Identification, Functional Characterization, and Regulon Prediction of the Zinc Uptake Regulator (zur) of Bacillus anthracis - An Insight Into the Zinc Homeostasis of the Pathogen.

Zinc has an abounding occurrence in the prokaryotes and plays paramount roles including catalytic, structural, and regulatory. Zinc uptake regulator (Zur), a Fur family transcriptional regulator, is connoted in maintaining zinc homeostasis in the pathogenic bacteria by binding to zinc and regulating the genes involved in zinc uptake and mobilization. Zinc homeostasis has been marginally scrutinized in Bacillus anthracis, the top-rated bio-terror agent, with no decipherment of the role of Zur. Of the three Fur family regulators in B. anthracis, BAS4181 is annotated as a zinc-specific transcriptional regulator. This annotation was further substantiated by our stringent computational and experimental analyses. The residues critical for zinc and DNA binding were delineated by homology modeling and sequence/structure analysis. ba zur existed as a part of a three-gene operon. Purified BaZur prodigiously existed in the dimeric form, indicated by size exclusion chromatography and blue native-polyacrylamide gel electrophoresis (PAGE). Computational and manual strategies were employed to decipher the putative regulon of ba zur, comprising of 11 genes, controlled by six promoters, each harboring at least one Zur box. The DNA binding capability of the purified BaZur to the upstream regions of the ba zur operon, yciC, rpmG, znuA, and genes encoding a GTPase cobalamine synthesis protein and a permease was ascertained by electrophoretic mobility shift assays. The regulon genes, implicated in zinc uptake and mobilization, were mostly negatively regulated by BaZur. The ba zur expression was downregulated upon exposure of cells to an excess of zinc. Conversely, it exhibited a marked upregulation under N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) mediated zinc-depleted environment, adding credence to its negative autoregulation. Moreover, an increase in the transcript levels of the regulon genes znuA, rpmG, and yciC upon exposure of cells to TPEN connoted their role in combating hypo-zincemic conditions by bringing about zinc uptake and mobilization. Thus, this study functionally characterizes Zur of B. anthracis and elucidates its role in maintaining zinc homeostasis.

Biochemical characterization of the GTP-sensing protein, CodY of Bacillus anthracis.

The pleiotropism of the GTP-sensing transcriptional regulator CodY is evident by the gamut of processes that it regulates in almost all low G+C Gram-positive bacteria, including general metabolism, biosynthesis of some amino acids and transport systems, nitrogen uptake, sporulation, biofilm formation, motility and virulence. The role of CodY in virulence has been established in Bacillus anthracis, the top rated bioterrorism agent. In this study, we investigated the biochemical attributes of this global regulator. Homology modeling and sequence/structure analysis revealed putative GTP-binding residues in CodY of B. anthracis. CodY exhibited an interaction with the GTP as tested by ultraviolet cross-linking experiments. It could autophosphorylate itself at a conserved Ser215 residue. This was further corroborated by the impairment of autophosphorylation activity in the CodYS215A mutant. Autophosphorylation may be speculated as an additional mechanism regulating CodY activity in the cell. The protein could also hydrolyze GTP, albeit weakly, as indicated by thin- layer chromatography and spectrophotometric quantification of its kinetic parameters. Altogether, these observations provide us an insight into the mechanism of action of this global regulator and a better understanding of its functional regulation.

Enolase of Mycobacterium tuberculosis is a surface exposed plasminogen binding protein.

BACKGROUND: Enolase, a glycolytic enzyme, has long been studied as an anchorless protein present on the surface of many pathogenic bacteria that aids in tissue remodeling and invasion by binding to host plasminogen. METHODS: Anti-Mtb enolase antibodies in human sera were detected using ELISA. Immunoelectron microscopy, immunofluorescence microscopy and flow cytometry were used to show surface localization of Mtb enolase. SPR was used to determine the affinity of enolase-plasminogen interaction. Plasmin formation upon plasminogen binding to enolase and Mtb surface was measured by ELISA. Mice challenge and histopathological studies were undertaken to determine the protective efficacy of enolase immunization. RESULTS: Enolase of Mtb is present on its surface and binds human plasminogen with high affinity. There was an average of 2-fold increase in antibody mediated recognition of Mtb enolase in human sera from TB patients with an active disease over control individuals. Substitution of C-terminal lysine to alanine in rEno decreased its binding affinity with human plasminogen by >2-folds. Enolase bound plasminogen showed urokinase mediated conversion into plasmin. Binding of plasminogen to the surface of Mtb and its conversion into fibrinolytic plasmin was significantly reduced in the presence of anti-rEno antibodies. Immunization with rEno also led to a significant decrease in lung CFU counts of mice upon infection with Mtb H37Rv. CONCLUSIONS: Mtb enolase is a surface exposed plasminogen binding protein which upon immunization confers significant protection against Mtb challenge. GENERAL SIGNIFICANCE: Plasminogen binding has been recognized for Mtb, however, proteins involved have not been characterized. We show here that Mtb enolase is a moonlighting plasminogen binding protein.

Role of the recognition helix of response regulator WalR from Bacillus anthracis in DNA binding and specificity.

