Scientific Report 2007

Molecular and Experimental Medicine

Division of Cellular Biology

Sensor Kinases That Regulate Sporulation and the Synthesis of Toxins

J.A. Hoch, M. Perego, T. Fukushima, F. Scaramozzino, H. Szurmant, C. Bongiorni, A. Wilson

The complex developmental program of sporulation is under the control of the spo0 genes, which control entry of the cell into sporulation and the production of toxins and virulence factors in pathogens such as Bacillus anthracis. The transcription factor Spo0A is the key master regulator of the initiation of developmental transcription. The activity of Spo0A is controlled by a reversible phosphorylation-dephosphorylation mechanism.

In B anthracis, expression of the anthrax toxin, and presumably the potential of the toxin to cause pathologic changes, requires a low level of phosphorylated Spo0A. We have discovered a unique pathogenesis mechanism associated with the pXO1 plasmid of B anthracis that regulates low-level phosphorylation. The pXO1 plasmid encodes 2 genes whose proteins are highly homologous to the sensor domain of the sporulation sensor kinase BA2291. Expression of these proteins from the plasmid titrates the signal to activate BA2291, converting it from a kinase to a phosphatase of phosphorylated Spo0A. This mechanism in conjunction with the Rap phosphatase also encoded by pXO1 prevents high-level phosphorylation of Spo0A and sporulation while maintaining a low level that enables toxin expression.

The target of Spo0A regulation is the transcription factor AtxA, which is a required activator of expression of the genes that encode the anthrax toxin. We initiated transposon mutagenesis studies to identify additional regulators of atxA gene expression as well as mechanisms independent of AtxA. These studies have revealed genes for a heme–cytochrome c pathway and 2 unique genes encoded on the pXO1 plasmid required for atxA expression in addition to Spo0A. The mechanism by which this pathway directly affects atxA expression is unknown.

AtxA is a protein composed of a helix-turn-helix DNA recognition domain and 2 phosphenolpyruvate–sugar phosphotransferase system regulation domains (PRDs). Our mutational studies have shown that the phosphorylation of the histidine residues in the PRDs regulates the activity of AtxA; one of the PRDs requires phosphorylation for activity and the other acts as an inhibitor when phosphorylated. This nonclassical activity of PRDs indicates that unique phosphorylation mechanisms may be acting on these domains and that the regulation of AtxA activity may be much more complex than originally imagined. Indeed, the results of our transposon studies have confirmed the complexity of the regulation: we identified a large number of genes in which a mutation prevents toxin synthesis while allowing normal atxA gene expression. The complexity of expression of the genes for anthrax toxin and the relationship of the gene expression to normal cellular processes interrupted by pathogenesis plasmid genes is now being revealed.

Computational Analysis of Molecular Specificity in 2-Component Signaling

J.A. Hoch, R.A. White, H. Szurmant, T. Hwa*

* University of California, San Diego, California

In both prokaryotes and eukaryotes, a large number of pathways with proteins with identical structural folds are used to interpret and propagate vastly different signals specific for unique targets. A central question in understanding signal transduction is how does a signaling protein distinguish its true partner from the much larger number of similar partners present in the cell. In collaboration with T. Hwa, University of California, San Diego, we have developed a sequence-based method, independent of structural considerations, for identifying specificity-determining interactions between proteins for which genomic data indicate a large number of examples of functionally coupled pairs. This method was applied to the phosphotransfer domains of 2-component signaling proteins. Using this method, we identified a network of residue-residue interactions and generated a 3-dimensional structure consistent with the exemplary cocrystal structure obtained for the Spo0B-Spo0F 2-component complex. We also identified an interaction network that links long-distance interactions with pair specificity of 2-component signaling proteins.

The method provides a simple scoring procedure that can be used to identify potential cross-phosphorylation between functional pairs and to assign orphan 2-component signaling proteins with no known mate to their signaling partners. Although currently we are applying the method to 2-component signal transduction systems for which structural and mutational data allow proof of principle, the method may generate interaction structures for less-characterized protein pairs if sufficient functional pairs exist in genomic data.

Molecular Dynamics of Response Regulators

J.A. Hoch, J. Cavanagh*

* North Carolina State University, Raleigh, North Carolina

Recognition specificity by sensor kinases in 2-component signal transduction depends on the composition of amino acid residues in the surface of the response regulator with which the sensor kinase interacts. Part of this surface of response regulators consists of several dynamic loops generated by the folding of strands and helices to form the core of the response regulator. Using nuclear magnetic resonance chemical-shift perturbation experiments with specific mutants of the Spo0F response regulator, we found that the conformation of the β4-β4 loop and the α4 helix dictates kinase specificity. The presentation of the loop, and therefore kinase recognition, can be altered by perturbations of core residues that propagate to the surface. These results further support our earlier hypothesis that molecular recognition processes are significantly influenced by intraprotein communication networks in the core of the response regulator. These networks are critical in providing the precise surface to the appropriate sensor kinase in signal transduction, for which hundreds of structurally similar proteins have evolved from gene duplication to carry out as many different signaling functions within the cell.

Negative Regulation of Development in Bacilli

M. Perego, C. Bongiorni

Initiation of spore formation in gram-positive bacilli is regulated by the phosphorelay signal transduction system. Multiple positive and negative signals are integrated by the phosphorelay through the opposing activities of histidine protein kinases and aspartyl phosphate phosphatases. The phosphatases belong to 2 families: Rap and Spo0E. Rap proteins act as negative regulators of the initiation of sporulation by dephosphorylating the Spo0F response regulator intermediate of the phosphorelay; the Spo0E proteins act as negative regulators of the phosphorelay by dephosphorylating the Spo0A response regulator and transcription factor for the initiation of sporulation.

