DegS-DegU Two-component System Research Articles

Enhanced surface colonisation and competition during bacterial adaptation to a fungus.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.

Viscous drag on the flagellum activates Bacillus subtilis entry into the K-state.

Bacillus subtilis flagella are not only required for locomotion but also act as sensors that monitor environmental changes. Although how the signal transmission takes place is poorly understood, it has been shown that flagella play an important role in surface sensing by transmitting a mechanical signal to control the DegS-DegU two-component system. Here we report a role for flagella in the regulation of the K-state, which enables transformability and antibiotic tolerance (persistence). Mutations impairing flagellar synthesis are inferred to increase DegU-P, which inhibits the expression of ComK, the master regulator for the K-state, and reduces transformability. Tellingly, both deletion of the flagellin gene and straight filament (hagA233V ) mutations increased DegU phosphorylation despite the fact that both mutants had wild type numbers of basal bodies and the flagellar motors were functional. We propose that higher viscous loads on flagellar motors result in lower DegU-P levels through an unknown signaling mechanism. This flagellar-load based mechanism ensures that cells in the motile subpopulation have a tenfold enhanced likelihood of entering the K-state and taking up DNA from the environment. Further, our results suggest that the developmental states of motility and competence are related and most commonly occur in the same epigenetic cell type.

An alternate route to phosphorylating DegU of Bacillus subtilis using acetyl phosphate.

BackgroundTwo-component signal transduction pathways allow bacteria to sense and respond to the environment. Typically such pathways comprise a sensor histidine kinase and a response regulator. Phosphorylation of the response regulator commonly results in its activation, allowing the protein to bind to target promoter elements to regulate transcription. Several mechanisms are used to prevent inappropriate phosphorylation of the response regulator, thereby ensuring a specific response. In Bacillus subtilis, the DegS-DegU two-component system controls transcription of target genes in a manner dependent on the level of the phosphorylated response regulator, DegU. Previous work has tentatively indicated that DegU, and DegU H12L, a DegU variant which displays enhanced stability of the phosphoryl moiety, can be phosphorylated in the absence of the kinase, DegS.ResultsThe data presented here reveal that DegU H12L requires aspartic acid 56 (D56), the identified DegU phosphorylation site, for its activity. By indirectly measuring the level of DegU ~ P in the cell by assessment of several well recognised DegU regulated processes it was shown that DegU H12L retains its activity in the absence of DegS, and that mutation of D56 produced an inactive protein. Further experiments designed to raise the level of acetyl phosphate within the cell suggest that DegU can be phosphorylated by acetyl phosphate in the absence of degS. Additionally, the phenotypic and biochemical experiments presented indicate that DegU H12L can reliably mimic high levels of phosphorylated DegU.ConclusionsThe ability of acetyl phosphate to modify DegU, and indeed DegU H12L, reveal an additional layer of regulation for DegU phosphorylation that will be relevant when the level of DegS is low or in the absence of degS. Given the number of processes that DegU can activate or inhibit, extensive regulation at a number of levels is required to ensure that the system is not inappropriately stimulated. DegS has both kinase and phosphatase activity and our findings demonstrate that the phosphatase activity of DegS is essential to control the level of DegU phosphate. Overall we contribute to our understanding of how the intricate signalling pathway DegS-DegU is regulated in B. subtilis.

Phosphorylation of DegU is essential for activation of amyE expression in Bacillus subtilis.

Alpha-amylase (amyE) is one of the major exo-enzymes secreted by Bacillus subtilis during the post-exponential phase. The DegS-DegU two-component system regulates expression of majority of post-exponentially expressed genes in B. subtilis. It has been demonstrated that varying levels of the phosphorylated form of DegU (DegU-P) control different cellular processes. Exo-protease production is observed when effective concentration of DegU-P rises in the cell, whereas swarming motility is favoured at very low amounts of DegU-P. In this study we show that like other exo-proteases, expression of amyE is positively regulated by increase in DegU-P levels in the cell. We also demonstrate that residues at the DNA-binding helix-turn-helix (HTH) motif of DegU are necessary for the amyE expression. This observation is further reinforced by demonstrating the direct interaction of DegU on amyE promoter.

Mutations Suppressing the Loss of DegQ Function in Bacillus subtilis (natto) Poly-γ-Glutamate Synthesis

The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.

