News and Publications

Division of Cellular Biology

James A. Hoch, Ph.D., Division Head

Signal Transduction in Cellular Differentiation

J.A. Hoch, M. Perego, C. Fabret, K. Kanamaru, M. Jiang, W. Song, W. Shao

Formation of endospores in Bacillus subtilis is a model for understanding the mechanism of developmentally programmed gene expression. Several dozen genetically dispersed sporulation operons are regulated coordinately as temporal classes during the time required to complete the formation of spores. This complex developmental program is under the control of the spo0 genes, which control entry of the cell into sporulation. The protein Spo0A is the key master regulator of the initiation of developmental transcription. The activity of the protein is controlled by a reversible phosphorylation-dephosphorylation mechanism. In its unphosphorylated form, Spo0A is inactive. In its phosphorylated form, it is both an activator of the transcription of sporulation genes and a negative regulator of genes that prevent sporulation.

The key to understanding the initiation of sporulation is understanding the mechanism of Spo0A phosphorylation. We showed that the pathway to Spo0A activation is a sequential series of phosphorylation reactions termed a multicomponent phosphorelay. The initial event in the phosphorelay is the activation of 1 of 2 kinases that phosphorylate the sporulation-specific response regulator Spo0F in response to environmental and metabolic signals. Spo0F acts as a second messenger, accumulating phosphate groups from developmentally activated kinases. Phosphorylated Spo0F (Spo0F~P) is the substrate for the Spo0B protein phosphotransferase that phosphorylates Spo0A. In this pathway, the signal transduction event is the activation (or perhaps the deinhibition) of the kinases to autophosphorylate. This step is followed by 3 sequential phosphotransferase reactions that produce Spo0A~P, the crucial transcription regulator for sporulation.

The flow of phosphate through the phosphorelay to Spo0A is highly controlled at several levels. Although the primary signals transduced by KinA and KinB, the 2 kinases responsible for Spo0F phosphorylation, remain obscure, genetic studies have revealed a series of genes unique for the activation of each kinase. The activity of each kinase is regulated by complex signal transduction pathways that respond to environmental, metabolic, and cell-cycle signals. Recently, we discovered a new type of signal transduction kinase inhibitor for KinA that binds to the ATP-binding site of the KinA catalytic domain. This protein, KipI, may also bind to another protein, KipA, that regulates the activity of KipI on KinA. This inhibition system may be regulated by the level of available nitrogen.

It is now clear that all the signals that affect sporulation cannot be processed by the kinases. Access to the phosphorelay for additional signals is provided by 2 families of phosphatases, which dephosphorylate either Spo0F~P or Spo0A~P and act to prevent sporulation. One of these phosphatases is controlled by the competence pathway, a finding that suggests that alternative physiologic processes induced at the end of exponential growth compete with sporulation by preventing activation of the major sporulation transcription factor.

Thus, the probability of initiating sporulation depends on the competition between kinases and phosphatases. The activities of the kinases and phosphatases on the phosphorelay act as a signal integration circuit, allowing input from a variety of environmental, metabolic, and cell-cycle sources with a single output, the cellular level of Spo0A~P. By placing the developmental fate of the cell in the cellular level of a single phosphorylated transcription factor and coupling all signal inputs to this level, a large number of signal inputs can be accommodated with incremental effects that lead to a level of Spo0A~P that reflects the sum of all positive and negative factors.

PUBLICATIONS

Dartois, V., Djavakhishvili, T., Hoch, J.A. KapB is a lipoprotein required for KinB signal transduction and activation of the phosphorelay to sporulation in Bacillus subtilis. Mol. Microbiol. 25:1097, 1997.

Dartois, V., Liu, J., Hoch, J.A. Alterations in the flow of one-carbon units affect KinB-dependent sporulation in Bacillus subtilis. Mol. Microbiol. 25:39, 1997.

Grimshaw, C.E., Huang, S., Hanstein, C.G., Strauch, M.A., Burbulys, D., Wang, L., Hoch, J.A., Whiteley, J.M. Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis. Biochemistry 37:1365, 1998.