In previous studies, we identified the Rap and Spo0E proteins that control the phosphorelay in Bacillus subtilis and Bacillus anthracis. Among the Rap proteins of B subtilis, RapA, RapB, and RapE inhibit initiation of sporulation by dephosphorylating the Spo0F protein. RapC and RapF inhibit the initiation of the early competence pathway to DNA transformation by preventing the ComA transcription factor from binding to its target DNA promoters. Recently, we characterized RapH as a dual-specificity protein that regulates both sporulation and competence. In in vivo and in vitro studies, we showed that RapH acts by dephosphorylating Spo0F and by inhibiting the DNA-binding activity of ComA. RapH creates a negative feedback loop that prevents sporulation while competence is in full development but also helps shut down competence to allow resumption of growth. In this way, RapH ensures the temporal separation of the 2 differentiation pathways of B subtilis, that is, competence and sporulation. Our current focus is to understand the molecular mechanism of the interactions of Rap proteins with their specific Phr peptide inhibitor or their target Spo0F.

Additionally, we have structurally characterized the Spo0E-family of proteins by determining the 3-dimensional structure of 2 members of the family from
B anthracis. The BA1655 and BA5174 proteins are each composed of 2 antiparallel α-helices flanked by flexible regions at the termini. BA5174 is a monomer, and BA1655 is a dimer. The signature motif of amino acids (serine–glutamine–glutamic acid–leucine–aspartic acid) involved in the interaction with the target Spo0A is situated in the middle of helix α2 with its polar residues projecting outward. The role of the signature motif in the phosphatase activity of Spo0E is being investigated.

Signal Transduction in Enterococcus faecalis

M. Perego

Enterococci are commensal bacteria within the intestinal tract in mammals but also can cause disease in compromised hosts. The acquisition of resistance to multiple antibiotics by enterococci makes infections caused by these microorganisms clinically challenging. The ability of the bacteria to adapt and respond to different environmental stimuli, including the host environment, led my group to investigate the role of 2-component signal transduction in the physiology and pathogenesis of Enterococcus faecalis.

We identified 17 2-component systems consisting of a sensory histidine kinase and a cognate response regulator. We inactivated each nonessential response regulator and tested the effect of the deletions on a number of physiologic conditions. We found defects in growth, antibiotic resistance, stress response, and formation of biofilms.

Analysis of the 2-component system encoded by the gene fsr revealed that this system is the only one that affects growth of enterococci as a biofilm on solid surfaces, because the system regulates the transcription of gelatinase, a zinc metalloprotease. In recent studies, we focused on the molecular characterization of the FsrA response regulator and transcription factor. We showed phosphorylation of FsrA by the FsrC histidine kinase in vitro and binding of FsrA to the promoter regions of fsrC, fsrB, and gelE, the gene that encodes gelatinase. Once we identify the conserved sequences for FsrA DNA-binding recognition, we will analyze the E faecalis genome sequence to complete the list of genes regulated by this transcription factor.

Publications

Bongiorni, C., Stoessel, R., Perego, M. Negative regulation of Bacillus anthracis sporulation by the Spo0E family of phosphatases. J. Bacteriol. 189:2637, 2007.

Grenha, R., Rzechorzek, N.J., Brannigan, J.A., de Jong, R.N., Ab, E., Diercks, T., Truffault, V., Ladds, J.C., Fogg, M.J., Bongiorni, C., Perego, M., Kaptein, R., Wilson, K.S., Folkers, G.E., Wilkinson, A.J. Structural characterization of Spo0E-like protein-aspartic acid phosphatases that regulate sporulation in bacilli. J. Biol. Chem. 281:37993, 2006.

McLaughlin, P.D., Bobay, B.G., Regel, E.J., Thompson, R.J., Hoch, J.A., Cavanagh, J. Predominantly buried residues in the response regulator Spo0F influence specific sensor kinase recognition. FEBS Lett. 581:1425, 2007.

Santelli, E., Liddington, R.C., Mohan, M.A., Hoch, J.A., Szurmant, H. The crystal structure of Bacillus subtilis YycI reveals a common fold for two members of an unusual class of sensor histidine kinase regulatory proteins. J. Bacteriol. 189:3290, 2007.

Smits, W.K., Bongiorni, C., Veening, J.W., Hamoen, L.W., Kuipers, O.P., Perego, M. Temporal separation of distinct differentiation pathways by a dual specificity Rap-Phr system in Bacillus subtilis. Mol. Microbiol. 65:103, 2007.

Szurmant, H., Mohan, M.A., Imus, P.M., Hoch, J.A. YycH and YycI interact to regulate the essential YycFG two-component system in Bacillus subtilis. J. Bacteriol. 189:3280, 2007.

Tsvetanova, B., Wilson, A.C., Bongiorni, C., Chiang, C., Hoch, J.A., Perego, M. Opposing effects of histidine phosphorylation regulate the AtxA virulence transcription factor in Bacillus anthracis. Mol. Microbiol. 63:644, 2007.

White, A.K., Hoch, J.A., Grynberg, M., Godzik, A., Perego, M. Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase. J. Bacteriol. 188:6354, 2006.