Publications by authors named "Praveen K Singh"

37 Publications

Quantitative image analysis of microbial communities with BiofilmQ.

Nat Microbiol 2021 Feb 4;6(2):151-156. Epub 2021 Jan 4.

Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ-a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time.
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http://dx.doi.org/10.1038/s41564-020-00817-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840502PMC
February 2021

Vibrio cholerae biofilm scaffolding protein RbmA shows an intrinsic, phosphate-dependent autoproteolysis activity.

IUBMB Life 2021 Feb 28;73(2):418-431. Epub 2020 Dec 28.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which biofilm communities are considered to be environmental reservoirs. In endemic regions, and after algal blooms, which may result from phosphate enrichment following agricultural runoff, the bacterium is released from biofilms resulting in seasonal disease outbreaks. However, the molecular mechanism by which V. cholerae senses its environment and switches lifestyles from the biofilm-bound state to the planktonic state is largely unknown. Here, we report that the major biofilm scaffolding protein RbmA undergoes autocatalytic proteolysis via a phosphate-dependent induced proximity activation mechanism. Furthermore, we show that RbmA mutants that are defective in autoproteolysis cause V. cholerae biofilms to grow larger and mechanically stronger, correlating well with the observation that RbmA stability directly affects microbial community homeostasis and rheological properties. In conclusion, our biophysical study characterizes a novel phosphate-dependent breakdown pathway of RbmA, while microbiological data suggest a new, sensory role of this biofilm scaffolding element.
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http://dx.doi.org/10.1002/iub.2439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898620PMC
February 2021

RNA-mediated control of cell shape modulates antibiotic resistance in Vibrio cholerae.

Nat Commun 2020 11 27;11(1):6067. Epub 2020 Nov 27.

Institute of Microbiology, Friedrich Schiller University, 07745, Jena, Germany.

Vibrio cholerae, the cause of cholera disease, exhibits a characteristic curved rod morphology, which promotes infectivity and motility in dense hydrogels. Periplasmic protein CrvA determines cell curvature in V. cholerae, yet the regulatory factors controlling CrvA are unknown. Here, we discover the VadR small RNA (sRNA) as a post-transcriptional inhibitor of the crvA mRNA. Mutation of vadR increases cell curvature, whereas overexpression has the inverse effect. We show that vadR transcription is activated by the VxrAB two-component system and triggered by cell-wall-targeting antibiotics. V. cholerae cells failing to repress crvA by VadR display decreased survival upon challenge with penicillin G indicating that cell shape maintenance by the sRNA is critical for antibiotic resistance. VadR also blocks the expression of various key biofilm genes and thereby inhibits biofilm formation in V. cholerae. Thus, VadR is an important regulator for synchronizing peptidoglycan integrity, cell shape, and biofilm formation in V. cholerae.
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http://dx.doi.org/10.1038/s41467-020-19890-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695739PMC
November 2020

Quality of life after distraction osteogenesis in TMJ ankylosis patients.

Oral Surg Oral Med Oral Pathol Oral Radiol 2021 Mar 15;131(3):295-303. Epub 2020 Sep 15.

Junior Resident, Department of Oral & Maxillofacial Surgery, King George Medical University, Lucknow, India.

Objective: The aim of our study was to evaluate the success of distraction osteogenesis in temporomandibular joint (TMJ) ankylosis patients with facial deformities at our maxillofacial unit; assess the psychosocial and well-being outcomes of distraction osteogenesis and its impact on oral health; and discriminate the differences in quality of life (QoL) with application of external or internal devices, unilateral or bilateral, linear or multivector, and maxillomandibular or mandibular distraction.

Study Design: QoL and the Oral Health Impact Profile (OHIP) were prospectively studied in 42 consecutive patients with facial deformities, planned for maxillofacial distraction osteogenesis, using 2 validated questionnaires, the Orthognathic Quality of Life Questionnaire and OHIP-14. Patients who had undergone any previous surgeries were excluded.

