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.

A protein complex supports the production of Spo0A-P and plays additional roles for biofilms and the K-state in Bacillus subtilis.

Bacillus subtilis can enter three developmental pathways to form spores, biofilms or K-state cells. The K-state confers competence for transformation and antibiotic tolerance. Transition into each of these states requires a stable protein complex formed by YlbF, YmcA and YaaT. We have reported that this complex acts in sporulation by accelerating the phosphorylation of the response regulator Spo0A. Phosphorelay acceleration was also predicted to explain their involvement in biofilm formation and the K-state. This view has been challenged in the case of biofilms, by the suggestion that the three proteins act in association with the mRNA degradation protein RNaseY (Rny) to destabilize the sinR transcript. Here, we reaffirm the roles of the three proteins in supporting the phosphorylation of Spo0A for all three developmental pathways and show that in their absence sinR mRNA is not stabilized. We demonstrate that the three proteins also play unknown Spo0A-P-independent roles in the expression of biofilm matrix and in the production of ComK, the master transcription factor for competence. Finally, we show that domesticated strains of B. subtilis carry a mutation in sigH, which influences the expression kinetics of the early spore gene spoIIG, thereby increasing the penetrance of the ylbF, ymcA and yaaT sporulation phenotypes.

The YmdB phosphodiesterase is a global regulator of late adaptive responses in Bacillus subtilis.

Bacillus subtilis mutants lacking ymdB are unable to form biofilms, exhibit a strong overexpression of the flagellin gene hag, and are deficient in SlrR, a SinR antagonist. Here, we report the functional and structural characterization of YmdB, and we find that YmdB is a phosphodiesterase with activity against 2',3'- and 3',5'-cyclic nucleotide monophosphates. The structure of YmdB reveals that the enzyme adopts a conserved phosphodiesterase fold with a binuclear metal center. Mutagenesis of a catalytically crucial residue demonstrates that the enzymatic activity of YmdB is essential for biofilm formation. The deletion of ymdB affects the expression of more than 800 genes; the levels of the σ(D)-dependent motility regulon and several sporulation genes are increased, and the levels of the SinR-repressed biofilm genes are decreased, confirming the role of YmdB in regulating late adaptive responses of B. subtilis.

Determinants of interaction specificity of the Bacillus subtilis GlcT antitermination protein: functionality and phosphorylation specificity depend on the arrangement of the regulatory domains.

The control of several catabolic operons in bacteria by transcription antitermination is mediated by RNA-binding proteins that consist of an RNA-binding domain and two reiterated phosphotransferase system regulation domains (PRDs). The Bacillus subtilis GlcT antitermination protein regulates the expression of the ptsG gene, encoding the glucose-specific enzyme II of the phosphotransferase system. In the absence of glucose, GlcT becomes inactivated by enzyme II-dependent phosphorylation at its PRD1, whereas the phosphotransferase HPr phosphorylates PRD2. However, here we demonstrate by NMR analysis and mass spectrometry that HPr also phosphorylates PRD1 in vitro but with low efficiency. Size exclusion chromatography revealed that non-phosphorylated PRD1 forms dimers that dissociate upon phosphorylation. The effect of HPr on PRD1 was also investigated in vivo. For this purpose, we used GlcT variants with altered domain arrangements or domain deletions. Our results demonstrate that HPr can target PRD1 when this domain is placed at the C terminus of the protein. In agreement with the in vitro data, HPr exerts a negative control on PRD1. This work provides the first insights into how specificity is achieved in a regulator that contains duplicated regulatory domains with distinct dimerization properties that are controlled by phosphorylation by different phosphate donors. Moreover, the results suggest that the domain arrangement of the PRD-containing antitermination proteins is under selective pressure to ensure the proper regulatory output, i.e. transcription antitermination of the target genes specifically in the presence of the corresponding sugar.

RNA processing in Bacillus subtilis: identification of targets of the essential RNase Y.

RNA processing and degradation is initiated by endonucleolytic cleavage of the target RNAs. In many bacteria, this activity is performed by RNase E which is not present in Bacillus subtilis and other Gram-positive bacteria. Recently, the essential endoribonuclease RNase Y has been discovered in B. subtilis. This RNase is involved in the degradation of bulk mRNA suggesting a major role in RNA metabolism. However, only a few targets of RNase Y have been identified so far. In order to assess the global impact of RNase Y, we compared the transcriptomes in response to the expression level of RNase Y. Our results demonstrate that processing by RNase Y results in accumulation of about 550 mRNAs. Some of these targets were substantially stabilized by RNase Y depletion, resulting in half-lives in the range of an hour. Moreover, about 350 mRNAs were less abundant when RNase Y was depleted among them the mRNAs of the operons required for biofilm formation. Interestingly, overexpression of RNase Y was sufficient to induce biofilm formation. The results presented in this work emphasize the importance of RNase Y as the global acting endoribonuclease for B. subtilis.

A novel factor controlling bistability in Bacillus subtilis: the YmdB protein affects flagellin expression and biofilm formation.

Cells of Bacillus subtilis can either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterized ymdB gene for bistable gene expression in B. subtilis. We observed a strong overexpression of the hag gene encoding flagellin and of other genes of the σ(D)-dependent motility regulon in the ymdB mutant, whereas the two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were not expressed. As a result, the ymdB mutant is unable to form biofilms. An analysis of the individual cells of a population revealed that the ymdB mutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of the ymdB mutant to form biofilms is suppressed by the deletion of the sinR gene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in a ymdB mutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of a ymdB mutation.

Prevention of cross-talk in conserved regulatory systems: identification of specificity determinants in RNA-binding anti-termination proteins of the BglG family.

Each family of signal transduction systems requires specificity determinants that link individual signals to the correct regulatory output. In Bacillus subtilis, a family of four anti-terminator proteins controls the expression of genes for the utilisation of alternative sugars. These regulatory systems contain the anti-terminator proteins and a RNA structure, the RNA anti-terminator (RAT) that is bound by the anti-terminator proteins. We have studied three of these proteins (SacT, SacY, and LicT) to understand how they can transmit a specific signal in spite of their strong structural homology. A screen for random mutations that render SacT capable to bind a RNA structure recognized by LicT only revealed a substitution (P26S) at one of the few non-conserved residues that are in contact with the RNA. We have randomly modified this position in SacT together with another non-conserved RNA-contacting residue (Q31). Surprisingly, the mutant proteins could bind all RAT structures that are present in B. subtilis. In a complementary approach, reciprocal amino acid exchanges have been introduced in LicT and SacY at non-conserved positions of the RNA-binding site. This analysis revealed the key role of an arginine side-chain for both the high affinity and specificity of LicT for its cognate RAT. Introduction of this Arg at the equivalent position of SacY (A26) increased the RNA binding in vitro but also resulted in a relaxed specificity. Altogether our results suggest that this family of anti-termination proteins has evolved to reach a compromise between RNA binding efficacy and specific interaction with individual target sequences.