Sporulation in prokaryotes and lower eukaryotes.

During the past year, highlights in sporulation research include the demonstration that phosphorylation of SpoOA is a critical factor in Bacillus subtilis development; the identification of C alpha proteins, adenylyl cyclase and protein kinase A genes in Dictyostelium; proof that an endogenous antisense RNA regulates gene expression in Dictyostelium; and characterization of a second type of differentiated cell in Myxococcus.

Signal transduction in Bacillus subtilis sporulation.

The initiation of sporulation in Bacillus subtilis is regulated by a signal transduction system leading to activation (by phosphorylation) of the Spo0A transcription factor. Activated Spo0A controls the expression of genes encoding different RNA polymerase sigma factors, whose synthesis and activities are related to morphological events and intercompartmental communication between the developing forespore and the mother cell.

Protein aspartate phosphatases control the output of two-component signal transduction systems.

Phosphorylation or dephosphorylation of an aspartate regulates the output activity of the response regulator of two-component signaling systems. Signal input in these systems is dependent on signal-transducing kinases, which can respond to a variety of signal ligands and, in some cases, to small phosphorylated metabolic intermediates. The kinase component of many two-component signaling systems also displays a response regulator-phosphate phosphatase activity that inactivates the response regulator in response to signals. Newly discovered kinase-independent phosphatases allow additional signals to influence the extent of response-regulator phosphorylation. Such phosphatases are prevalent in signal transduction systems controlling complex processes, such as the initiation of development in microorganisms.

Initiation of bacterial development.

The discovery of a mitotic apparatus in bacteria has led to significant recent progress being made in understanding the regulatory connections between the cell cycle, chromosome segregation and the onset of developmental processes in sporulation. The control of developmental transcription by antagonism between protein kinase and protein phosphatase reached a new level of complexity with the discovery of peptide inhibitors of protein phosphatases that cycle between the interior and exterior cell surface as information messengers. New mechanisms of developmental regulation are being uncovered in a variety of microbial systems.

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.

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.

Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis.

BACKGROUND: Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS: We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS: The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

A clinical evaluation of serum C3DP levels in individuals with malignant diseases.

A human serum DNA-binding protein (C3DP) derived from complement component C3 has been found in elevated concentrations in the sera of individuals with malignant diseases. An assay system has been devised which reveals quantitative values of serum C3DP levels. Results obtained using this system indicate that normal human sera have an average C3DP level of 242 mug/ml (range, 40 to 146), whereas sera from individuals with active carcinomas have an average C3DP level of 242 mug/ml (range, 146 to 400). Sera from individuals with active leukemias, lymphomas, and melanomas all had elevated levels of C3DP, whereas sera from individuals with polycythemia vera or other nonmalignant diseases had normal or only slightly elevated C3DP concentrations. No tissue specificity seems to be required for malignant growths to result in elevated C3DP serum concentrations. The levels of C3DP in 79% of the individuals who experienced disease remission were found to decline to normal values, concurrent with the disease regression. Patients who did not respond to therapy regimens retained high C3DP levels.

Regulation of lactate dehydrogenase synthesis in Bacillus subtilis.

The regulation of lactate dehydrogenase in Bacillus subtilis was determined under a variety of growth conditions and in mutants blocked in the citric acid cycle. The synthesis of lactate dehydrogenase increased sharply concomitantly upon the exhaustion of glucose from the medium and the onset of the stationary phase. The synthesis of lactate dehydrogenase may be under catabolite repression control. Studies with mutants blocked in the citric acid cycle showed that lactate dehydrogenase is regulated independently of either the oxidative or reductase branches of the cycle. Certain citric acid cycle mutants, e.g., aconitase or succinate dehydrogenase, exhibited very low levels of lactate dehydrogenase while others, e.g., malate dehydrogenase or isocitrate dehydrogenase, showed normal levels. A stage O sporulation mutant expressed levels of lactate dehydrogenase more than one thousand-fold higher than the low group of citric acid cycle mutants. The induction of lactate dehydrogenase was shown to be independent of the accumulation of its substrate, pyruvate.

Molecular recognition in signal transduction: the interaction surfaces of the Spo0F response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis.

The phosphorelay, a signal transduction pathway composed of two-component regulatory proteins, mediates the initiation of sporulation in Bacillus subtilis. Environmental and physiological signals activate the autophosphorylation of histidine kinases, KinA and KinB, which transfer the phosphoryl group to Spo0F, a single domain homolog of the two-component response regulator. Phosphorylated Spo0F passes the phosphate to the final transcriptional regulator, Spo0A, through a phosphotransferase, Spo0B. Spo0F shares significant homology with other members of the response regulator family. It displays a (beta/alpha)5-barrel scaffold with the active site situated at the carboxyl end of the beta strands. The molecular recognition of Spo0F with its cognate proteins was investigated using a comprehensive strategy termed alanine-scanning mutagenesis. Of the total 124 residues, 79 in the region of helices and loops were individually changed to alanine using site-directed mutagenesis. The mutants with notable in vivo sporulation phenotypes were further examined in vitro to identify the corresponding effect in each protein-protein interaction. This study revealed that most, if not all, protein-protein interactions involve the residues in the vicinity of the active site. The surface-exposed residues critical for the interactions with KinA or Spo0B were identified. Surprisingly, although these interaction proteins are very different, they recognize subsets of residues comprising a common surface of Spo0F.