Results: Among these patients, 16 were female, 26 male; mean age was 14.98 ± 4.88 years, and all had prearthroplastic distraction. The shortening in the mandible was in the proportion 29:01:12 in the body, ramus, and ramus-body, respectively. Mean QoL scores before and after distraction were 68.52 ± 9.50 and 26.62 ± 3.51; and mean OHIP scores before and after distraction were 33.88 ± 6.26 and 15.36 ± 2.54, a highly significant difference (P < .001) suggesting improvement. Significant improvement was identified on all QoL and OHIP questions after distraction (P < .01). The postdistraction overall mean QoL score among patients with extraoral or intraoral distractor did not have a significant difference (P = .32), but facial appearance in the bilateral distraction group; jaw function and overall well-being in the multivector distraction group; and facial appearance, jaw function, and overall well-being in maxillomandibular distraction group had significant improvements (P < .05).

Conclusions: Distraction osteogenesis considerably improves oral health and health-related QoL in patients with TMJ ankylosis with facial deformities. The use of an external or internal distractor did not make any difference in the QoL; however, bilateral distraction, multivector distraction, and maxillomandibular distraction resulted in better QoL outcomes.
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http://dx.doi.org/10.1016/j.oooo.2020.09.005DOI Listing
March 2021

Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20.

Nucleic Acids Res 2020 11;48(19):10785-10801

Centro de Biología Molecular "Severo Ochoa'' (CSIC-UAM), C. Nicolás Cabrera 1, Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.

Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.
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http://dx.doi.org/10.1093/nar/gkaa797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641744PMC
November 2020

Multicellular and unicellular responses of microbial biofilms to stress.

Biol Chem 2020 11;401(12):1365-1374

Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 16, D-35043Marburg, Germany.

Biofilms are a ubiquitous mode of microbial life and display an increased tolerance to different stresses. Inside biofilms, cells may experience both externally applied stresses and internal stresses that emerge as a result of growth in spatially structured communities. In this review, we discuss the spatial scales of different stresses in the context of biofilms, and if cells in biofilms respond to these stresses as a collection of individual cells, or if there are multicellular properties associated with the response. Understanding the organizational level of stress responses in microbial communities can help to clarify multicellular functions of biofilms.
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http://dx.doi.org/10.1515/hsz-2020-0213DOI Listing
November 2020

A tyrosine phosphoregulatory system controls exopolysaccharide biosynthesis and biofilm formation in Vibrio cholerae.

PLoS Pathog 2020 08 25;16(8):e1008745. Epub 2020 Aug 25.

Department of Microbiology and Environmental Toxicology, University of California-Santa Cruz, Santa Cruz, California, United States of America.

Production of an extracellular matrix is essential for biofilm formation, as this matrix both secures and protects the cells it encases. Mechanisms underlying production and assembly of matrices are poorly understood. Vibrio cholerae, relies heavily on biofilm formation for survival, infectivity, and transmission. Biofilm formation requires Vibrio polysaccharide (VPS), which is produced by vps gene-products, yet the function of these products remains unknown. Here, we demonstrate that the vps gene-products vpsO and vpsU encode respectively for a tyrosine kinase and a cognate tyrosine phosphatase. Collectively, VpsO and VpsU act as a tyrosine phosphoregulatory system to modulate VPS production. We present structures of VpsU and the kinase domain of VpsO, and we report observed autocatalytic tyrosine phosphorylation of the VpsO C-terminal tail. The position and amount of tyrosine phosphorylation in the VpsO C-terminal tail represses VPS production and biofilm formation through a mechanism involving the modulation of VpsO oligomerization. We found that tyrosine phosphorylation enhances stability of VpsO. Regulation of VpsO phosphorylation by the phosphatase VpsU is vital for maintaining native VPS levels. This study provides new insights into the mechanism and regulation of VPS production and establishes general principles of biofilm matrix production and its inhibition.
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http://dx.doi.org/10.1371/journal.ppat.1008745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485978PMC
August 2020

Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells.

Hepatology 2020 Nov;72(5):1528-1540

Immunocore Ltd, Abingdon, United Kingdom.