Hoch, J.A. Initiation of bacterial development. Curr. Opin. Microbiol. 1:170, 1998.

Hoch, J.A., Losick, R. Panspermia, spores and the Bacillus subtilis genome. Nature 390:237, 1997.

Manson, M.D., Armitage, J.P., Hoch, J.A., Macnab, R.M. Bacterial locomotion and signal transduction. J. Bacteriol. 180:1009, 1998.

Wang, L., Grau, R., Perego, M., Hoch, J.A. A novel histidine kinase inhibitor regulating development in Bacillus subtilis. Genes Dev. 11:2569, 1997.

Molecular Recognition in Signal Transduction

J.A. Hoch, Y.-L. Tzeng, Madhusudan, K.I. Varughese

Two-component signal transduction systems are the major mechanism for environmental signal recognition in bacteria and the induction of virulence in pathogens. Microorganisms such as Escherichia coli and Bacillus subtilis contain about 50 different pairs of kinase response regulator 2-component systems that are highly related in sequence and structure and that process a variety of signals. Each signal activates a specific kinase that phosphorylates only the response regulator to which the kinase is mated despite the presence of many response regulators of identical structure. This circuitry is kept intact by the exquisite recognition properties of the kinase for its response regulator. We are using a multifaceted approach of mutational and structural analysis to study the molecular basis of recognition between the kinases and response regulators.

The Spo0F response regulator is the key intermediate in the signal transduction phosphorelay. It interacts with 2 different kinases, at least 2 phosphatases, and the Spo0B response regulator phosphotransferase. The complete structure of Spo0F was determined with both crystallography and nuclear magnetic resonance. This structure was the basis for studies that used the alanine-scanning technique to determine the roles of amino acid side chains in molecular recognition. All the surface residues of the Spo0F protein were changed individually to alanine, and we determined how deleting the side chain past the ß carbon affected the interaction of Sp0F with kinases, phosphatases, and Spo0B. These studies revealed that residues important for recognition by all these proteins are clustered around the aspartate triad active site of phosphorylation. Furthermore, individual residues could be identified that are specific for interaction with 1 or more kinases, phosphatases, or Spo0B.

Spo0B is a unique phosphotransferase capable of transferring phosphate from one response regulator to another with specificity for both donor and recipient. The alanine-scanning studies of the donor phosphorylated Spo0F protein are being complemented by structural analyses of its primary phosphate recipient, Spo0B. Mechanistic studies of the Spo0B phosphotransferase reaction revealed that a histidine-phosphate is an obligatory intermediate in the reaction, and the identity of this histidine was established. Spo0B was crystallized with 2 molecules in the asymmetric unit. Diffraction data to a resolution of 2.6 Å were acquired for a native crystal and for 5 heavy-atom derivatives. The interface of the dimer is formed by a 4-helix bundle, 2 helices from each monomer.

PUBLICATIONS

Madhusudan, Zapf, J., Hoch, J.A., Whiteley, J.M., Xuong, N.H., Varughese, K.I. A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: Crystal structure of Spo0F from Bacillus subtilis. Biochemistry 36:42, 1998.

Zapf, J., Madhusudan, Grimshaw, C.E., Hoch, J.A., Varughese, K.I., Whiteley, J.M. A source of response regulator autophosphatase activity: The critical role of a residue adjacent to the Spo0F autophosphorylation active site. Biochemistry 37:7725, 1998.

Molecular Dynamics of Response Regulators

J.A. Hoch, Y.-L. Tzeng, V. Feher*, J. Cavanagh*

* New York State Department of Health, Albany, NY

Spo0F belongs to a large class of proteins, the response regulators, that participate in many different bacterial signal transduction pathways. Response regulators have diverse functions, although common to all is a regulatory domain of about 120 residues that becomes phosphorylated at a conserved aspartate residue in a magnesium-dependent reaction with a histidine autokinase. The overall fold of Spo0F consists of 5 -helices surrounding 5 parallel ß-strands, forming a hydrophobic sheet, as is the case in other response regulators. The fold brings 3 aspartic acid residues into proximity to form the binding pocket to accept the phosphoryl group. From structural studies, we determined the orientation of secondary structure elements in the putative recognition surfaces and the relative charge distribution of residues surrounding the site of phosphorylation.

