppression (tryptophan and vitamin D3) were among the most critical effectors. Intracellular metabolic mechanisms were assessed, identifying a suppressive role for de-novo nucleotide synthesis. Finally, underlying metabolic mechanisms of macrophage activation are identified by analyzing multi-omic data obtained from LPS-stimulated RAW cells in the context of our flux-based predictions. Our study demonstrates metabolism’s role in regulating activation may be greater than previously anticipated and elucidates underlying connections between activation and metabolic effectors. [ABSTRACT FROM AUTHOR]

In this work we describe the identification of a copper-inducible regulon in Mycobacterium tuberculosis ( Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five-locus regulon, which includes a gene that encodes the copper-protective metallothionein MymT, was highly induced in wild-type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper-resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for egulated n opper epressor. Intriguingly, several of the RicR-regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper-responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host. [ABSTRACT FROM AUTHOR]

This study evaluated the validity of monitoring for Escherichia coli in lieu of Cryptosporidium to assess the vulnerability of source waters to the presence of Cryptosporidium oocysts. Over a period of seven years, four large water systems in Utah collected Cryptosporidium, E. coli, and turbidity data from seven water treatment plants that treat both reservoir and stream sources. The data represented analyses completed by two US Environmental Protection Agency-approved protozoan laboratories and local state-certified laboratories for E. coli. Results of the statistical analyses indicated poor correlation between Crypto sporidium and E. coli and between Cryptosporidium and turbidity in all monitored water sources throughout the entire monitoring period. The analyses indicate that elevated concentrations of E. coli are not indicative of the presence of Cryptosporidium in surface water. Both E. coli and turbidity are poor surrogates for occurrence of Cryptosporidium at treatment plant intakes. Further investigation is needed to establish appropriate surrogates for Cryptosporidium occurrence. [ABSTRACT FROM AUTHOR]

The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the ‘death effector’. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm. [ABSTRACT FROM AUTHOR]

The HspR protein functions as a negative regulator of chaperone and protease gene expression in a diversity of bacteria. Here we have identified, cloned and deleted theDeinococcus radioduransHspR homologue, DR0934.ΔhspRmutants exhibit moderate growth defects when shifted to mild heat shock temperatures, but are severely impaired for survival at 48°C. Using quantitative reverse transcription polymerase chain reaction and global transcriptional analysis, we have identified 14 genes that are derepressed in the absence of stress in theΔhspRbackground, 11 of which encode predicted chaperones and proteases, includingdnaKJgrpE,ftsH,lonB,hsp20andclpB. Promoter mapping indicated that the transcription of these genes initiates from a promoter bearing aσ70-type consensus, and that putative HspR binding sites (HAIR) were present in the 5′-untranslated regions. Electrophoretic mobility shift assays indicated that HspR binds to these promoters at the HAIR sitein vitro. These results strongly suggest that DR0934 encodes the HspR-like global negative regulator ofD. radioduransthat directly represses chaperone and protease gene expression by binding to the HAIR site in close proximity to promoter regions. [ABSTRACT FROM AUTHOR]

Most clinical isolates ofStreptococcus pneumoniaeconsist of heterogeneous populations of at least two colony phenotypes, opaque and transparent, selected for in the bloodstream and nasopharynx, respectively. Microarray analysis revealed 24orfsthat demonstrated differences in expression greater than twofold between variants of independent strains. Twenty-one of these showed increased expression in the transparent variants, including 11 predicted to be involved in sugar metabolism. A single genomic region contains seven of these loci including the gene that encodes the neuraminidase, NanA. In contrast to previous studies, there was no contribution of NanA to adherence ofS. pneumoniaeto epithelial cells or colonization in an animal model. However, we observed NanA-dependent desialylation of human airway components that bind to the organism and may mediate bacterial clearance. Targets of desialylation included human lactoferrin, secretory component, and IgA2 that were shown to be present on the surface of the pneumococcusin vivoduring pneumococcal pneumonia. The efficiency of desialylation was increased in the transparent variants and enhanced for host proteins binding to the surface ofS. pneumoniae. Because deglycosylation affects the function of many host proteins, NanA may contribute to a protease-independent mechanism to modify bound targets and facilitate enhanced survival of the bacterium. [ABSTRACT FROM AUTHOR]

Natural genetic transformation in Streptococcus pneumoniae is controlled in part by a quorum-sensing system mediated by a peptide pheromone called competence-stimulating peptide (CSP), which acts to coordinate transient activation of genes required for competence. To characterize the transcriptional response and regulatory events occurring when cells are exposed to competence pheromone, we constructed DNA microarrays and analysed the temporal expression profiles of 1817 among the 2129 unique predicted open reading frames present in the S. pneumoniae TIGR4 genome (84%). After CSP stimulation, responsive genes exhibited four temporally distinct expression profiles: early, late and delayed gene induction, and gene repression. At least eight early genes participate in competence regulation including comX , which encodes an alternative sigma factor. Late genes were dependent on ComX for CSP-induced expression, many playing important roles in transformation. Genes in the delayed class (third temporal wave) appear to be stress related. Genes repressed during the CSP response include ribosomal protein loci and other genes involved in protein synthesis. This study increased the number of identified CSP-responsive genes from approximately 40 to 188. Given the relatively large number of induced genes (6% of the genome), it was of interest to determine which genes provide functions essential to transformation. Many of the induced loci were subjected to gene disruption mutagenesis, allowing us to establish that among 124 CSP-inducible genes, 67 were individually dispensable for transformation, whereas 23 were required for transformation. [ABSTRACT FROM AUTHOR]

Provides information on a study which used a computational modeling approach to test one possible explanation for the limited capacity of the subitizing phenomenon. Experiments; Methods; Results and discussion; General discussion.