Background And Aims: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA).

Approach And Results: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA.

Conclusions: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.
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http://dx.doi.org/10.1002/hep.31503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702151PMC
November 2020

Inactivation of the dimeric RappLS20 anti-repressor of the conjugation operon is mediated by peptide-induced tetramerization.

Nucleic Acids Res 2020 08;48(14):8113-8127

ALBA Synchrotron Light Source, C. de la Llum 2-26, Cerdanyola del Vallès, 08290 Barcelona, Spain.

Quorum sensing allows bacterial cells to communicate through the release of soluble signaling molecules into the surrounding medium. It plays a pivotal role in controlling bacterial conjugation in Gram-positive cells, a process that has tremendous impact on health. Intracellular regulatory proteins of the RRNPP family are common targets of these signaling molecules. The RRNPP family of gene regulators bind signaling molecules at their C-terminal domain (CTD), but have highly divergent functionalities at their N-terminal effector domains (NTD). This divergence is also reflected in the functional states of the proteins, and is highly interesting from an evolutionary perspective. RappLS20 is an RRNPP encoded on the Bacillus subtilis plasmid pLS20. It relieves the gene repression effectuated by RcopLS20 in the absence of the mature pLS20 signaling peptide Phr*pLS20. We report here an in-depth structural study of apo and Phr*pLS20-bound states of RappLS20 at various levels of atomic detail. We show that apo-RappLS20 is dimeric and that Phr*pLS20-bound Rap forms NTD-mediated tetramers. In addition, we show that RappLS20 binds RcopLS20 directly in the absence of Phr*pLS20 and that addition of Phr*pLS20 releases RcopLS20 from RappLS20. This allows RcopLS20 to bind the promotor region of crucial conjugation genes blocking their expression.
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http://dx.doi.org/10.1093/nar/gkaa540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430634PMC
August 2020

Flow-Induced Symmetry Breaking in Growing Bacterial Biofilms.

Phys Rev Lett 2019 Dec;123(25):258101

Department of Mathematics, Massachusetts Institute of Technology, Cambridge Massachusetts 02139-4307, USA.

Bacterial biofilms represent a major form of microbial life on Earth and serve as a model active nematic system, in which activity results from growth of the rod-shaped bacterial cells. In their natural environments, ranging from human organs to industrial pipelines, biofilms have evolved to grow robustly under significant fluid shear. Despite intense practical and theoretical interest, it is unclear how strong fluid flow alters the local and global architectures of biofilms. Here, we combine highly time-resolved single-cell live imaging with 3D multiscale modeling to investigate the mechanisms by which flow affects the dynamics of all individual cells in growing biofilms. Our experiments and cell-based simulations reveal three quantitatively different growth phases in strong external flow and the transitions between them. In the initial stages of biofilm development, flow induces a downstream gradient in cell orientation, causing asymmetrical dropletlike biofilm shapes. In the later developmental stages, when the majority of cells are sheltered from the flow by the surrounding extracellular matrix, buckling-induced cell verticalization in the biofilm core restores radially symmetric biofilm growth, in agreement with predictions of a 3D continuum model.
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http://dx.doi.org/10.1103/PhysRevLett.123.258101DOI Listing
December 2019

Surface Exclusion Revisited: Function Related to Differential Expression of the Surface Exclusion System of Plasmid pLS20.

Front Microbiol 2019 10;10:1502. Epub 2019 Jul 10.

Laboratory 402, Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Department of Virology and Microbiology, Autonomous University of Madrid, Madrid, Spain.

