Spatial regulation of histidine kinases governing biofilm formation in Bacillus subtilis

Bacillus subtilis is able to form architecturally complex biofilms on solid medium due to the production of an extracellular matrix. A master regulator that controls the expression of the genes involved in matrix synthesis is Spo0A, which is activated by phosphorylation via a phosphorelay involving multiple histidine kinases. Here we report that four kinases, KinA, KinB, KinC, and KinD, help govern biofilm formation but that their contributions are partially masked by redundancy. We show that the kinases fall into two categories and that the members of each pair (one pair comprising KinA and KinB and the other comprising KinC and KinD) are partially redundant with each other. We also show that the kinases are spatially regulated: KinA and KinB are active principally in the older, inner regions of the colony, and KinC and KinD function chiefly in the younger, outer regions. These conclusions are based on the morphology of kinase mutants, real-time measurements of gene expression using luciferase as a reporter, and confocal microscopy using a fluorescent protein as a reporter. Our findings suggest that multiple signals from the older and younger regions of the colony are integrated by the kinases to determine the overall architecture of the biofilm community.     

In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis

The phosphorylated form of the response regulator Spo0A (Spo0A~P) is required for the initiation of sporulation in Bacillus subtilis. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0A~P-specific phosphatase, on sporulation of strains containing the spo0A mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed medium-dependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants.     

Osmotic pressure can regulate matrix gene expression in Bacillus subtilis

Many bacteria organize themselves into structurally complex communities known as biofilms in which the cells are held together by an extracellular matrix. In general, the amount of extracellular matrix is related to the robustness of the biofilm. Yet, the specific signals that regulate the synthesis of matrix remain poorly understood. Here we show that the matrix itself can be a cue that regulates the expression of the genes involved in matrix synthesis in Bacillus subtilis. The presence of the exopolysaccharide component of the matrix causes an increase in osmotic pressure that leads to an inhibition of matrix gene expression. We further show that non‐specific changes in osmotic pressure also inhibit matrix gene expression and do so by activating the histidine kinase KinD. KinD, in turn, directs the phosphorylation of the master regulatory protein Spo0A, which at high levels represses matrix gene expression. Sensing a physical cue such as osmotic pressure, in addition to chemical cues, could be a strategy to non‐specifically co‐ordinate the behaviour of cells in communities composed of many different species.     

Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis.

The kinA (spoIIJ) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems. The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the Spo0A and Spo0F sporulation regulatory proteins. Spo0F protein was a much better phosphoreceptor for this kinase than Spo0A protein in vitro. Mutants with deletion mutations in the kinA gene were delayed in their sporulation. They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain. Such mutations had no effect on the regulation of the abrB gene or on the timing of subtilisin expression and therefore did not impair the repression function of the Spo0A protein. Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spo0B, spo0E, and spo0F mutations but not of spo0A mutations. The results suggest that the spo0B-, spo0E-, and spo0F-dependent pathway of activation (phosphorylation) of the Spo0A regulator may be by-passed through the kinA gene product if it is present at sufficiently high intracellular concentration. The results suggest that multiple kinases exist for the Spo0A protein.     

Structural characterization of Spo0E-like protein-aspartic acid phosphatases that regulate sporulation in bacilli

Spore formation is an extreme response of many bacterial species to starvation. In the case of pathogenic species of Bacillus and Clostridium, it is also a component of disease transmission. Entry into the pathway of sporulation in Bacillus subtilis and its relatives is controlled by an expanded two-component system in which starvation signals lead to the activation of sensor kinases and phosphorylation of the master sporulation response regulator Spo0A. Accumulation of threshold concentrations of Spo0A approximately P heralds the commitment to sporulation. Countering the activities of the sensor kinases are phosphatases such as Spo0E, which dephosphorylate Spo0A approximately P and inhibit sporulation. Spo0E-like protein-aspartic acid-phosphate phosphatases, consisting of 50-90 residues, are conserved in sporeforming bacteria and unrelated in sequence to proteins of known structure. Here we determined the structures of the Spo0A approximately P phosphatases BA1655 and BA5174 from Bacillus anthracis using nuclear magnetic resonance spectroscopy. Each is composed of two anti-parallel alpha-helices flanked by flexible regions at the termini. The signature SQELD motif (SRDLD in BA1655) is situated in the middle of helix alpha2 with its polar residues projecting outward. BA5174 is a monomer, whereas BA1655 is a dimer. The four-helix bundle structure in the dimer is reminiscent of the phosphotransferase Spo0B and the chemotaxis phosphatase CheZ, although in contrast to these systems, the subunits in BA1655 are in head-to-tail rather than head-to-head apposition. The implications of the structures for interactions between the phosphatases and their substrate Spo0A approximately P are discussed.