A Life in Bacillus subtilis Signal Transduction.

This is a tale of how technology drove the discovery of the molecular basis for signal transduction in the initiation of sporulation in Bacillus subtilis and in bacterial two-component systems. It progresses from genetics to cloning and sequencing to biochemistry to structural biology to an understanding of how proteins evolve interaction specificity and to identification of interaction surfaces by statistical physics. This is about how the people in my laboratory accomplished this feat; without them little would have been done.

Evidence that YycJ is a novel 5'-3' double-stranded DNA exonuclease acting in Bacillus anthracis mismatch repair.

The most important system for correcting replication errors that survive the built in editing system of DNA polymerase is the mismatch repair (MMR) system. We have identified a novel mutator strain yycJ in Bacillus anthracis. Mutations in the yycJ gene result in a spontaneous mutator phenotype with a mutational frequency and specificity comparable to that of MMR-deficient strains such as those with mutations in mutL or mutS. YycJ was annotated as a metallo-ß-lactamase (MßL) super family member with unknown activity. In this study we carried out a biochemical characterization of YycJ and demonstrated that a recombinant YycJ protein possesses a 5'-3' exonuclease activity at the 5' termini and at nicks of double-stranded DNA. This activity requires a divalent metal cofactor Mn2+ and is stimulated by 5'-phosphate ends of duplex DNA. The mutagenesis of conserved amino acid residues revealed that in addition to the five MßL family conserved motifs, YycJ appears to have its specific motifs that can be used to distinguish YycJ from other closely related MßL family members. A phylogenetic survey showed that putative YycJ homologs are present in several bacterial phyla as well as in members of the Methanomicrobiales and Thermoplasmales from Archaea. We propose that YycJ represents a new group of MßL fold exonucleases, which is likely to act in the recognition of MMR entry point and subsequent removal of the mismatched base in certain MutH-less bacterial species.

Statistical analyses of protein sequence alignments identify structures and mechanisms in signal activation of sensor histidine kinases.

Statistical analyses of genome sequence-derived protein sequence data can identify amino acid residues that interact between proteins or between domains of a protein. These statistical methods are based on evolution-directed amino acid variation responding to structural and functional constraints in proteins. The identified residues form a basis for determining structure and folding of proteins as well as inferring mechanisms of protein function. When applied to two-component systems, several research groups have shown they can be used to identify the amino acid interactions between response regulators and histidine kinases and the specificity therein. Recently, statistical studies between the HisKA and HATPase-ATP-binding domains of histidine kinases identified amino acid interactions for both the inactive and the active catalytic states of such kinases. The identified interactions generated a model structure for the domain conformation of the active state. This conformation requires an unwinding of a portion of the C-terminal helix of the HisKA domain that destroys the inactive state residue contacts and suggests how signal-binding determines the equilibrium between the inactive and active states of histidine kinases. The rapidly accumulating protein sequence databases from genome, metagenome and microbiome studies are an important resource for functional and structural understanding of proteins and protein complexes in microbes.

Structural basis of histidine kinase autophosphorylation deduced by integrating genomics, molecular dynamics, and mutagenesis.

Signal transduction proteins such as bacterial sensor histidine kinases, designed to transition between multiple conformations, are often ruled by unstable transient interactions making structural characterization of all functional states difficult. This study explored the inactive and signal-activated conformational states of the two catalytic domains of sensor histidine kinases, HisKA and HATPase. Direct coupling analyses, a global statistical inference approach, was applied to >13,000 such domains from protein databases to identify residue contacts between the two domains. These contacts guided structural assembly of the domains using MAGMA, an advanced molecular dynamics docking method. The active conformation structure generated by MAGMA simultaneously accommodated the sequence derived residue contacts and the ATP-catalytic histidine contact. The validity of this structure was confirmed biologically by mutation of contact positions in the Bacillus subtilis sensor histidine kinase KinA and by restoration of activity in an inactive KinA(HisKA):KinD(HATPase) hybrid protein. These data indicate that signals binding to sensor domains activate sensor histidine kinases by causing localized strain and unwinding at the end of the C-terminal helix of the HisKA domain. This destabilizes the contact positions of the inactive conformation of the two domains, identified by previous crystal structure analyses and by the sequence analysis described here, inducing the formation of the active conformation. This study reveals that structures of unstable transient complexes of interacting proteins and of protein domains are accessible by applying this combination of cross-validating technologies.

Dynamics and activation in response regulators: the ß4-α4 loop.