WalRK two-component system of Bacillus anthracis potentially regulates multiple genes spanning diverse cellular functions. Its constituent response regulator (RR), WalR belongs to the OmpR/PhoB family which possesses a winged helix-turn-helix motif for DNA binding. An in silico knowledge based model of WalR C-terminal DNA binding domain in complex with its ftsE promoter region binding motif was used to identify specific residues of the recognition helix important for DNA binding. The model was validated by mutagenesis in conjunction with in vitro DNA binding analysis. The ftsE promoter region DNA binding motif was also varied. Optimal binding of WalR to DNA required the presence of both half-sites in its binding motif. Substitution of invariant bases of WalR DNA binding motif abrogated the binding whereas changes at variable motif positions governed affinity. D199 was not in direct contact with the DNA but its substitution modified the WalR-DNA specificity indicating the importance of contact avoidance by this residue for DNA specificity. This represents the first in-depth study of RR-DNA interaction from B. anthracis.

Identification, Functional Characterization and Regulon Prediction of a Novel Two Component System Comprising BAS0540-BAS0541 of Bacillus anthracis.

Two component systems (TCSs) can be envisaged as complex molecular devices that help the bacteria to sense its environment and respond aptly. 41 TCSs are predicted in Bacillus anthracis, a potential bioterrorism agent, of which only four have been studied so far. Thus, the intricate signaling network contributed by TCSs remains largely unmapped in B. anthracis and needs comprehensive exploration. In this study, we functionally characterized one such system composed of BAS0540 (Response regulator) and BAS0541 (Histidine kinase). BAS0540-BAS0541, the closest homolog of CiaRH of Streptococcus in B. anthracis, forms a functional TCS with BAS0541 displaying autophosphorylation and subsequent phosphotransfer to BAS0540. BAS0540 was also found to accept phosphate from physiologically relevant small molecule phosphodonors like acetyl phosphate and carbamoyl phosphate. Results of qRT-PCR and immunoblotting demonstrated that BAS0540 exhibits a constitutive expression throughout the growth of B. anthracis. Regulon prediction for BAS0540 in B. anthracis was done in silico using the consensus DNA binding sequence of CiaR of Streptococcus. The predicted regulon of BAS0540 comprised of 23 genes, which could be classified into 8 functionally diverse categories. None of the proven virulence factors were a part of the predicted regulon, an observation contrasting with the regulon of CiaRH in Streptococci. Electrophoretic mobility shift assay was used to show direct binding of purified BAS0540 to the upstream regions of 5 putative regulon candidates- BAS0540 gene itself; a gene predicted to encode cell division protein FtsA; a self-immunity gene; a RND family transporter gene and a gene encoding stress (heat) responsive protein. A significant enhancement in the DNA binding ability of BAS0540 was observed upon phosphorylation. Overexpression of response regulator BAS0540 in B. anthracis led to a prodigious increase of ~6 folds in the cell length, thereby conferring it a filamentous phenotype. Furthermore, the sporulation titer of the pathogen also decreased markedly by ~16 folds. Thus, this study characterizes a novel TCS of B. anthracis and elucidates its role in two of the most important physiological processes of the pathogen: cell division and sporulation.

Overexpression of the pleiotropic regulator CodY decreases sporulation, attachment and pellicle formation in Bacillus anthracis.

CodY, a global transcriptional regulator, primarily functions as a nutrient and energy sensor. It is activated by metabolic effectors like BCAA and GTP. In low G + C Gram positive bacteria, it facilitates coupling of changes in the cellular metabolite pool with those required in the transcriptome of the cell. This pleiotropic regulator controls the expression of a vast number of genes as the cell transits from exponential to the stationary phase. Earlier studies have shown that CodY is required for the virulence of Bacillus anthracis. We sought to investigate the effect of its overexpression on the physiology of B. anthracis. In our study, we found that cellular CodY levels were unchanged during this phase-transition. Expression of endogenous CodY remained the same in different nutrient limiting conditions. Immunoblotting studies revealed CodY presence in the whole spore lysate of B. anthracis indicating it to be a component of the spore proteome. We could also detect CodY in the secretome of B. anthracis. Further, CodY was overexpressed in B. anthracis Sterne strain and this led to a 100-fold decrease in the sporulation titer and a 2.5-fold decrease in the in vitro attachment ability of the bacteria. We also observed a decrease in the pellicle formation by CodY overexpressed strain when compared to wildtype bacilli. The CodY overexpressed strain showed chaining phenotype during growth in liquid media and pellicle.

WalRK two component system of Bacillus anthracis responds to temperature and antibiotic stress.

WalRK Two Component System (TCS) of Bacillus anthracis forms a functional TCS. This report elaborates upon the WalRK genomic architecture, promoter structure, promoter activity and expression under various stress conditions in B. anthracis. 5' RACE located the WalRK functional promoter within 317 bp region upstream of WalR. Reporter gene assays demonstrated maximal promoter activity during early growth phases indicating utility in exponential stages of growth. qRT-PCR showed upregulation of WalRK transcripts during temperature and antibiotic stress. However, WalR overexpression did not affect the tested antibiotic MIC values in B. anthracis. Collectively, these results confirm that WalRK responds to cell envelope stress in B. anthracis.