Mutational Analysis of the Helix-Turn-Helix Region of Bacillus subtilis Response Regulator DegU, and Identification of cis-Acting Sequences for DegU in the aprE and comK Promoters

The DegS-DegU two-component system in Bacillus subtilis regulates exoprotease production and competence development. Phosphorylated and unphosphorylated forms of DegU are required for activation of aprE and comK, respectively. Alanine-scanning mutagenesis of the helix-turn-helix region of DegU and in vivo examination of 27 DegU variants revealed five common mutants that showed severe reduction of gene expression of both aprE and comK because of reduced DNA-binding activity. This observation suggested that the DegU-recognized cis-sequences might not be considerably changed for either promoter. We identified a DegU-recognized inverted repeat in the comK promoter using various mutant comK-lacZ fusions. Inspection of the aprE promoter sequence revealed a tandem repeat consisting of short AT-rich sequences containing a consensus one, 5'-TAAAT-3', which was found in the downstream half of the inverted repeat involved in comK activation. Oligonucleotide-directed replacement of the short AT-rich sequences located in the center of each motif decreased DegU-dependent aprE expression, implying that the repeat is required for the activation of aprE. Based on these results, it was concluded that DegU would function through the inverted repeat in the comK promoter and the tandem repeat in the aprE promoter.

DegU-P represses expression of the motility fla-che operon in Bacillus subtilis.

Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the sigmaA-dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the PA promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.

Binding of response regulator DegU to the aprE promoter is inhibited by RapG, which is counteracted by extracellular PhrG in Bacillus subtilis

We screened the putative rap-phr (response regulator aspartyl-phosphate phosphatase-phosphatase regulator) systems identified in the Bacillus subtilis genome for a rap gene that affects aprE (alkaline protease gene) expression by using a multicopy plasmid. We found that rapG was involved in the regulation of aprE, which belongs to the regulon of DegU, the response regulator of the DegS-DegU two-component system. Disruption of rapG and phrG resulted in enhancement and reduction of aprE-lacZ expression, respectively, suggesting that PhrG inhibits RapG activity. Addition of 1-30 nM of a synthetic pentapeptide (PhrG; NH2-EKMIG-COOH) to the phrG disruptant completely rescued aprE-lacZ expression, indicating that the PhrG peptide is indeed involved in aprE-lacZ expression. Surprisingly, either introduction of multicopy phrG or addition of the PhrG peptide at high concentrations (100-300 nM) to the phrG cells decreased aprE-lacZ expression. These results are reminiscent of the previous observation that at higher concentrations the PhrC peptide inhibits srfA-lacZ expression directed by ComA, the regulator of the ComP-ComA two-component system. Because the Rap proteins belong to a family of aspartyl protein phosphatases, we tried to investigate the possible influence of RapG on dephosphorylation of DegU-P (phosphorylated DegU) in vitro. RapG, however, did not affect dephosphorylation of DegU-P under the adopted experimental conditions. Therefore, we hypothesized that RapG might inhibit the binding activity of DegU to the target promoters. We analysed the interaction of DegU and RapG using the aprE promoter and another target, a comK promoter. Gel shift analysis revealed that RapG served as the inhibitor of DegU binding to the promoter regions of aprE and comK and that this inhibition was counteracted by the PhrG peptide.

Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis.

Growth under conditions of salt stress has important effects on the synthesis of degradative enzymes in Bacillus subtilis. Salt stress strongly stimulates the expression of sacB, encoding levansucrase (about ninefold), and downregulates the expression of aprE, encoding alkaline protease (about sixfold). It is suggested that the DegS-DegU two-component system is involved in sensing salt stress. Moreover, it has been shown that the level of sacB expression strongly depends on the growth conditions; its expression level is about eightfold higher in cells grown on agar plates than in cells grown in liquid medium.

DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ

Production of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis is regulated at the transcriptional level by a signal transduction pathway which includes the DegS-DegU two-component system and at least two additional regulatory genes, degQ and degR, encoding polypeptides of 46 and 60 amino acids, respectively. Expression of degQ was shown to be controlled by DegS-DegU. This expression is decreased in the presence of glucose and increased under any of the following conditions: growth with poor carbon sources, amino acid deprivation, phosphate starvation, and growth in the presence of decoyinine, a specific inhibitor of GMP synthetase. In addition, expression of degQ is shown to be positively regulated by the ComP-ComA two-component system. Separate targets for regulation of degQ gene expression by DegS-DegU and ComP-ComA were located by deletion analysis between positions -393 and -186 and between positions -78 and -40, respectively. Regulation of degQ expression by amino acid deprivation was shown to be dependent upon ComA. Regulation by phosphate starvation, catabolite repression, and decoyinine was independent of the two-component systems and shown to involve sequences downstream from position -78. The ComP-ComA and DegS-DegU two-component systems seem to be closely related, sharing several target genes in common, such as late competence genes, as well as the degQ regulatory gene. Sequence analysis of the degQ region revealed the beginning of an open reading frame directly downstream from degQ. Disruption of this gene, designated comQ, suggests that it also controls expression of degQ and is required for development of genetic competence.