Two-component signal transduction systems of microbes are a primary means to respond to signals emanating from environmental and metabolic fluctuations as well as to signals coordinating the cell cycle with macromolecular syntheses, among a large variety of other essential roles. Signals are recognized by a sensor domain of a histidine kinase which serves to convert signal binding to an active transmissible phosphoryl group through a signal-induced ATP-dependent autophosphorylation reaction directed to histidine residue. The sensor kinase is specifically mated to a response regulator, to which it transfers the phosphoryl group that activates the response regulator's function, most commonly gene repression or activation but also interaction with other regulatory proteins. Two-component systems have been genetically amplified to control a wide variety of cellular processes; for example, both Escherichia coli and Pseudomonas aeruginosa have 60 plus confirmed and putative two-component systems. Bacillus subtilis has 30 plus and Nostoc punctiformis over 100. As genetic amplification does not result in changes in the basic structural folds of the catalytic domains of the sensor kinase or response regulators, each sensor kinase must recognize its partner through subtle changes in residues at the interaction surface between the two proteins. Additionally, the response regulator must prepare itself for efficient activation by the phosphorylation event. In this short review, we discuss the contributions of the critical ß4-α4 recognition loop in response regulators to their function. In particular, we focus on this region's microsecond-millisecond timescale dynamics propensities and discuss how these motions play a major role in response regulator recognition and activation.

Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum.

The phosphorylated Spo0A transcription factor controls the initiation of endospore formation in Clostridium acetobutylicum, but genes encoding key phosphorelay components, Spo0F and Spo0B, are missing in the genome. We hypothesized that the five orphan histidine kinases of C. acetobutylicum interact directly with Spo0A to control its phosphorylation state. Sequential targeted gene disruption and gene expression profiling provided evidence for two pathways for Spo0A activation, one dependent on a histidine kinase encoded by cac0323, the other on both histidine kinases encoded by cac0903 and cac3319. Purified Cac0903 and Cac3319 kinases autophosphorylated and transferred phosphoryl groups to Spo0A in vitro, confirming their role in Spo0A activation in vivo. A cac0437 mutant hyper-sporulated, suggesting that Cac0437 is a modulator that prevents sporulation and maintains cellular Spo0A∼P homeostasis during growth. Accordingly, Cac0437 has apparently lost the ability to autophosphorylate in vitro; instead it catalyses the ATP-dependent dephosphorylation of Spo0A∼P releasing inorganic phosphate. Direct phosphorylation of Spo0A by histidine kinases and dephosphorylation by kinase-like proteins may be a common feature of the clostridia that may represent the ancestral state before the great oxygen event some 2.4 billion years ago, after which additional phosphorelay proteins were recruited in the evolutionary lineage that led to the bacilli.

A role for the essential YycG sensor histidine kinase in sensing cell division.

The YycG sensor histidine kinase co-ordinates cell wall remodelling with cell division in Gram-positive bacteria by controlling the transcription of genes for autolysins and their inhibitors. Bacillus subtilis YycG senses cell division and is enzymatically activated by associating with the divisome at the division septum. Here it is shown that the cytoplasmic PAS domain of this multi-domain transmembrane kinase is a determining factor translocating the kinase to the division septum. Furthermore, translocation to the division septum, per se, is insufficient to activate YycG, indicating that specific interactions and/or ligands produced there are required to stimulate kinase activity. N-terminal truncations of YycG lose negative regulation of their activity inferring that this regulation is accomplished through its transmembrane and extramembrane domains interacting with the membrane associated YycH and YycI proteins that do not localize to the divisome. The data indicate that YycG activity in non-dividing cells is suppressed by its interaction with YycH and YycI and its activation is co-ordinated to cell division in dividing cells by specific interactions that occur within the divisome.

Computational modeling of phosphotransfer complexes in two-component signaling.

Two-component signal transduction systems enable cells in bacteria, fungi, and plants to react to extracellular stimuli. A sensor histidine kinase (SK) detects such stimuli with its sensor domains and transduces the input signals to a response regulator (RR) by trans-phosphorylation. This trans-phosphorylation reaction requires the formation of a complex formed by the two interacting proteins. The complex is stabilized by transient interactions. The nature of the transient interactions makes it challenging for experimental techniques to gain structural information. X-ray crystallography requires stable crystals, which are difficult to grow and stabilize. Similarly, the mere size of these systems proves problematic for NMR. Theoretical methods can, however, complement existing data. The statistical direct coupling analysis presented in the previous chapter reveals the interacting residues at the contact interface of the SK/RR pair. This information can be combined with the structures of the individual proteins in molecular dynamical simulation to generate structural models of the complex. The general approach, referred to as MAGMA, was tested on the sporulation phosphorelay phosphotransfer complex, the Spo0B/Spo0F pair, delivering crystal resolution accuracy. The MAGMA method is described here in a step-by-step explanation. The developed parameters are transferrable to other SK/RR systems.

