RESEARCH
Signal Transduction in Cellular Differentiation
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 over 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 (Fig.1). 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.
Figure 1
The cell cycle of Bacillus subtilis. Mutations in the spo0 genes block the initiation of the sporulation process.
The key to understanding the initiation of sporulation is understanding the mechanism of Spo0A phosphorylation. We have shown that the pathway to Spo0A activation is a sequential series of phosphorylation reactions termed a multicomponent phosphorelay (Fig.2). The initial event in the phosphorelay is the activation of multiple kinases that phosphorylate the sporulation-specific response regulator Spo0F in response to environmental and metabolic signals. Spo0F acts as a secondary 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 of the kinases to autophosphorylate. This is followed by three sequential phosphotransferase reactions that produce Spo0A~P, the crucial transcription regulator for sporulation.
Figure 2
The phosphorelay for sporulation initiation in B. subtilis. In the phosphorelay, the activity of five sporulation kinases (KinA, B, C, D and E) is counteracted by six response regulator aspartyl phosphate phosphatases (RapA, B, C, Spo0E, YisI and YnzD).
The flow of phosphate through the phosphorelay to Spo0A is highly controlled at several levels. Although the primary signals transduced by the two kinases, KinA and KinB, 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. It is now clear that all the signals that affect sporulation cannot be processed by the kinases alone. Access to the phosphorelay for additional signals is provided by two 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 physiological 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 activity of the kinases and phosphatases on the phosphorelay acts 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.
Molecular Dynamics of Response Regulators 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, though common to all of these proteins is a regulatory domain of ~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 five a -helices surrounding five parallel b -strands, forming a hydrophobic sheet; a structure in common with other response regulators (Fig.3). The fold brings three aspartic acid residues into close proximity to form the binding pocket to accept the phosphoryl group. From structural studies, we have been able to determine in the orientation of secondary structure elements in the putative recognition surfaces and the relative charge distribution of residues surrounding the site of phosphorylation.
Figure 3 Crystal structure of the Spo0F response regulator.
We have been studying the backbone dynamics of the Spo0F protein. In conjunction with alanine scanning mutagenesis studies, our dynamics studies have allowed 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 its cognate kinase following 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, which move in a dynamically-concerted fashion, driven by the motion of the imidazole ring of His101. We are proposing 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 conformation change in the aspartic acid binding pocket upon phosphorylation. Movement of the ring disrupts packing interactions which alters the topology of the kinase recognition site, thereby causing the kinase to dissociate. This model represents one of the first connections between protein dynamics and its specific biological function.
Regulatory Circuits Coordinating Chromosome Segregation and Development
The mechanism by which bacterial chromosomes are equipartitioned into daughter cells at division had remained obscure for decades until recent studies showed that bacterial nucleoids undergo abrupt movements reminiscent of mitosis in eukaryotes. The mechanisms of chromosome segregation are distinct from eukaryotes mainly by the lack of obvious cytoskeleton and mitotic spindle apparatus. Nevertheless active chromosome segregation mechanisms exist in bacteria that accomplish the same task. Two of the proteins involved in regulation of the fidelity of chromosome segregation are the Spo0JA and Spo0JB proteins that are related to the ParA and ParB proteins encoded by extrachromosomal elements that ensure their equal partition to daughter cells. Spo0JB binds to the origin region of the chromosome and migrates with the origin during the mitotic process (Fig.4). Mutants lacking this protein are severely affected in the ability to initiate sporulation development and this deficiency is due to the Spo0JA protein. This 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 have 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 is likely to be much more complex than is apparent at this time.