During conjugation a genetic element is transferred from a bacterial donor to a recipient cell via a connecting channel. It is the major route responsible for the spread of antibiotic resistance. Conjugative elements can contain exclusion system(s) that inhibit its transfer to a cell already harboring the element. Our limited knowledge on exclusion systems is mainly based on plasmids of Gram-negative bacteria. Here we studied the conjugative plasmid pLS20 of the Gram-positive . We demonstrate that pLS20 contains an exclusion system and identified the single gene responsible for exclusion, named , which is embedded in the conjugation operon. Ses is the founding member of a novel family of surface exclusion proteins encoded by conjugative elements of Gram-positive origin. We show that the extent of surface exclusion correlates with the level of expression, and that is expressed at basal low-levels in all donor cells but becomes highly expressed in conjugating cells. Accordingly, the transfer of pLS20 from a conjugation-primed donor cell to an un-primed or conjugation-primed donor is inhibited moderately and very efficiently, respectively. The consequences of this differential regulation, which appears to be a conserved feature of surface exclusion systems of Gram-positive and Gram-negative origin, are discussed.
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http://dx.doi.org/10.3389/fmicb.2019.01502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635565PMC
July 2019

Emergence of three-dimensional order and structure in growing biofilms.

Nat Phys 2019 Apr 26;15(3):251-256. Epub 2018 Nov 26.

Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, DE.

Surface-attached bacterial biofilms are self-replicating active liquid crystals and the dominant form of bacterial life on earth (1-4). In conventional liquid crystals and solid-state materials, the interaction potentials between the molecules that comprise the system determine the material properties. However, for growth-active biofilms it is unclear whether potential-based descriptions can account for the experimentally observed morphologies, and which potentials would be relevant. Here, we overcame previous limitations of single-cell imaging techniques (5,6) to reconstruct and track all individual cells inside growing three-dimensional (3D) biofilms with up to 10,000 individuals. Based on these data, we identify, constrain, and provide a microscopic basis for an effective cell-cell interaction potential, which captures and predicts the growth dynamics, emergent architecture, and local liquid crystalline order of biofilms. Furthermore, we show how external fluid flows control the microscopic structure and 3D morphology of biofilms. Our analysis implies that local cellular order and global biofilm architecture in these active bacterial communities can arise from mechanical cell-cell interactions, which cells can modulate by regulating the production of particular matrix components. These results establish an experimentally validated foundation for improved continuum theories of active matter and thereby contribute to solving the important problem of controlling biofilm growth.
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http://dx.doi.org/10.1038/s41567-018-0356-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544526PMC
April 2019

Draft Genome Sequences of Sporulation-Impaired Bacillus pumilus Strain NRS576 and Its Native Plasmid p576.

Microbiol Resour Announc 2019 Apr 18;8(16). Epub 2019 Apr 18.

Department of Virology and Microbiology, Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma Madrid, Madrid, Spain

spores can cause foodborne poisonings. strain NRS576 forms spores with a very reduced efficiency due to the presence of a plasmid, named p576. Here, we report the genome sequence of strain NRS576 and its plasmid p576.
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http://dx.doi.org/10.1128/MRA.00089-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6473135PMC
April 2019

Learning the space-time phase diagram of bacterial swarm expansion.

Proc Natl Acad Sci U S A 2019 01 11;116(5):1489-1494. Epub 2019 Jan 11.

Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany;

Coordinated dynamics of individual components in active matter are an essential aspect of life on all scales. Establishing a comprehensive, causal connection between intracellular, intercellular, and macroscopic behaviors has remained a major challenge due to limitations in data acquisition and analysis techniques suitable for multiscale dynamics. Here, we combine a high-throughput adaptive microscopy approach with machine learning, to identify key biological and physical mechanisms that determine distinct microscopic and macroscopic collective behavior phases which develop as swarms expand over five orders of magnitude in space. Our experiments, continuum modeling, and particle-based simulations reveal that macroscopic swarm expansion is primarily driven by cellular growth kinetics, whereas the microscopic swarming motility phases are dominated by physical cell-cell interactions. These results provide a unified understanding of bacterial multiscale behavioral complexity in swarms.
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http://dx.doi.org/10.1073/pnas.1811722116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358709PMC
January 2019

Revalidation of the neuroprotective effects of a United States patented polyherbal formulation on scopolamine induced learning and memory impairment in rats.

Biomed Pharmacother 2018 Jan 9;97:1046-1052. Epub 2017 Nov 9.