We are also studying the backbone dynamics of the Spo0F protein. In conjunction with alanine-scanning mutagenesis studies, our dynamics studies have enabled us to propose a model in which communication of information through the core of the protein, between buried and surface-bound residues, is responsible for the dissociation of the cognate kinase of the protein after phosphorylation. The helix-4--strand-5 loop contains a primary recognition site for the kinase involving residues Tyr84, Glu86, and Leu87. The structural and dynamics studies show that this region contains a propensity for multiple conformers. We defined a region on the protein, including helix-4, part of helix-3, strand-5, and the helix-4--strand-5 loop, that moves in a dynamically concerted fashion, driven by the motion of the imidazole ring of His101.

We propose that the imidazole ring moves from a buried position under the helix-4--strand-5 loop to a more solvent, exposed position in response to a conformational change in the aspartic acid binding pocket upon phosphorylation. Movement of the ring disrupts packing interactions, a condition that alters the topology of the kinase recognition site, thereby causing the kinase to dissociate. This model is one of the first connections between the dynamics of a protein and its specific biological function.

PUBLICATIONS

Feher, V.A., Tzeng, Y.-L., Hoch, J.A., Cavanagh, J. Identification of communication networks in Spo0F: A model for phosphorylation-induced conformational change and implications for activation of multiple domain bacterial response regulators. FEBS Lett. 425:1, 1998.

Feher, V.A., Zapf, J.W., Hoch, J.A., Whiteley, J.M., McIntosh, L.P., Rance, M., Skelton, N.J., Dahlquist, F.W., Cavanagh, J. High-resolution NMR structure and backbone dynamics of the Bacillus subtilis response regulator, Spo0F: Implications for phosphorylation and molecular recognition. Biochemistry 36:10015, 1997.

Tzeng, Y.-L., Hoch, J.A. Molecular recognition in signal transduction: The interaction surfaces of the Spo0F response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis. J. Mol. Biol. 272:200, 1997.

Regulatory Circuits Coordinating Chromosome Segregation and Development

J.A. Hoch, M. Perego, J. Errington,* G.B. Spiegelman**

* University of Oxford, Oxford, England
** University of British Columbia, Vancouver, British Columbia

The mechanism by which bacterial chromosomes are equipartitioned into daughter cells at division remained obscure for decades until recent studies showed that bacterial nucleoids undergo abrupt movements reminiscent of mitosis in eukaryotes. The mechanisms of chromosome segregation in bacteria are distinct from those in eukaryotes, mainly because bacteria lack an obvious cytoskeleton and a mitotic spindle apparatus. Nevertheless mechanisms of active chromosome segregation exist in bacteria that accomplish the same task as a cytoskeleton and a mitotic spindle do in eukaryotes.

Two of the proteins involved in regulation of the fidelity of chromosome segregation in Bacillus subtilis are Spo0JA and Spo0JB, which are related to the ParA and ParB proteins encoded by extrachromosomal elements that ensure equal partition of the elements to daughter cells. Spo0JB binds to the origin region of the chromosome and migrates with the origin during the mitotic process. Mutations in this protein severely affect the ability of the bacteria to initiate sporulation, and this deficiency is due to the Spo0JA protein. This finding suggests that Spo0JA and Spo0JB communicate with each other, forming a direct regulatory link between mitosis and development.