The Bacillus subtilis response regulator Spo0A stimulates transcription of the spoIIG operon through modification of RNA polymerase promoter complexes.

Sporulation in Bacillus subtilis is dependent on the response regulator Spo0A, which both represses and activates transcription in vitro. The activity of Spo0A is increased by phosphorylation. We previously demonstrated that the phosphorylation increased the ability of Spo0A to stimulate in vivo transcription from the promoter for the spoIIG operon, one of the operons known to be regulated by Spo0A in vivo. In the work reported here we have examined the kinetics of transcription initiation at the spoIIG operon promoter using a single round transcription assay and the kinetics of formation of spoIIG promoter-RNA polymerase complexes using DNase I footprinting. Both the kinetic assays and the footprint assays indicated that the initial binding of the polymerase to the template was not dependent on the presence of Spo0A. The phosphorylated form of Spo0A stimulated the rate of initiation by affecting a step that occurred after the initial interaction of the polymerase with the template. Phosphorylation of Spo0A may stimulate transcription by modifying preinitiation complexes containing the polymerase and the promoter.

Chromosomal location of pleiotropic negative sporulation mutations in Bacillus subtilis.

Genetic analysis by PBS-1 transduction and transformation of a large group of pleiotropic negative sporulation mutants has shown that mutations of this phenotype may be located in five genetically distinct regions. The first group of mutant sites, spoA mutations, is located in the terminal region of the chromosome and linked to the lys-1 marker by PBS-1 transduction. The second group, spoB mutations, is located between phe-1 and the attachment site for the lysogenic bacteriophage varphi 105. Fine structure analysis of the mutant sites within the spoB locus has been accomplished. A third location for mutants of this phenotype, spoE mutants, was found between the metC3 and ura-1 markers. Two mutants were found at this site and both were capable of sporulation, in contrast to the rest of the pleiotropic sporulation mutants. A fourth chromosomal site, spoH mutations, was found near the ribosomal and RNA polymerase loci. A large group of mutant sites, spoF mutations, was found to be linked to each other by recombination index analysis in transformation but unlinked to any of the known auxotrophic mutations comprising the chromosomal map. All mutants analyzed showing a pleiotropic negative phenotype were found to map within one of these five regions. Interspecific transformation with Bacillus amyloliquefaciens as donor has shown that all of the pleiotropic negative sporulation mutations are conserved relative to a selected group of auxotrophic markers. The degree of conservation in decreasing order is: spoH > spoF = spoB > spoA.

Characterization of antigens on human lymphocytes coded by messenger RNA translated in vitro.

Translation and immunoprecipitation were used to identify messenger RNAs (mRNAs) coding for surface antigens expressed on human lymphoblastoid cells. The mRNAs were extracted from several human lymphoid cell lines as well as from fibroblastoid lines. These mRNAs were translated in vitro, and the translation products were reacted with xenoantisera raised against the antigens on human lymphoid cells. Products immunoprecipitated by these antisera were analysed by electrophoresis and fluorography. Four antisera immunoprecipitated a polypeptide with a mol. wt (MW) of approximately 32,000 (p32) from translations programmed with mRNA extracted from all the cell lines. Two antisera immunoprecipitated, in addition to p32, another polypeptide with a MW of approximately 25,000 (p25) only from translations programmed with RNA from lymphoid cell lines. p25 mRNA in the different lymphoid cell lines fell into three basic abundance classes as determined by in vitro translation and immunoprecipitation. Cells from two Burkitt's lymphomas (Raji and Daudi) did not express detectable p25 mRNA. Two T-lymphoblastoid lines (Molt-4 and 1301) contained five- to 10-fold less p25 mRNA than the B-lymphoid cell lines (Victor, RPMI-8866, RPMI-6410, RPMI-8226 and RPMI-1788). Both p32 and p25 were expressed on the cell surface inasmuch as lymphoblastoid cells adsorbed antibodies to both polypeptides. Human fibroblast, Raji or Daudi cells adsorbed anti-p32 antibodies from the antiserum but not anti-p25. Quantitative absorptions of the antiserum with T- or B-lymphoblastoid cells was used to determine the relative amounts of p32 and p25 expressed on the cell surface. B-lymphoblastoid cells were found to express two- to five-fold more p25 on the cell surface then T-lymphoblastoid cells. p25 does not represent an immunoglobulin light-chain precursor inasmuch as a 1000-fold excess of unlabeled human Ig did not compete with p25 translated in vitro for binding by its respective antibody.

1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.

