James A. Hoch

Initiation of Sporulation in B. subtilis Is Controlled by a Multicomponent Phosphorelay

Stage 0 sporulation (spo0) mutants of Bacillus subtilis are defective in the signal transduction system initiating sporulation. Two of the products of these genes, Spo0A and Spo0F, are related to response regulator components of two-component regulatory systems used to control environmental responses in bacteria. The Spo0F response regulator was found to be the primary substrate for phosphorylation by the sporulation-specific protein kinase, KinA. Phosphorylated Spo0F was the phosphodonor for a phosphotransferase, Spo0B, which transferred the phosphate group to the second response regulator, the transcription regulatory protein Spo0A. This phosphorelay provides a mechanism for signal gathering from several protein kinases using Spo0F as a secondary messenger. These divergent signals are integrated through Spo0B phosphotransferase to activate the Spo0A transcription factor. This system provides for many levels of control to prevent capricious induction of sporulation.

Two-component and phosphorelay signal transduction.

Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Recent results have implicated these systems in the regulation of a variety of essential processes including cell-cycle progression, pathogenicity, and developmental pathways. Elucidation of the structures of the interacting domains is leading to an understanding of the mechanisms of molecular recognition and phosphotransfer in these systems.

Identification of direct residue contacts in protein–protein interaction by message passing

Understanding the molecular determinants of specificity in protein–protein interaction is an outstanding challenge of postgenome biology. The availability of large protein databases generated from sequences of hundreds of bacterial genomes enables various statistical approaches to this problem. In this context covariance-based methods have been used to identify correlation between amino acid positions in interacting proteins. However, these methods have an important shortcoming, in that they cannot distinguish between directly and indirectly correlated residues. We developed a method that combines covariance analysis with global inference analysis, adopted from use in statistical physics. Applied to a set of >2,500 representatives of the bacterial two-component signal transduction system, the combination of covariance with global inference successfully and robustly identified residue pairs that are proximal in space without resorting to ad hoc tuning parameters, both for heterointeractions between sensor kinase (SK) and response regulator (RR) proteins and for homointeractions between RR proteins. The spectacular success of this approach illustrates the effectiveness of the global inference approach in identifying direct interaction based on sequence information alone. We expect this method to be applicable soon to interaction surfaces between proteins present in only 1 copy per genome as the number of sequenced genomes continues to expand. Use of this method could significantly increase the potential targets for therapeutic intervention, shed light on the mechanism of protein–protein interaction, and establish the foundation for the accurate prediction of interacting protein partners.

The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation

Bacillus subtilis spo0K mutants are blocked at the first step in sporulation. The spo0K strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromosome. The mutation linked to trpS was responsible for the sporulation defect (spo‐). The unlinked mutation enhanced this sporulation deficiency but had no phenotype on its own. The spo‐ mutation was located in an operon of five genes highly homologous to the oligopeptide transport (Opp) system of Gram‐negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide‐transport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP‐binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide‐binding protein, which is periplasmically located in Gram‐negative species, has a signal sequence characteristic of lipo‐proteins with an amino‐terminal lipo‐amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in stationary phase. A major role of the Opp system in Gram‐negative bacteria is the recycling of cell‐wall peptides as they are released from the growing peptidoglycan. We postulate that the accumulation of such peptides may play a signaling role in the initiation of sporulation, and that the sporulation defect in opp mutants results from an inability to transport these peptides.

Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis

Protein homology studies identified five kinases potentially capable of phosphorylating the Spo0F response regulator and initiating sporulation in Bacillus subtilis. Two of these kinases, KinA and KinB, were known from previous studies to be responsible for sporulation in laboratory media. In vivo studies of the activity of four of the kinases, KinA, KinC, KinD (ykvD) and KinE (ykrQ), using abrB transcription as an indicator of Spo0A∼P level, revealed that KinC and KinD were responsible for Spo0A∼P production during the exponential phase of growth in the absence of KinA and KinB. In vitro, all four kinases dephosphorylated Spo0F∼P with the production of ATP at approximately the same rate, indicating that they possess approximately equal affinity for Spo0F. All the kinases were expressed during growth and early stationary phase, suggesting that the differential activity observed in growth and sporulation results from differential activation by signal ligands unique to each kinase.

Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis

Sporulation begins coincidentally with the expression of several stationary‐phase‐associated gene products during the transition state of a culture from exponential to stationary phase. Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation. Suppressor mutations in the abrB gene, in a spo0 background, restore stationary‐phase‐associated gene expression but not sporulation. The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene. The abrB gene coded for a 96‐amino‐acid protein (molecular weight 10773) and contained a helix‐turn‐helix structure common to DNA binding proteins. Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene. Primer extension analysis of abrB gene mRNA revealed two initiation sites. The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A. Five abrB mutant alleles were cloned and sequenced. One mutation, abrB 4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters. A promoter mutation, abrB 15, reduced transcription from the downstream promoter but not the upstream promoter. Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression. The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation. The level of this regulator is, in turn, controlled by the spo0A gene. The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator.

Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis.

The initiation of sporulation in B. subtilis is regulated by the Spo0A transcription factor, which is activated by phosphorylation to control developmental switching from the vegetative to the sporulation state. The level of phosphorylation of Spo0A is regulated by the phosphorelay, a signal transduction system based on the protein-histidine kinase-response regulator two-component paradigm. To initiate sporulation, the cell must recognize and interpret a large variety of environmental, metabolic, and cell cycle signals that influence the phosphorylation level of Spo0A. We describe here a family of protein-aspartate phosphatases with activity on Spo0F approximately P, a response regulator component of the phosphorelay, that provide a mechanism for signal recognition and interpretation. These phosphatases function to drain the phosphorelay, lower Spo0A approximately P levels, and prevent sporulation. The integration of diverse environmental signals that affect the initiation of sporulation likely occurs through the competition between opposing activities of protein kinases and protein phosphatases.

The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein.

The product of the abrB gene of Bacillus subtilis is an ambiactive repressor and activator of the transcription of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Purified AbrB protein binds specifically in a highly co‐operative fashion to fragments of DNA containing the promoters it affects. DNase I footprints of the binding regions in these promoters revealed large protected areas of 50‐120 nucleotides or more depending on the promoter. Methylation protection experiments gave protected guanine residues on only one face of the DNA helix. A consensus sequence could be deduced around these guanine residues that was not found around non‐protected guanine residues in the footprint region. The results suggested that stationary phase functions and sporulation are repressed during active growth by AbrB and other transition state regulators by binding to the affected promoters in a concentration‐dependent manner.

Transition‐state regulators: sentinels of Bacillus subtilis post‐exponential gene expression

When Bacillus subtilis encounters a nutrient‐depleted environment, it expresses a wide variety of genes that encode functions in alternative pathways of metabolism and energy production. Expression of these genes first occurs during the transition from active growth into stationary phase and is controlled by a class of proteins termed transition‐state regulators. In several instances, a given gene is redundantly controlled by two or more of these regulators and many of these regulators control genes in numerous different pathways. The AbrB, Hpr and Sin proteins are the best‐studied examples of these regulatory molecules. Their role is to prevent inappropriate and possibly detrimental functions from being expressed during exponential growth when they are not needed. They serve as elements integrating sporulation with ancillary stationary‐phase phenomena and appear to participate in the timing of early sporulation events and in fine‐tuning the magnitude of gene expression in response to specific environmental conditions.

A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction.

BACKGROUND Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F. Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. RESULTS Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques. The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer. Upon complex formation, the Spo0B conformation remains unchanged. Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations. CONCLUSIONS The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.