Collaborative Programme, Institute of Medical Science, BHU, Varanasi, India; Department of Kriya Sharir, Faculty of Ayurveda, IMS, BHU, Varanasi, India. Electronic address:

Objective: Alzheimer's disease (AD) is the most common cause of dementia yet treatment options are extremely limited. The disease is associated with cognitive impairment as well as structural irregularities, accumulation of plaques and neurofibrillary tangles, diminished levels of acetylcholine, oxidative stress, and inflammation in the brain. We have previously reported on the positive effects of a united states patented (US 7,273,626 B2) poly herbal test formulation, consisting of Bacopa monnieri, Hippophae rhamnoides and Dioscorea bulbifera extracts, on cognitive deficits in AD patients. The present study was conducted to investigate the mechanism(s) of action of the formulation using scopolamine treated rats as an AD model.

Method: The formulation was administered daily along with scopolamine for a period of 14days following which the elevated plus maze, passive avoidance, and Morris water maze tests were performed to assess learning and memory. Rats treated with scopolamine or vehicle only were also included in the experiment. Acetylcholine levels and activities of acetylcholinesterase (AChE) and anti-oxidant enzymes in the brain were also measured at the end of the treatment period.

Results: The study demonstrate that scopolamine treatment resulted in learning and memory deficits which were partially and significantly ameliorated by the formulation. The formulation also counteracted scopolamine-induced decreases in acetylcholine levels, increases in AChE activity, and decreases in activities of the antioxidant enzymes.

Conclusion: The study demonstrates the ability of the test formulation to reverse scopolamine-induced learning and memory deficits in rats which may at least partially be explained by the reversal of scopolamine-induced reductions in brain acetylcholine levels and antioxidant activities by the test formulation.
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http://dx.doi.org/10.1016/j.biopha.2017.11.008DOI Listing
January 2018

Dynamic biofilm architecture confers individual and collective mechanisms of viral protection.

Nat Microbiol 2018 Jan 30;3(1):26-31. Epub 2017 Oct 30.

Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

In nature, bacteria primarily live in surface-attached, multicellular communities, termed biofilms . In medical settings, biofilms cause devastating damage during chronic and acute infections; indeed, bacteria are often viewed as agents of human disease . However, bacteria themselves suffer from diseases, most notably in the form of viral pathogens termed bacteriophages , which are the most abundant replicating entities on Earth. Phage-biofilm encounters are undoubtedly common in the environment, but the mechanisms that determine the outcome of these encounters are unknown. Using Escherichia coli biofilms and the lytic phage T7 as models, we discovered that an amyloid fibre network of CsgA (curli polymer) protects biofilms against phage attack via two separate mechanisms. First, collective cell protection results from inhibition of phage transport into the biofilm, which we demonstrate in vivo and in vitro. Second, CsgA fibres protect cells individually by coating their surface and binding phage particles, thereby preventing their attachment to the cell exterior. These insights into biofilm-phage interactions have broad-ranging implications for the design of phage applications in biotechnology, phage therapy and the evolutionary dynamics of phages with their bacterial hosts.
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http://dx.doi.org/10.1038/s41564-017-0050-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739289PMC
January 2018

Vibrio cholerae Combines Individual and Collective Sensing to Trigger Biofilm Dispersal.

Curr Biol 2017 Nov 19;27(21):3359-3366.e7. Epub 2017 Oct 19.

Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, Marburg 35043, Germany; Department of Physics, Philipps-Universität Marburg, Renthof 6, Marburg 35032, Germany. Electronic address:

