Modulation of the ComA-dependent quorum response in Bacillus subtilis by multiple Rap proteins and Phr peptides

In Bacillus subtilis, extracellular peptide signaling regulates several biological processes. Secreted Phr signaling peptides are imported into the cell and act intracellularly to antagonize the activity of regulators known as Rap proteins. B. subtilis encodes several Rap proteins and Phr peptides, and the processes regulated by many of these Rap proteins and Phr peptides are unknown. We used DNA microarrays to characterize the roles that several rap-phr signaling modules play in regulating gene expression. We found that rapK-phrK regulates the expression of a number of genes activated by the response regulator ComA. ComA activates expression of genes involved in competence development and the production of several secreted products. Two Phr peptides, PhrC and PhrF, were previously known to stimulate the activity of ComA. We assayed the roles that PhrC, PhrF, and PhrK play in regulating gene expression and found that these three peptides stimulate ComA-dependent gene expression to different levels and are all required for full expression of genes activated by ComA. The involvement of multiple Rap proteins and Phr peptides allows multiple physiological cues to be integrated into a regulatory network that modulates the timing and magnitude of the ComA response.     

Molecular analysis of Phr peptide processing in Bacillus subtilis

In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F approximately P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.     

The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains

Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.     

Identification of subtilisin, Epr and Vpr as enzymes that produce CSF, an extracellular signalling peptide of Bacillus subtilis

Cell-cell communication regulates many important processes in bacteria. Gram-positive bacteria use peptide signals for communication, such as the Phr pentapeptides of Bacillus subtilis. The Phr pentapeptides are secreted with a pro domain that is cleaved to produce an active signalling peptide. To identify the protease(s) involved in production of the mature Phr signalling peptides, we developed assays for detecting cleavage of one of the B. subtilis Phr pentapeptides, CSF, from the proCSF precursor. Using both a cellular and a mass spectrometric approach, we determined that a sigma-H-regulated, secreted, serine protease(s) cleaved proCSF to CSF. Mutants lacking the three proteases that fit these criteria, subtilisin, Epr and Vpr, had a defect in CSF production. Purified subtilisin and Vpr were shown to be capable of processing proCSF as well as at least one other Phr peptide produced by B. subtilis, PhrA, but they were not able to process the PhrE signalling peptide of B. subtilis, indicating that there are probably other unidentified proteases involved in Phr peptide production. Subtilisin, Epr and Vpr are members of the subtilisin family of proteases that are widespread in bacteria, suggesting that many bacterial species may be capable of producing Phr signalling peptides.     

The intracellular function of extracellular signaling peptides

A novel class of extracellular signaling peptides has been identified in Gram-positive bacteria that are actively transported into the cell to interact with intracellular receptors. The defining members of this novel class of signaling peptides are the Phr peptides of Bacillus subtilis and the mating pheromones of Enterococcus faecalis. These peptides are small and unmodified, gene encoded, and secreted by the bacterium. Most of these peptides diffuse into the extracellular medium, and when their concentration is sufficiently high, they are then actively transported into the cell by an oligopeptide permease (Opp). Once inside the cell, these peptides interact with an array of intracellular receptors. In B. subtilis, the Phr peptides regulate development of environmentally resistant spores and genetically competent cells (i.e. the natural ability to take up exogenous DNA). In E. faecalis, the mating pheromones regulate cell-cell transfer of plasmids, many of which encode antibiotic resistance or virulence factors. At least one component of the signaling pathway for these peptides is conserved in many bacteria, Opp. Opp is a non-specific transporter that transports peptides for use as carbon and nitrogen sources. The possibility that other bacteria could possess similar intracellularly functioning signaling peptides is discussed.     

Cross-talk between the general stress response and sporulation initiation in Bacillus subtilis - the σ(B) promoter of spo0E represents an AND-gate

The general stress response and the decision-making processes of sporulation initiation are interconnected pathways in the regulatory network of Bacillus subtilis. In a previous study we provided evidence for a mechanism capable of impairing sporulation by σ(B) -dependent induction of spo0E, encoding a phosphatase specifically inactivating the sporulation master regulator Spo0A～P. Here we show that the σ(B) promoter (Pσ(B) ) of spo0E is responsive to sub-inhibitory levels of ethanol stress, producing a σ(B) -dependent sporulation deficient phenotype. In addition to positive regulation by σ(B) , we identified Rok, the repressor of comK, to be a direct repressor of spo0E expression from Pσ(B) . This constellation provides the possibility to integrate signals negatively acting on sporulation initiation through the σ(B) branch as well as a positive feedback loop acting on Pσ(B) by Rok that is most likely a direct consequence of Spo0A～P activity. Thus, the molecular mechanism described here offers the opportunity for cross-talk between the general stress response and sporulation initiation in the adaptational gene expression network of B.?subtilis.     

Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions.

The system previously used to study recombination of nonreplicating UV-irradiated phage lambda DNA was adapted to study UV repair. Irradiated phages infected undamaged homoimmune lysogens. Pyrimidine dimer content (by treatment with Micrococcus luteus UV endonuclease and alkaline sucrose sedimentation) and a biological activity endpoint (infectivity in transfection of uvrB recA recB spheroplasts) were followed. Unless room light was excluded during DNA extraction procedures, photoreactivation (Phr function) was significant. In uvr delta phr bacteria, repair, by both assays, was very low but not zero. Even when light was totally excluded, Phr function appeared to play a role in Uvr-mediated excision repair: both dimer removal and restoration of infectivity were two to five times as efficient in uvr+ phr+ bacteria as in uvr+ delta phr bacteria. Similarly, UV-irradiated phages plated with higher efficiencies on phr+ than delta phr bacteria even under totally dark conditions. In uvr phr+ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr+ infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system.     

PHR1, a PH domain-containing protein expressed in primary sensory neurons

Previously, we identified PHR1 as an abundantly expressed gene in photoreceptors and showed that it encodes four isoforms, each with N-terminal pleckstrin homology (PH) and C-terminal transmembrane domains. To better understand PHR1 function and expression, we made a Phr1 null mouse by inserting a beta-galactosidase/neor cassette into exon 3. In addition to photoreceptors, we found abundant expression of specific Phr1 splice forms in olfactory receptor neurons and vestibular and cochlear hair cells. We also found Phr1 expression in cells with a possible sensory function, including peripheral retinal ganglion cells, cochlear interdental cells, and neurons of the circumventricular organ. Despite this discrete expression in known and putative sensory neurons, mice lacking PHR1 do not have overt sensory deficits.     

A negative feedback loop that limits the ectopic activation of a cell type-specific sporulation sigma factor of Bacillus subtilis

Two highly similar RNA polymerase sigma subunits, σ(F) and σ(G), govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σ(F) drives synthesis of σ(G) but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σ(G) while discriminating between σ(F) and σ(G) in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σ(G) (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σ(G) in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σ(G). CsfB is normally produced in the forespore, under σ(F) control, but sigGN45E mutant cells also expressed csfB and did so in a σ(G)-dependent manner, autonomously from σ(F). Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σ(G) activity. N45 is invariant in the homologous position of σ(G) orthologues, whereas its functional equivalent in σ(F) proteins, E39, is highly conserved. While CsfB does not bind to wild-type σ(F), a E39N substitution in σ(F) resulted in efficient binding of CsfB to σ(F). Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σ(F) in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis.     

A new quorum‐sensing system (TprA/PhrA) for Streptococcus pneumoniae D39 that regulates a lantibiotic biosynthesis gene cluster

The Phr peptides of the Bacillus species mediate quorum sensing, but their identification and function in other species of bacteria have not been determined. We have identified a Phr peptide quorum‐sensing system (TprA/PhrA) that controls the expression of a lantibiotic gene cluster in the Gram‐positive human pathogen, Streptococcus pneumoniae. Lantibiotics are highly modified peptides that are part of the bacteriocin family of antimicrobial peptides. We have characterized the basic mechanism for a Phr‐peptide signaling system in S. pneumoniae and found that it induces the expression of the lantibiotic genes when pneumococcal cells are at high density in the presence of galactose, a main sugar of the human nasopharynx, a highly competitive microbial environment. Activity of the Phr peptide system is not seen when pneumococcal cells are grown with glucose, the preferred carbon source and the most prevalent sugar encountered by S. pneumoniae during invasive disease. Thus, the lantibiotic genes are expressed under the control of both cell density signals via the Phr peptide system and nutritional signals from the carbon source present, suggesting that quorum sensing and the lantibiotic machinery may help pneumococcal cells compete for space and resources during colonization of the nasopharynx.