Quorum-sensing regulators in Gram-positive bacteria: 'cherchez le peptide'.

Gram-positive bacteria can regulate gene expression at the population level via a mechanism known as quorum sensing. Oligopeptides serve as the signaling molecules; they are secreted and then are either detected at the bacterial surface by two-component systems or reinternalized via an oligopeptide transport system. In the latter case, imported peptides interact with cognate regulators (phosphatases or transcriptional regulators) that modulate the expression of target genes. These regulators help control crucial functions such as virulence, persistence, conjugation and competence and have been reported in bacilli, enterococci and streptococci. They form the rapidly growing RRNPP group. In this issue of Molecular Microbiology, Hoover et al. (2015) highlight the group's importance: they have identified a new family of regulators, Tprs (Transcription factor regulated by a Phr peptide), which work with internalized Phr-like peptides. The mechanisms underlying the expression of the genes that encode these internalized peptides are poorly documented. However, Hoover et al. (2015) have provided a new insight: an environmental molecule, glucose, can inhibit expression of the Phr-like peptide gene via catabolic repression. This previously undescribed regulatory pathway, controlling the production of a bacteriocin, might influence Streptococcus pneumonia's fitness in the nasopharynx, where galactose is present.

Rgg proteins associated with internalized small hydrophobic peptides: a new quorum-sensing mechanism in streptococci.

We identified a genetic context encoding a transcriptional regulator of the Rgg family and a small hydrophobic peptide (SHP) in nearly all streptococci and suggested that it may be involved in a new quorum-sensing mechanism, with SHP playing the role of a pheromone. Here, we provide further support for this hypothesis by constructing a phylogenetic tree of the Rgg and Rgg-like proteins from Gram-positive bacteria and by studying the shp/rgg1358 locus of Streptococcus thermophilus LMD-9. We identified the shp1358 gene as a target of Rgg1358, and used it to confirm the existence of the steps of a quorum-sensing mechanism including secretion, maturation and reimportation of the pheromone into the cell. We used surface plasmon resonance to demonstrate interaction between the pheromone and the regulatory protein and performed electrophoretic mobility shift assays to assess binding of the transcriptional regulator to the promoter regions of its target genes. The active form of the pheromone was identified by mass spectrometry. Our findings demonstrate that the shp/rgg1358 locus encodes two components of a novel quorum-sensing mechanism involving a transcriptional regulator of the Rgg family and a SHP pheromone that is detected and reimported into the cell by the Ami oligopeptide transporter.

Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Expression of a new operon from Bacillus subtilis, ykzB-ykoL, under the control of the TnrA and PhoP-phoR global regulators.

The ykzB and ykoL genes encode two peptides, of 51 and 60 amino acids, the functions of which are unknown. The ykzB and tnrA genes are contiguous and transcribed divergently. Expression of ykzB and ykoL is induced by glutamate and is under the control of the TnrA global regulator of nitrogen utilization. TnrA regulated its own synthesis in glutamate minimal medium. Two DNA sequences (TnrAB1 and TnrAB2) homologous to the TnrA binding site are present in the region between tnrA and ykzB. Deletion mapping indicated that the TnrAB2 binding site was involved in activation of the ykzB promoter. In addition, transcription of tnrA depends on the presence of the TnrAB1 binding site. The ykzB and ykoL genes are probably in the same transcriptional unit. A single promoter involved in transcription in the presence of glutamate was mapped by primer extension. ykoL expression was induced by phosphate limitation and depended on the PhoP-PhoR two-component regulatory system. Its promoter was mapped to the region between ykoL and ykzB. Four boxes similar to the PhoP binding site are present upstream from the ykoL promoter. These boxes are probably recognized by PhoP approximately P during the activation of transcription in phosphate limitation conditions.

Role of bkdR, a transcriptional activator of the sigL-dependent isoleucine and valine degradation pathway in Bacillus subtilis.

A new gene, bkdR (formerly called yqiR), encoding a regulator with a central (catalytic) domain was found in Bacillus subtilis. This gene controls the utilization of isoleucine and valine as sole nitrogen sources. Seven genes, previously called yqiS, yqiT, yqiU, yqiV, bfmBAA, bfmBAB, and bfmBB and now referred to as ptb, bcd, buk, lpd, bkdA1, bkdA2, and bkdB, are located downstream from the bkdR gene in B. subtilis. The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1alpha (dehydrogenase), E1beta (decarboxylase), and E2 (dihydrolipoamide acyltransferase). Isoleucine and valine utilization was abolished in bcd and bkdR null mutants of B. subtilis. The seven genes appear to be organized as an operon, bkd, transcribed from a -12, -24 promoter. The expression of the bkd operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family. Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene bkdR. Deletion analysis showed that upstream activating sequences are involved in the expression of the bkd operon and are probably the target of bkdR. Transcription of the bkd operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions.