Inference of direct residue contacts in two-component signaling.

Since the onset of the genomic era more than 1000 bacterial genomes have been sequenced and several fold more are expected to be completed in the near future. These genome sequences supply a wealth of information that can be exploited by statistical methods to gain significant insights into cellular processes. In Volume 422 of Methods in Enzymology we described a covariance-based method, which was able to identify coevolving residue pairs between the ubiquitous bacterial two-component signal transduction proteins, the sensor kinase and the response regulator. Such residue position pairs supply interaction specificity in the light of highly amplified but structurally conserved two-component systems in a typical bacterium and are enriched with interaction surface residue pairings. In this chapter we describe an extended version of this method, termed "direct coupling analysis" (DCA), which greatly enhances the predictive power of traditional covariance analysis. DCA introduces a statistical inference step to covariance analysis, which allows to distinguish coevolution patterns introduced by direct correlations between two-residue positions, from those patterns that arise via indirect correlations, that is, correlations that are introduced by covariance with other residues in the respective proteins. This method was shown to reliably identify residue positions in spatial proximity within a protein or at the interface between two interaction partners. It is the goal of this chapter to allow an experienced programmer to reproduce our techniques and results so that DCA can soon be applied to new targets.

Long range dynamic effects of point-mutations trap a response regulator in an active conformation.

When a point-mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point-mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This 'conformational trapping' results in functionally hyperactive Spo0F. Consequently, point-mutations are seen to affect functionally critical motions both close to and far from the mutational site.

Interaction fidelity in two-component signaling.

Two component signal transduction systems and phosphorelays have been adapted and amplified by bacteria to respond to a multitude of environmental, metabolic and cell cycle signals while maintaining essentially identical structures for the domains responsible for recognition and phosphotransfer between the sensor histidine kinase and the response regulator. Co-crystal structures of these domains have revealed the variable residues at the interaction surface of the two components responsible for interaction specificity in signal transfer. This information has formed the basis for the development and validation of statistical methods to identify interaction residues and surfaces from compiled databases of interacting proteins and holds forth the promise of determining structures of multi-protein complexes and signaling networks.

Crystal structure of the transcriptional repressor PagR of Bacillus anthracis.

PagR is a transcriptional repressor in Bacillus anthracis that controls the chromosomal S-layer genes eag and sap, and downregulates the protective antigen pagA gene by direct binding to their promoter regions. The PagR protein sequence is similar to those of members of the ArsR repressor family involved in the repression of arsenate-resistance genes in numerous bacteria. The crystal structure of PagR was solved using multi-wavelength anomalous diffraction (MAD) techniques and was refined with 1.8 A resolution diffraction data. The PagR molecules form dimers, as observed in all SmtB/ArsR repressor family proteins. In the crystal lattice four PagR dimers pack together to form an inactive octamer. Model-building studies suggest that the dimer binds to a DNA duplex with a bend of around 4 degrees.

Two small c-type cytochromes affect virulence gene expression in Bacillus anthracis.

Regulated expression of the genes for anthrax toxin proteins is essential for the virulence of the pathogenic bacterium Bacillus anthracis. Induction of toxin gene expression depends on several factors, including temperature, bicarbonate levels, and metabolic state of the cell. To identify factors that regulate toxin expression, transposon mutagenesis was performed under non-inducing conditions and mutants were isolated that untimely expressed high levels of toxin. A number of these mutations clustered in the haem biosynthetic and cytochrome c maturation pathways. Genetic analysis revealed that two haem-dependent, small c-type cytochromes, CccA and CccB, located on the extracellular surface of the cytoplasmic membrane, regulate toxin gene expression by affecting the expression of the master virulence regulator AtxA. Deregulated AtxA expression in early exponential phase resulted in increased expression of toxin genes in response to loss of the CccA-CccB signalling pathway. This is the first function identified for these two small c-type cytochromes of Bacillus species. Extension of the transposon screen identified a previously uncharacterized protein, BAS3568, highly conserved across many bacterial and archeal species, as involved in cytochrome c activity and virulence regulation. These findings are significant not only to virulence regulation in B. anthracis, but also to analysis of virulence regulation in many pathogenic bacteria and to the study of cytochrome c activity in Gram-positive bacteria.

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.

A unique GTP-dependent sporulation sensor histidine kinase in Bacillus anthracis.

