Sppl Forms a Membrane Protein Complex with SppA and Inhibits Its Protease Activity in Bacillus subtilis

The membrane protease SppA of Bacillus subtilis was first described as a signal peptide peptidase and later shown to confer resistance to lantibiotics. Here, we report that SppA forms octameric complexes with YteJ, a membrane protein of thus-far-unknown function. Interestingly, sppA and yid deletion mutants exhibited no protein secretion defects. However, these mutant strains differed significantly in their resistance to antimicrobial peptides. In particular, sppA mutant cells displayed increased sensitivity to the lantibiotics nisin and subtilin and the human lysozyme-derived cationic antimicrobial peptide LP9. Importantly, YteJ was shown to antagonize SppA activity both in vivo and in vitro, and this SppA-inhibitory activity involved the C-terminal domain of YteJ, which was therefore renamed Sppl. Most likely, Sppl-mediated control is needed to protect B. subtilis against the potentially detrimental protease activity of SppA since a mutant overexpressing sppA by itself displayed defects in cell division. Altogether, we conclude that the SppA-Sppl complex of B. subtills has a major role in protection against antimicrobial peptides. IMPORTANCE Our study presents new insights into the molecular mechanism that regulates the activity of SppA, a widely conserved bacterial membrane protease. We show that the membrane proteins SppA and Sppl form a complex in the Gram-positive model bacterium B. subtilis and that Sppl inhibits SppA protease activity in vitro and in vivo. Furthermore, we demonstrate that the C-terminal domain of Sppl is involved in SppA inhibition. Since SppA, through its protease activity, contributes directly to resistance to lantibiotic peptides and cationic antibacterial peptides, we propose that the conserved SppA-Sppl complex could play a major role in the evasion of bactericidal peptides, including those produced as part of human innate immune defenses

“Interactive Technology Assessment” and Beyond: the Field Trial of Genetically Modified Grapevines at INRA-Colmar

International audienc

Blue and Red Light Modulates SigB-Dependent Gene Transcription, Swimming Motility and Invasiveness in Listeria monocytogenes

Background: In a number of gram-positive bacteria, including Listeria, the general stress response is regulated by the alternative sigma factor B (SigB). Common stressors which lead to the activation of SigB and the SigB-dependent regulon are high osmolarity, acid and several more. Recently is has been shown that also blue and red light activates SigB in Bacillus subtilis. Methodology/Principal Findings: By qRT-PCR we analyzed the transcriptional response of the pathogen L. monocytogenes to blue and red light in wild type bacteria and in isogenic deletion mutants for the putative blue-light receptor Lmo0799 and the stress sigma factor SigB. It was found that both blue (455 nm) and red (625 nm) light induced the transcription of sigB and SigB-dependent genes, this induction was completely abolished in the SigB mutant. The blue-light effect was largely dependent on Lmo0799, proving that this protein is a genuine blue-light receptor. The deletion of lmo0799 enhanced the red-light effect, the underlying mechanism as well as that of SigB activation by red light remains unknown. Blue light led to an increased transcription of the internalin A/B genes and of bacterial invasiveness for Caco-2 enterocytes. Exposure to blue light also strongly inhibited swimming motility of the bacteria in a Lmo0799- and SigB-dependent manner, red light had no effect there. Conclusions/Significance: Our data established that visible, in particular blue light is an important environmental signal with an impact on gene expression and physiology of the non-phototrophic bacterium L. monocytogenes. In natural environments these effects will result in sometimes random but potentially also cyclic fluctuations of gene activity, depending on the light conditions prevailing in the respective habitat

Interval-Based approach for uncertainty quantification of Energy Consumption modeling in Digital Twin

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Phosphorylation and RsbX-Dependent Dephosphorylation of RsbR in the RsbR-RsbS Complex of Bacillus subtilis

In the pathway that controls σ(B) activity, the RsbR-RsbS complex plays an important role by trapping RsbT, a positive regulator of σ(B) of Bacillus subtilis. We have proposed that at the onset of stress, RsbR becomes phosphorylated, resulting in an enhanced activity of RsbT towards RsbS. RsbT is then free to interact with and activate RsbU, which in turn ultimately activates σ(B). In this study with purified proteins, we used mutant RsbR proteins to analyze the role of its phosphorylatable threonine residues. The results show that the phosphorylation of either of the two RsbT-phosphorylatable threonine residues (T171 and T205) in RsbR enhanced the kinase activity of RsbT towards RsbS. However, it appeared that RsbT preferentially phosphorylates T171. We also present in vitro evidence that identifies RsbX as a potential phosphatase for RsbR T205

The dynamic protein partnership of RNA polymerase in <em>Bacillus subtilis</em>

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Binding of σ(A) and σ(B) to Core RNA Polymerase after Environmental Stress in Bacillus subtilis

In Bacillus subtilis, the alternative sigma factor σ(B) is activated in response to environmental stress or energy depletion. The general stress regulon under the control of σ(B) provides the cell with multiple stress resistance. Experiments were designed to determine how activated σ(B) replaces σ(A) as a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the σ(A)-dependent stress gene clpE under σ(B)-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type. The relative affinities of σ(A) and σ(B) for binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of σ(B) for E was 60-fold lower than that of σ(A). Western blot analyses with antibodies against σ(A), σ(B), and E showed that, after exposure to ethanol stress, the concentration of σ(B) was only twofold higher than those of σ(A) and E. Thus, the concentration of σ(B) after stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of σ(B) to E after stress

MOESM2 of Revisiting the in vivo GlnR-binding sites at the genome scale in Bacillus subtilis

Additional file 2: Table S1. Mapping of GlnR DNA binding sites by ChIP-on-chip. ChIP-on-chip experiments were performed and data were analysed as previously described [32] using the method described by Reppas et al. [34]. GlnR was purified in two biological replicates for each condition of growth. This table lists all significantly enriched DNA regions by ChIP-on-chip experiment performed with a GlnRSPA expressing Bacillus subtilis strain