Role of the transcriptional activator RocR in the arginine-degradation pathway of Bacillus subtilis.

In Bacillus subtilis, genes involved in arginine and ornithine catabolism constitute two operons, rocABC and rocDEF. Inducible expression of these two operons is SigL-dependent and requires the transcriptional activator RocR. RocR is a member of the NtrC/NifA family of regulators. To study the molecular mechanisms leading to the activation of RocR, we constructed a series of mutants affected in various steps of arginine catabolism. Results obtained using these mutants strongly suggest that the true inducer is ornithine or citrulline. Constitutive mutants of rocR containing either missense mutations, frameshift mutations resulting from deletions, or in-frame deletions leading to the synthesis of N-terminal truncated RocR polypeptides were obtained. Analysis of these mutants indicates that the N-terminal part of RocR is an intramolecular repressor domain. AhrC is a second positive regulatory protein of the rocABC and rocDEF operons. Two missense mutations modifying the N-terminal domain of RocR led to high constitutive expression of the Roc regulon in the absence of AhrC. Constitutive RocR proteins still require the presence of UAS1 and therefore probably bending of the DNA region located between the UAS1 and the promoter, suggesting that AhrC is not involved in DNA bending which facilitates interaction between RocR and sigma54-RNA polymerase. We suggest that the positive role of AhrC involves protein-protein interaction with RocR.

Expression of the rocDEF operon involved in arginine catabolism in Bacillus subtilis.

Three genes called rocD, rocE and rocF were found near the rocR gene in B. subtilis. The product of rocD is similar to eukaryotic ornithine aminotransferases. The product of rocE shares similarity with the product of B. subtilis rocC and with the product of E. coli lysP. The rocE gene may encode an arginine permease. The rocF gene encodes a polypeptide similar to several arginases. Heterologous expression in E. coli indicated that rocD encodes an ornithine aminotransferase and that rocF encodes an arginase. Arginine utilization was abolished in both rocD and rocF mutants of B. subtilis confirming the role of these genes in arginine catabolism. The rocDEF genes form an operon transcribed from a -12, -24 promoter almost identical to the -12, -24 promoter of the rocABC operon. The expression of the rocDEF operon was induced by the presence of arginine, ornithine or proline in the growth medium and depended on the presence of the sigma factor SigL. Transcription of this operon was also abolished in a B. subtilis strain containing a null mutation in the regulatory gene rocR. Two tandemly repeated upstream activating sequences very similar to those previously identified in the rocABC system were found centered at positions -120 and -70, respectively, upstream from the transcription start site of rocDEF. Deletion analysis showed that at least one upstream activating sequence is involved in the expression of the rocDEF operon. These sequences are probably the target of RocR. Analysis of a rocR'-'lacZ fusion strain showed that the expression of rocR is not induced by arginine and is negatively autoregulated.

RocR, a novel regulatory protein controlling arginine utilization in Bacillus subtilis, belongs to the NtrC/NifA family of transcriptional activators.

Bacillus subtilis can use ammonium and various amino acids as sole nitrogen sources. The utilization of arginine or ornithine is abolished in a sigma L-deficient strain of B. subtilis, indicating that one or several genes involved in this pathway are transcribed by a sigma L-RNA polymerase holoenzyme. Three B. subtilis genes, called rocA, rocB, and rocC, which seem to form an operon, were found near the sacTPA locus (P. Glaser, F. Kunst, M. Arnaud, M.-P. Coudart, W. Gonzales, M.-F. Hullo, M. Ionescu, B. Lubochinsky, L. Marcelino, I. Moszer, E. Presecan, M. Santana, E. Schneider, J. Schweizer, A. Vertes, G. Rapport, and A. Danchin, Mol. Microbiol. 10:371-384, 1993). The expression of this putative operon is induced by arginine and is sigma L dependent. Mutants impaired in the transcription of rocA were obtained. One of these mutants was used as recipient to clone and sequence a new regulatory gene, called rocR. This gene encodes a polypeptide of 52 kDa which belongs to the NtrC/NifA family of transcriptional activators. Upstream activating sequences highly similar to those of NtrC in Escherichia coli were also identified upstream from the rocABC genes. A B. subtilis strain containing a rocR null mutation is unable to use arginine as the sole nitrogen source, indicating that RocR is a positive regulator of arginine catabolism. After LevR, RocR is the second example of an activator stimulating sigma 54-dependent promoters in gram-positive bacteria.