How does Spo0JA regulate development, and what is the nature of the signal transfer from chromosome segregation mediated by Spo0JB? We now understand how Spo0JA acts to control developmental transcription. In in vitro studies, we found that Spo0JA specifically dissociates transcription initiation complexes formed by the phosphorylated developmental transcription factor Spo0A and a developmental promoter. Thus, the status of chromosome replication and segregation is signaled via Spo0JB, which controls the repressor activity of Spo0JA. What remains unknown is whether Spo0JB contacts Spo0JA directly or through other intermediate regulators. This regulatory circuit most likely is much more complex than is now apparent.

PUBLICATIONS

Cervin, M.A., Spiegelman, G.B., Raether, B., Ohlsen, K., Perego, M., Hoch, J.A. A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis. Mol. Microbiol. 29:85, 1998.

Glaser, P., Sharpe, M.E., Raether, B., Perego, M., Ohlsen, K., Errington, J. Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning. Genes Dev. 11:1160, 1997.

Antibacterial Agents That Inhibit 2-Component Signal Transduction Systems

J.A. Hoch, J.M. Whiteley, J.F. Barrett*

* The R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ

Infectious disease is the No. 1 cause of mortality in the world despite the large number of antibacterial agents developed in the past half century. During this time, bacterial strains have evolved that are recalcitrant to our best efforts to destroy them, and they have begun to compromise the treatment of infectious diseases, particularly in the hospital setting. Resistance to antibacterials takes many forms and is a probable consequence of widespread use and misuse of antibiotics. Therefore, the search for new antibacterials with new targets is not only a continual process but also, at this time, an urgent necessity. Few attempts have been made to specifically target the mechanisms by which pathogenic bacteria establish an infection within the host. In particular, inhibition of expression of bacterial virulence factors offers an opportunity for specific intervention at the level of host invasion through biochemical processes that are clearly unique to the bacterial cell.

Virulence is an adaptive genetic response that requires the induction of genes that encode virulence factors. This response implies that the infectious agent must be able to sense when it is in position to invade. Much of this environmental sensing occurs through 2-component signal transduction systems with a common phosphorylation-dependent mechanism of signal transduction that apparently is nearly ubiquitous in bacteria. Four features in particular make the 2-component family attractive as a potential target for antimicrobial agents: (1) Significant homology is shared among kinase and response regulator proteins of different genera of bacteria, particularly in those amino acid residues located near active sites. (2) Pathogenic bacteria use 2-component signal transduction to regulate expression of essential virulence factors that are required for survival inside the host. (3) Bacteria contain many 2-component systems, and some of the systems are essential for viability. (4) Signal transduction in mammals occurs by a different mechanism.

In a collaborative effort between TSRI and the R.W. Johnson Pharmaceutical Research Institute, a class of antibacterials has been discovered that inhibits the growth of gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, is a potent bactericidal compound, and it inhibits the autophosphorylation of kinase A of the KinA-Spo0F 2-component signal transduction system in vitro. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria, and this ability correlates directly with their activity as kinase A inhibitors. In a laboratory passage experiment, the development of drug resistance was less in bacteria treated with RWJ-49815 than in bacteria treated with the potent quinolone ciprofloxacin. Inhibition of the histidine protein kinase response regulator 2-component signal transduction pathways may present an opportunity to depress the emergence of drug resistance by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that may be a breakthrough in antibacterial therapy.

PUBLICATIONS

Barrett, J.F., Goldschmidt, R.M., Lawrence, L.E., Foleno, B., Chen, R., Demers, J.P., Johnson, S., Kanojia, R., Fernandez, J., Bernstein, J., Licata, L., Donetz, A., Huang, S., Hlasta, D.J., Macielag, M.J., Ohemeng, K., Frechiette, R., Froscoo, M.D., Klaubert, D.H., Whiteley, J.M., Wang, L., Hoch, J.A. Antibacterial agents that inhibit two-component signal transduction systems. Proc. Natl. Acad. Sci. U.S.A. 95:5317, 1998.

Barrett, J.F., Hoch, J.A. Two-component signal transduction as a target for microbial anti-infective therapy. Antimicrob. Agents Chemother. 42:1529, 1998.