The Bacillus anthracis BA2291 gene codes for a sensor histidine kinase involved in the induction of sporulation. Genes for orthologs of the sensor domain of the BA2291 kinase exist in virulence plasmids in this organism, and these proteins, when expressed, inhibit sporulation by converting BA2291 to an apparent phosphatase of the sporulation phosphorelay. Evidence suggests that the sensor domains inhibit BA2291 by titrating its activating signal ligand. Studies with purified BA2291 revealed that this kinase is uniquely specific for GTP in the forward reaction and GDP in the reverse reaction. The G1 motif of BA2291 is highly modified from ATP-specific histidine kinases, and modeling this motif in the structure of the kinase catalytic domain suggested how guanine binds to the region. A mutation in the putative coiled-coil linker between the sensor domain and the catalytic domains was found to decrease the rate of the forward autophosphorylation reaction and not affect the reverse reaction from phosphorylated Spo0F. The results suggest that the activating ligand for BA2291 is a critical signal for sporulation and in a limited concentration in the cell. Decreasing the response to it either by slowing the forward reaction through mutation or by titration of the ligand by expressing the plasmid-encoded sensor domains switches BA2291 from an inducer to an inhibitor of the phosphorelay and sporulation.

The bicarbonate transporter is essential for Bacillus anthracis lethality.

In the pathogenic bacterium Bacillus anthracis, virulence requires induced expression of the anthrax toxin and capsule genes. Elevated CO2/bicarbonate levels, an indicator of the host environment, provide a signal ex vivo to increase expression of virulence factors, but the mechanism underlying induction and its relevance in vivo are unknown. We identified a previously uncharacterized ABC transporter (BAS2714-12) similar to bicarbonate transporters in photosynthetic cyanobacteria, which is essential to the bicarbonate induction of virulence gene expression. Deletion of the genes for the transporter abolished induction of toxin gene expression and strongly decreased the rate of bicarbonate uptake ex vivo, demonstrating that the BAS2714-12 locus encodes a bicarbonate ABC transporter. The bicarbonate transporter deletion strain was avirulent in the A/J mouse model of infection. Carbonic anhydrase inhibitors, which prevent the interconversion of CO2 and bicarbonate, significantly affected toxin expression only in the absence of bicarbonate or the bicarbonate transporter, suggesting that carbonic anhydrase activity is not essential to virulence factor induction and that bicarbonate, and not CO2, is the signal essential for virulence induction. The identification of this novel bicarbonate transporter essential to virulence of B. anthracis may be of relevance to other pathogens, such as Streptococcus pyogenes, Escherichia coli, Borrelia burgdorferi, and Vibrio cholera that regulate virulence factor expression in response to CO2/bicarbonate, and suggests it may be a target for antibacterial intervention.

Dual promoters control expression of the Bacillus anthracis virulence factor AtxA.

The AtxA virulence regulator of Bacillus anthracis is required for toxin and capsule gene expression. AtxA is a phosphotransferase system regulatory domain-containing protein whose activity is regulated by phosphorylation/dephosphorylation of conserved histidine residues. Here we report that transcription of the atxA gene occurs from two independent promoters, P1 (previously described by Dai et al. [Z. Dai, J. C. Sirard, M. Mock, and T. M. Koehler, Mol. Microbiol. 16:1171-1181, 1995]) and P2, whose transcription start sites are separated by 650 bp. Both promoters have -10 and -35 consensus sequences compatible with recognition by sigma(A)-containing RNA polymerase, and neither promoter depends on the sporulation sigma factor SigH. The dual promoter activity and the extended untranslated mRNA suggest that as-yet-unknown regulatory mechanisms may act on this region to influence the level of AtxA in the cell.

Co-evolving motions at protein-protein interfaces of two-component signaling systems identified by covariance analysis.

Short-lived protein interactions determine signal transduction specificity among genetically amplified, structurally identical two-component signaling systems. Interacting protein pairs evolve recognition precision by varying residues at specific positions in the interaction surface consistent with constraints of charge, size, and chemical properties. Such positions can be detected by covariance analyses of two-component protein databases. Here, covariance is shown to identify a cluster of co-evolving dynamic residues in two-component proteins. NMR dynamics and structural studies of both wild-type and mutant proteins in this cluster suggest that motions serve to precisely arrange the site of phosphoryl transfer within the complex.

Virulence gene expression is independent of ResDE-regulated respiration control in Bacillus anthracis.

The ResDE two-component system regulates the synthesis of several components of the aerobic and anaerobic respiratory pathways in bacilli. The ResD response regulator transcription factor has been implicated in the regulation of virulence factors in a number of gram-positive species, including Bacillus anthracis. The precise deletions of resD and resE in B. anthracis that retained the classical respiratory phenotypes did not affect the expression of the gene for the protective antigen of the anthrax toxin, pagA, or that of the toxin regulator, atxA. The results indicate that the loss of ResDE-controlled respiratory capacity does not affect the synthesis of anthrax toxin.