Mechanism of DNA cleavage by the endonuclease SauUSI: a major barrier to horizontal gene transfer and antibiotic resistance in Staphylococcus aureus.

Acquisition of foreign DNA by Staphylococcus aureus, including vancomycin resistance genes, is thwarted by the ATP-dependent endonuclease SauUSI. Deciphering the mechanism of action of SauUSI could unravel the reason how it singularly plays a major role in preventing horizontal gene transfer (HGT) in S. aureus. Here, we report a detailed biochemical and structural characterization of SauUSI, which reveals that in the presence of ATP, the enzyme can cleave DNA having a single or multiple target site/s. Remarkably, in the case of multiple target sites, the entire region of DNA flanked by two target sites is shred into smaller fragments by SauUSI. Crystal structure of SauUSI reveals a stable dimer held together by the nuclease domains, which are spatially arranged to hydrolyze the phosphodiester bonds of both strands of the duplex. Thus, the architecture of the dimeric SauUSI facilitates cleavage of either single-site or multi-site DNA. The structure also provides insights into the molecular basis of target recognition by SauUSI. We show that target recognition activates ATP hydrolysis by the helicase-like ATPase domain, which powers active directional movement (translocation) of SauUSI along the DNA. We propose that a pile-up of multiple translocating SauUSI molecules against a stationary SauUSI bound to a target site catalyzes random double-stranded breaks causing shredding of the DNA between two target sites. The extensive and irreparable damage of the foreign DNA by shredding makes SauUSI a potent barrier against HGT.

Diguanylate Cyclases in Vibrio cholerae: Essential Regulators of Lifestyle Switching.

Biofilm formation in Vibrio cholerae empowers the bacteria to lead a dual lifestyle and enhances its infectivity. While the formation and dispersal of the biofilm involves multiple components-both proteinaceous and non-proteinaceous, the key to the regulatory control lies with the ubiquitous secondary signaling molecule, cyclic-di-GMP (c-di-GMP). A number of different cellular components may interact with c-di-GMP, but the onus of synthesis of this molecule lies with a class of enzymes known as diguanylate cyclases (DGCs). DGC activity is generally associated with proteins possessing a GGDEF domain, ubiquitously present across all bacterial systems. V. cholerae is also endowed with multiple DGCs and information about some of them have been pouring in over the past decade. This review summarizes the DGCs confirmed till date in V. cholerae, and emphasizes the importance of DGCs and their product, c-di-GMP in the virulence and lifecycle of the bacteria.

The conserved aspartate in motif III of b family AdoMet-dependent DNA methyltransferase is important for methylation.

S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) are involved in diverse cellular functions. These enzymes show little sequence conservation but have a conserved structural fold. The DNA MTases have characteristic motifs that are involved in AdoMet binding, DNA target recognition and catalysis. Motif III of these MTases have a highly conserved acidic residue, often an aspartate, whose functional significance is not clear. Here, we report a mutational study of the residue in the ß family MTase of the Type III restriction-modification enzyme EcoP15I. Replacement of this residue by alanine affects its methylation activity. We propose that this residue contributes to the affinity of the enzyme for AdoMet. Analysis of the structures of DNA, RNA and protein MTases reveal that the acidic residue is conserved in all of them, and interacts with N6 of the adenine moiety of AdoMet. Interestingly, in the SET-domain protein lysine MTases, which have a fold different from other AdoMet-dependent MTases, N6 of the adenine moiety is hydrogen bonded to the main chain carbonyl group of the histidine residue of the highly conserved motif III. Our study reveals the evolutionary conservation of a carbonyl group in DNA, RNA and protein AdoMet-dependent MTases for specific interaction by hydrogen bond with AdoMet, despite the lack of overall sequence conservation.

Structure of the active GGEEF domain of a diguanylate cyclase from Vibrio cholerae.

Cyclic-di-GMP (c-di-GMP) synthesized by diguanylate cyclases has been an important and ubiquitous secondary messenger in almost all bacterial systems. In Vibrio cholerae, c-di-GMP plays an intricate role in the production of the exopolysaccharide matrix, and thereby, in biofilm formation. The formation of the surface biofilm enables the bacteria to survive in aquatic bodies, when not infecting a human host. Diguanylate cyclases are the class of enzymes which synthesize c-di-GMP from two molecules of GTP and are endowed with a GGDEF or, a GGEEF signature domain. The VC0395_0300 protein from V. cholerae, has been established as a diguanylate cyclase with a necessary role in biofilm formation. Here we present the structure of an N-terminally truncated form of VC0395_0300, which retains the active GGEEF domain for diguanylate cyclase activity but lacks 160 residues from the poorly organized N-terminal domain. X-ray diffraction data was collected from a crystal of VC0395_0300(161-321) to a resolution of 1.9 Å. The structure displays remarkable topological similarity with diguanylate cyclases from other bacterial systems, but lacks the binding site for c-di-GMP present in its homologues. Finally, we demonstrate the ability of the truncated diguanylate cyclase VC0395_0300(161-321) to produce c-di-GMP, and its role in biofilm formation for the bacteria.

DNA binding, cleavage and cytotoxicity studies of three mononuclear Cu(II) chloro-complexes containing N-S donor Schiff base ligands.