Figure 4
Localization of SpoOJB in B. subtilis cells by immunofluorescence (for details see Glaser, et al., 1997, Genes & Development 11:1160-1168). Antibacterial agents that inhibit two-component signal transduction systems
Infectious disease is the number one cause of mortality in the world despite the armamentarium of antibacterial agents developed over the last half century. Bacterial strains have evolved during this time that are recalcitrant to our best efforts to destroy them, and they have begun to compromise the treatment of infectious disease particularly in the hospital setting. Resistance to antibacterials takes many forms and is a probable consequence of widespread use and misuse of antibiotics. Thus, the search for new antibacterials directed toward new targets is not only a continuous 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, the inhibition of expression of bacterial virulence factors offers an opportunity for specific intervention at the level of host invasion through biochemical processes, which are clearly unique to the bacterial cell. It is now clear that virulence is an adaptive genetic response requiring the induction of genes coding for 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 two-component signal transduction systems employing a common phosphorylation-dependent mechanism of signal transduction that appears nearly ubiquitous in bacteria. Four features in particular make the two-component family attractive as a potential target for antimicrobials: (i) 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; (ii) pathogenic bacteria use two-component signal transduction to regulate expression of essential virulence factors that are required for survival inside the host; (iii) bacteria contain many two-component systems, and some of them are essential for viability and (iv) signal transduction in mammals occurs by different mechanisms. The yycG and yycF genes of B. subtilis encode a two-component signal transduction system that is essential for viability and appears to regulate cell division. This system is highly conserved in Gram-positive pathogens of the genera Staphylococcus, Streptococcus, and Enterococcus. Previous collaborative studies with the R.W. Johnson Pharmaceutical Research Institute identified a class of antibacterials effective against Gram-positive pathogens that were potent inhibitors of the kinase reaction in two-component systems. The relationship between kinase inhibition and the anti-bacterial properties of such molecules is under investigation. A search for new classes of inhibitors is underway.
Kinase - phosphatase competition in Bacterial Development
Recognition of the diverse signals involved in the initiation of sporulation is carried out by proteins that alter the level of phosphorylation of the Spo0A transcription factor. The pathway to Spo0A activation is a sequential series of phosphorylation and phosphotransfer reactions that occur in the multi-component phosphorelay. The phosphorelay signal transduction system is the province of signal integration and its multicomponent structure provides multiple entries for regulatory signals affecting the final goal of producing the appropriate level of Spo0A~P. The opposing activities of kinases and phosphatases are the major players influencing the output of the system in a manner similar to eukaryotic signal transduction systems. The Spo0E family of phosphatases specifically dephosphorylates the Spo0A~P component of the phosphorelay while three members of the Rap family of phosphatases specifically dephosphorylate the Spo0F~P intermediate. Because transcription of phosphatase-coding genes is under control of physiological states alternative to sporulation, protein phosphatases provide access to the phosphorelay for additional regulatory signals (Fig.2). In addition, the phosphatase activity of Rap proteins is differentially regulated by effector molecules. Phosphatase activity of RapA is regulated by a contrascribed gene, phrA, that encodes for a 44 amino acid protein. This PhrA protein is exported from the cell by the SecA dependent system, processed down to the carboxyl-terminal pentapeptide (ARNQT) and reimported through the oligopeptide transport system. Through in vivo and in vitro study we have shown that the pentapeptide specifically and directly inhibits the phosphatase activity of RapA. The journey of the phrA gene product to its final pentapeptide form is defined as an export-import control circuit with the function of a timing device that coordinates alternative physiological functions with sporulation initiation (Fig.5).
Figure 5
The export-import control circuit for PhrA processing. Once internalized by the oligopeptide system, the PhrA pentapeptide directly inhibits the dephosphorylation activity of RapA on Spo0F~P. This allows production of Spo0A~P and thus the initiation of sporulation.
While RapB does not appear to be controlled by a cognate Phr peptide, the activity of RapE is directly inhibited by a PhrE pentapeptide (SRNVT) which, unlike PhrA, is located nine amino acids from the C-terminal end. The Rap family of phosphatases consists of eleven chromosomal genes. The Rap proteins show 30-50% identities in amino acid residues and seven of them are associated with a PhrA-like protein. Preliminary studies indicate that the remaining Rap phosphatases are likely to have roles in processes, other than sporulation, that are controlled by two-component signal transduction systems.
