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.

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.

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.

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.

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.

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.

An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteins.

Two-component signal transduction systems with membrane-embedded sensor histidine kinases are believed to recognize environmental signals and transduce this information over the cellular membrane to influence the activity of a transcription factor to which they are mated. The YycG sensor kinase of Bacillus subtilis, containing two transmembrane helices, is subject to a complicated activity-control circuit involving two other proteins with N-terminal transmembrane helices, YycH and YycI. Truncation studies of YycH and YycI demonstrated that the individual transmembrane helices of these proteins are sufficient to adjust YycG activity, indicating that this control is achieved at the membrane level. A replica exchange molecular dynamics computational approach generated in silico structural models of the transmembrane helix complex that informed mutagenesis studies of the YycI transmembrane helix supporting the accuracy of the in silico model. The results predict that signal recognition by any of the extracellular domains of the sensor histidine kinase YycG or the associated proteins YycH and YycI is transmitted across the cellular membrane by subtle alterations in the positions of the helices within the transmembrane complex of the three proteins.

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.

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.

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.