We report the biological activity of three Cu(II) complexes [Cu(pabt)Cl] (1), [Cu(pma)Cl] (2), and [Cu(pdta)Cl]Cl (3) (pabt = N-(2-mercaptophenyl)-2'-pyridylmethylenimine, pma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta = 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide). 1-3 display four-line EPR multiplet in solution at RT suggesting that these are mononuclear. DNA-binding studies using spectrophotometric titration of these complexes with calf thymus DNA showed binding through intercalation mode which was found to be consistent with the observation of increased viscosity of DNA and quenching of fluorescence of ethidium bromide bound DNA in the presence of these complexes. All three complexes were found to be efficient in bringing about oxidative and hydrolytic cleavage of DNA. The proposed mechanism of hydrolytic DNA cleavage has been discussed. MTT assay showed remarkable cytotoxicity on cervical cancer HeLa cell line and the IC50 values were 1.27, 4.13, and 3.92 µM for 1, 2 and 3, respectively, as compared to the IC50 value (13 µM) reported for cisplatin in HeLa cells. AO/PI and Annexin-V/PI assay suggest the induction of cell death primarily via apoptotic pathway. Nuclear staining using DAPI was used to assess changes in nuclear morphology during apoptotic cell death. The role of reactive oxygen species (ROS) for induction of apoptotic cell death was studied using H2DCF-DA assay and the result suggests that the generation of ROS by the complexes may be a possible cause for their antiproliferative activity. TUNEL assay showed DNA fragmentation in apoptotic cells. Cell cycle analysis using flow cytometry showed significant increase in the G2/M phase in HeLa cells by the compounds 1-3. Mononuclear Cu(II) complexes display remarkable cytotoxicity against cervical cancer HeLa cell line. The generation of ROS by the complexes may be a cause of their antiproliferative activity. Fluorescent images from DAPI staining assay revealed that the cells undergoing apoptosis displayed typical features like cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. TUNEL assay showed DNA fragmentation in apoptotic cells.

Subtle Changes Due to Mutations in the GGDEF Domain Result in Loss of Biofilm Forming Activity in the VC0395_0300 Protein from Vibrio cholerae, but No Major Change in the Overall Structure.

BACKGROUND: Cyclic-di-GMP (c-di-GMP) is a ubiquitous secondary messenger molecule in bacteria synthesized by diguanylate cyclases. This universal messenger regulates diverse cellular functions in bacteria at the transcriptional, translational and posttranslational levels. The cellular functions regulated by c-di-GMP include cell motility, cell cycle progression, virulence, biofilm formation, antibiotic production and other unknown functions. The VC0395_0300 protein from the chromosome I of the Vibrio cholerae classical strain O395, serotype O1 has been established to be a diguanylate cyclase with a necessary role in biofilm formation. OBJECTIVE: Mutations in the central position of the GGEEF active site of VC0395_0300 protein have been created by site-directed mutagenesis. The conditions for maximum production of mutated protein have been optimized. While there is a significant loss-of-biofilm-forming activity in the mutants, the basis for the same needed an investigation at the structural level. METHODS: Subsequently, the mutant proteins have been characterized using spectrofluorimetry and circular dichroism spectroscopy. RESULTS: While the unfolding pattern of the mutant proteins shows some changes with respect to the wild type, the overall structure of the protein does not show significant changes due to the mutagenesis, despite the absence of biofilm formation in the mutants. CONCLUSION: This led us to conclude that whatever changes that occur in the mutated proteins, do not disturb the GGEEF domain architecture, but are restricted to the local architecture, and are hence, subtle in nature.

Putative protein VC0395_0300 from Vibrio cholerae is a diguanylate cyclase with a role in biofilm formation.

The hallmark of the lifecycle of Vibrio cholerae is its ability to switch between two lifestyles - the sessile, non-pathogenic form and the motile, infectious form in human hosts. One of these changes is in the formation of surface biofilms, when in sessile aquatic habitats. The cell-cell interactions within a V. cholerae biofilm are stabilized by the production of an exopolysachharide (EPS) matrix, which in turn is regulated by the ubiquitous secondary messenger, cyclic di-GMP (c-di-GMP), synthesized by proteins containing GGD(/E)EF domains in all prokaryotic systems. Here, we report the functional role of the VC0395_0300 protein (Sebox3) encoded by the chromosome I of V. cholerae, with a GGEEF signature sequence, in the formation of surface biofilms. In our study, we have shown that Escherichia coli containing the full-length Sebox3 displays enhanced biofilm forming ability with cellulose production as quantified and visualized by multiple assays, most notably using FEG-SEM. This has also been corroborated with the lack of motility of host containing Sebox3 in semi-solid media. Searching for the reasons for this biofilm formation, we have demonstrated in vitro that Sebox3 can synthesize c-di-GMP from GTP. The homology derived model of Sebox3 displayed significant conservation of the GGD(/E)EF architecture as well. Hence, we propose that the putative protein VC0395_0300 from V. cholerae is a diguanylate cyclase which has an active role in biofilm formation.

Effect of site-directed mutagenesis at the GGEEF domain of the biofilm forming GGEEF protein from Vibrio cholerae.

Vibrio cholerae, the cause of seven noted pandemics, leads a dual lifecycle-one in the human host in its virulent form, and the other as a sessile, non-virulent bacterium in aquatic bodies in surface biofilms. Surface biofilms have been attributed to be associated with a ubiquitous protein domain present in all branches of bacteria, known as the GGD(/E)EF domain. While the diguanlyate cyclase activities of these proteins are universally established, the role of these proteins as diguanlyate-specific phosphodiesterases in conjunction with a EAL domain has also been reported. The VC0395_0300 protein from V. cholerae which shows biofilm forming abilities also acts as a phosphodiesterase. Interestingly, this GGD(/E)EF protein contains a EAL site in the reverse orientation. We attempted to mutate the GGEEF signature along the sequence by site-directed mutagenesis. The resultant mutants (Sebox5-7) did not show much difference in phosphodiesterase activity in comparison with the wild type protein (Sebox3), indicating the independence of the phosphodiesterase activity of the protein from the GGD(/E)EF domain. However, the ability of the mutants to form surface biofilm was significantly lesser in the case of mutations in the three central positions of the signature domain.