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.     

Expression level of a chimeric kinase governs entry into sporulation in Bacillus subtilis

Upon starvation, Bacillus subtilis cells switch from growth to sporulation. It is believed that the N-terminal sensor domain of the cytoplasmic histidine kinase KinA is responsible for detection of the sporulation-specific signal(s) that appears to be produced only under starvation conditions. Following the sensing of the signal, KinA triggers autophosphorylation of the catalytic histidine residue in the C-terminal domain to transmit the phosphate moiety, via phosphorelay, to the master regulator for sporulation, Spo0A. However, there is no direct evidence to support the function of the sensor domain, because the specific signal(s) has never been found. To investigate the role of the N-terminal sensor domain, we replaced the endogenous three-PAS repeat in the N-terminal domain of KinA with a two-PAS repeat derived from Escherichia coli and examined the function of the resulting chimeric protein. Despite the introduction of a foreign domain, we found that the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. Further, by using chemical cross-linking, we showed that the chimeric protein exists predominantly as a tetramer, mediated by the N-terminal domain, as was found for KinA. These results suggest that tetramer formation mediated by the N-terminal domain, regardless of the origin of the protein, is important and sufficient for the kinase activity catalyzed by the C-terminal domain. Taken together with our previous observations, we propose that the primary role of the N-terminal domain of KinA is to form a functional tetramer, but not for sensing an unknown signal.     

The histidine kinase inhibitor Sda binds near the site of autophosphorylation and may sterically hinder autophosphorylation and phosphotransfer to Spo0F

Histidine kinases are widely used by bacteria, fungi and plants to sense and respond to changing environmental conditions. Signals in addition to those directly sensed by the kinase are often integrated by proteins that fine-tune the biological response by modulating the activity of the kinase or its targets. The Bacillus subtilis histidine kinase KinA promotes the initiation of sporulation when nutrients are limiting, but sporulation can be delayed by two inhibitors of KinA, Sda (when DNA replication is perturbed) or KipI (under unknown conditions). We have identified residues in the dimerization/histidine-phosphotransfer (DHp) domain of KinA that are functionally important for inhibition by Sda and KipI and overlapping surface-exposed residues that lie close to or comprise the Sda binding site. Sda inhibits the intermolecular transfer of phosphate from the catalytic ATP-binding (CA) domain of KinA to the autophosphorylation site in the DHp domain when the domains are split into separate polypeptides, either by steric hindrance or by altering the conformation of the DHp domain. Sda also slows the rate of phosphotransfer from KinA∼P to its target, Spo0F, consistent with our finding that a KinA residue important for Sda function overlaps with the predicted Spo0F binding site on KinA.     

Histidine kinase regulation by a cyclophilin-like inhibitor

The sensor histidine kinase A (KinA) from Bacillus subtilis triggers a phosphorelay that activates sporulation. The antikinase KipI prevents sporulation by binding KinA and inhibiting the autophosphorylation reaction. Using neutron contrast variation, mutagenesis, and fluorescence data, we show that two KipI monomers bind via their C-domains at a conserved proline in the KinA dimerization and histidine-phosphotransfer (DHp) domain. Our crystal structure of the KipI C-domain reveals the binding motif has a distinctive hydrophobic groove formed by a five-stranded antiparallel β-sheet; a characteristic of the cyclophilin family of proteins that bind prolines and often act as cis-trans peptidyl-prolyl isomerases. We propose that the DHp domain of KinA transmits conformational signals to regulate kinase activity via this proline-mediated interaction. Given that both KinA and KipI homologues are widespread in the bacterial kingdom, this mechanism has broad significance in bacterial signal transduction.     

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.     

A Secreted Factor Coordinates Environmental Quality with Bacillus Development

Entry into sporulation is governed by the master regulator Spo0A. Spo0A accumulates in its active form, Spo0A-P, as cells enter stationary phase. Prior reports have shown that the acute induction of constitutively active Spo0A during exponential growth does not result in sporulation. However, a subsequent study also found that a gradual increase in Spo0A-P, mediated through artificial expression of the kinase, KinA, during exponential growth, is sufficient to trigger sporulation. We report here that sporulation via KinA induction depends on the presence of an extracellular factor or factors (FacX) that only accumulates to active levels during post-exponential growth. FacX is retained by dialysis with a cutoff smaller than 500 Dalton, can be concentrated, and is susceptible to proteinase K digestion, similar to described quorum-sensing peptides shown to be involved in promoting sporulation. However, unlike previously characterized peptides, FacX activity does not require the Opp or App oligopeptide transporter systems. In addition, FacX activity does not depend on SigH, Spo0A, or ComX. Importantly, we find that in the presence of FacX, B. subtilis can be induced to sporulate following the artificial induction of constitutively active Spo0A. These results indicate that there is no formal requirement for gradual Spo0A-P accumulation and instead support the idea that sporulation requires both sufficient levels of active Spo0A and at least one other signal or condition.     

Characterization of sporulation histidine kinases of Bacillus anthracis

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.     

Structural alterations in the Bacillus subtilis Spo0A regulatory protein which suppress mutations at several spo0 loci

Secondary site mutations that restore sporulation to sporulation-defective spo0F or spo0B deletion mutants were found to reside in the spo0A gene. Sequence analysis of 23 such sof mutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator Spo0A protein. Changes were observed in codons 12, 14, 60, 92, and 121. The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the same topological face as the active site aspartate residues. The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested. All of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations. sof mutants segregated Spo- colonies at high frequency. Five of these Spo- mutants were found to result from mutations in the spo0A locus that reversed the effect of the sof mutatation. One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105. The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell. The results suggested that the sof mutations increase the avidity for or reactivity with transmitter kinases in an allele-specific manner, although in some cases it is possible that the sof mutations obviate the need for phosphorylation to activate the Spo0A protein. An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.