Mutational dissociation of the positive and negative regulatory properties of the Spo0A sporulation transcription factor of Bacillus subtilis.

The Spo0A regulatory protein controls the onset of stationary phase and sporulation by controlling transcription in both a negative and a positive manner depending on the promoter affected. Missense mutations, e.g., spo0A9V, which result in alterations in the eleventh amino acid preceding the C terminus of the Spo0A protein, give rise to a protein active as a negative regulator of the abrB gene but unable to activate transcription of the spoIIA gene. Second-site suppressors of spo0A9V occurred within the spo0A gene at codons 162 and 174. These suppressors did not suppress a spo0F mutation, indicating that the suppressed protein still requires phosphorylation for activity. The results suggest that the C terminus of Spo0A interacts with the transcription complex to activate transcription.

Identification of communication networks in Spo0F: a model for phosphorylation-induced conformational change and implications for activation of multiple domain bacterial response regulators.

Fundamental to understanding the mechanism by which phosphorylation activates bacterial signal transduction response regulator proteins is the identification of regions and residues that are responsible for the phosphorylation-induced conformational change. Here we review results from structural and protein dynamics investigations, and combine them with mutagenesis studies on the response regulator protein SpoOF to suggest a model in which a network of buried and surface residues link surface regions required for protein:protein interactions to the site of phosphorylation. The network described for SpoOF may provide pathways through which information is transmitted from the site of phosphorylation, propagating a conformational change many angstroms away. The general applicability of the communication network model for all bacterial response regulator proteins is discussed.

Developing inhibitors to selectively target two-component and phosphorelay signal transduction systems of pathogenic microorganisms.

Two-component signal transduction systems and their expanded variants known as phosphorelays are integral elements of the virulence and antimicrobial resistance responses of a wide range of pathogenic bacteria and fungi and also regulate essential functions. As a consequence, two-component systems and phosphorelays are recognized targets for the development of novel antimicrobial agents and a number of chemically synthesized inhibitors from different chemical classes have been identified by compound library screens. However, in the majority of cases these compounds do not appear to be selective for signal transduction pathways and exert their effect by multiple mechanisms of action. The key to designing molecules to selectively disrupt signal transduction may lie with the conserved features of response regulators and the structural analysis of complexes of signaling proteins.

Spo0A activates and represses its own synthesis by binding at its dual promoters.

The Spo0A protein of Bacillus subtilis controls the onset of sporulation by regulating transcription of various genes in both positive and negative manners depending on the promoters affected. The expression of the spo0A gene occurs from two promoters (Pv,Ps), separated by 148 bp, and transcription switches from Pv to Ps early in the sporulation program. DNase I footprint analysis of the spo0A promoter region revealed three distinct sites of Spo0A binding: -4 to +19 relative to Pv, -17 to +1 relative to Ps, and a region between Pv and Ps. The Pv region and the region between the two promoters was sufficient for repression of Pv. Induction of Ps also required these regions which are upstream of -52 relative to Ps. Mutant Spo0A proteins containing asp----asn mutations at asp10 and asp56 were inactive in repression of the abrB promoter in vivo yet still retained DNA-binding activity. The results presented are consistent with a model in which the phosphorylated form of Spo0A acts directly at its promoters to achieve induction of Ps and repression of Pv. These effects at the spo0A promoter were independent of the presence of the major kinase, KinA.

The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis.

The spoIIA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in Bacillus subtilis. We have used a single round in vitro transcription assay to probe requirements for transcription initiation at the spoIIA promoter. Core RNA polymerase or holoenzyme containing sigma A was reconstituted with sigma H protein and used to transcribe the spoIIA promoter. Formation of heparin resistant transcription initiation complexes required that the spoIIA template be supercoiled. Topoisomers of the spoIIA template were created and transcribed at various temperatures. Changes in the superhelicity of template DNA had a significant influence on the amount of transcription complexes formed at the spoIIA promoter.

Histidine kinase-mediated signal transduction systems of pathogenic microorganisms as targets for therapeutic intervention.

Pathogenic bacteria must be able to sense and respond rapidly to signals emanating from the host environment and use the signals to modulate the expression of genes required for the infection process. Two-component signal transduction systems, and their more complex variants known as phosphorelays, are woven within the fabric of bacterial cellular regulatory processes and are used to regulate the expression of genes involved in the virulence and antibiotic resistance responses of a large number of pathogens of major public health concern. The emergence of strains of pathogenic bacteria that are resistant to multiple antibiotics has driven the search for new targets and/or modes of action for anti-microbial agents. The presence of essential two-component systems in bacteria and the central role that these regulatory systems play in virulence and antibiotic resistance has meant that two-component systems and phosphorelays have been recognized as targets for antimicrobial intervention. This review will discuss the role of these signal transduction pathways in virulence responses and antibiotic sensitivity of pathogenic microorganisms and their potential use as targets for antimicrobial therapy. In addition, the current status on the development of inhibitors specific for two-component systems will be discussed.