Phosphorelay Proteins: Structure and Function
Phosphoryl group transfers from one protein to another is the basis for propagating information in phosphorylation activated signal transduction. Bacteria and many lower eukaryotes (e.g., yeast, fungi and plants) have adapted the phosphorylation-dependent two-component paradigm to couple signal recognition, signal transduction and signal response. The initiation of developmental gene expression in the sporulation of Bacillus subtilis is directly controlled by a more complex version of a two-component system termed a phosphorelay. In a phosphorelay, the histidine kinases are dephosphorylated by a common response regulator, Spo0F. Phosphorylated Spo0F is the substrate for the Spo0B phosphotransferase which serves to mediate phosphoryl transfer from Spo0F to Spo0A, the ultimate transcription factor. In recent years, we have determined the crystal structures of Spo0F and Spo0B. Phosphoryl transfer between the Spo0B phosphotransferase and the response regulators is achieved through a transient interaction and this interaction has been trapped in the crystal lattice of a molecular complex of these two proteins. Spo0B is dimer and the two monomers associate through the formation of a four helix bundle; the active histidines protrude from the four-helix bundle. Thus Spo0B has two active sites for binding Spo0F. Two Spo0F molecules associate with a Spo0B dimer bringing the active aspartates and active histidines in close proximity for catalysis (Fig.6). The environment resulting from the complex formation is favorable for stabilizing the pentacordinated reaction intermediate.
Figure 6
A view of the Spo0B:Spo0F complex along the four-helix bundle. The active histidines of Spo0B and active aspartates of Spo0F are in close proximity for phosphoryl transfer. The basis of recognition and response regulator specificity has been established. Recognition is achieved through the complementarity in shape and specificity through interactions of the residues surrounding the active site and the first a helix of Spo0F. The interacting residues of Spo0F are conserved in Spo0A to a high degree and therefore Spo0A also must interact with Spo0B in a similar manner. The interaction surface differs from that used by the chemotaxis protein CheY to interact with the P2 domain of the CheA suggesting several surfaces of the response regulators are involved in molecular recognition.
Signal transduction in Gram-positive pathogenic microorganisms
Gram-positive pathogens in the genera Streptococcus, Staphylococcus and Enterococcus are leading causes of human infections, causing diseases such as pneumonia, meningitis, bacterema, endocarditis and necrotizing fascitis. Some spore-forming Gram-positive organisms such as Bacillus anthracis and Clostridium botulinum are not only pathogens but also potential bioterror agents. To develop a maximally effective response to the constant threat posed by these organisms and the risk of bioterrorist attacks, the scientific and medical community will require new knowledge of the organisms, their genetics and pathogenesis in order to improve diagnostic techniques, phophylactic and therapeutic regimens.
Genomic sequencing of pathogens is providing an enormous amount of information that will facilitate developments toward the understanding of bacteria and their pathogenesis pathways, identification of common themes in virulence, and new targets to exploit toward the development of new defenses. Bacterial two-component signal transduction systems play a pivotal role in the process of pathogenesis. These systems enable bacterial cells to mount an adoptive response and cope with diverse environmental stresses, including nutrient deprivation and phogocytosis. These systems are also key players in the overwhelming emergence of antibiotic resistance that poses an additional serious threat to human health.
We are interested in the understanding of the molecular mechanisms of bacterial pathogenesis controlled by two-component signal transduction systems. The availability of the Enterococcus faecalis and Bacillus anthracis genome sequence allowed us to identify the key components of the bacterial regulatory mechanisms carried out by two-component signal transduction. By means of genetics, molecular biology, biochemistry and structural biology, we aim at defining the signaling and regulatory network responsible for pathogenicity in these organisms.