Bacteria can generate benefits for themselves and their kin by living in multicellular, matrix-enclosed communities, termed biofilms, which are fundamental to microbial ecology and the impact bacteria have on the environment, infections, and industry [1-6]. The advantages of the biofilm mode of life include increased stress resistance and access to concentrated nutrient sources [3, 7, 8]. However, there are also costs associated with biofilm growth, including the metabolic burden of biofilm matrix production, increased resource competition, and limited mobility inside the community [9-11]. The decision-making strategies used by bacteria to weigh the costs between remaining in a biofilm or actively dispersing are largely unclear, even though the dispersal transition is a central aspect of the biofilm life cycle and critical for infection transmission [12-14]. Using a combination of genetic and novel single-cell imaging approaches, we show that Vibrio cholerae integrates dual sensory inputs to control the dispersal response: cells use the general stress response, which can be induced via starvation, and they also integrate information about the local cell density and molecular transport conditions in the environment via the quorum sensing apparatus. By combining information from individual (stress response) and collective (quorum sensing) avenues of sensory input, biofilm-dwelling bacteria can make robust decisions to disperse from large biofilms under distress, while preventing premature dispersal when biofilm populations are small. These insights into triggers and regulators of biofilm dispersal are a key step toward actively inducing biofilm dispersal for technological and medical applications, and for environmental control of biofilms.
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http://dx.doi.org/10.1016/j.cub.2017.09.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678073PMC
November 2017

Structural dynamics of RbmA governs plasticity of biofilms.

Elife 2017 08 1;6. Epub 2017 Aug 1.

Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, United States.

Biofilm formation is critical for the infection cycle of exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity.
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http://dx.doi.org/10.7554/eLife.26163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605196PMC
August 2017

Discovery of a new family of relaxases in Firmicutes bacteria.

PLoS Genet 2017 02 16;13(2):e1006586. Epub 2017 Feb 16.

Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.

Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.
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http://dx.doi.org/10.1371/journal.pgen.1006586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313138PMC
February 2017

Unraveling the molecular basis of oxidative stress management in a drought tolerant rice genotype Nagina 22.

BMC Genomics 2016 10 4;17(1):774. Epub 2016 Oct 4.

ICAR-National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, Pusa Campus, New Delhi, 110 012, India.

Background: Drought stress tolerance for crop improvement is an important goal worldwide. Drought is a complex trait, and it is vital to understand the complex physiological, biochemical, and molecular mechanisms of drought tolerance to tackle it effectively. Osmotic adjustment, oxidative stress management (OSM), and cell membrane stability (CMS) are major components of cellular tolerance under drought stress. In the current study, we explored the molecular basis of OSM in the drought tolerant rice variety, Nagina 22 and compared it with the popular drought sensitive rice variety, IR 64, under drought imposed at the reproductive stage, to understand how the parental polymorphisms correlate with the superiority of Nagina 22 and tolerant bulk populations under drought.

Results: We generated recombinant inbred lines (RIL) from contrasting parents Nagina 22 and IR 64 and focussed on spikelet fertility (SF), in terms of its correlation with OSM, which is an important component of drought tolerance in Nagina 22. Based on SF under drought stress and its correlations with other yield related traits, we used superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) activity assays to establish the relationship between SF and OSM genes in the tolerant and sensitive lines. Among the OSM enzymes studied, GR had a significant and positive correlation with single plant yield (SPY) under drought stress. GR was also positively correlated with APX but negatively so with SOD. Interestingly, none of the enzyme-morphology correlations were significant under irrigated control (IC). Through genome-wide SNP analysis of the 21 genes encoding for OSM enzymes, we identified the functional polymorphisms between the parents and identified superior alleles. By using network analysis of OSM genes in rice, we identified the genes that are central to the OSM network.

Conclusions: From the biochemical and morphological data and the SNP analysis, the superiority of Nagina 22 in spikelet fertility under drought stress is because of its superior alleles for SOD (SOD2, SODCC1, SODA) and GR (GRCP2) rather than for APX, for which IR 64 had the superior allele (APX8). Nagina 22 can bypass APX8 by directly interacting with SODA. For nine of the 11 genes present in the central network, Nagina 22 had the superior alleles. We propose that Nagina 22 tolerance could mainly be because of SODA which is a reactive oxygen scavenger in mitochondria which is directly associated with spikelet fertility.
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http://dx.doi.org/10.1186/s12864-016-3131-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050613PMC
October 2016

Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta.

Biochemistry 2016 05 4;55(19):2713-21. Epub 2016 May 4.

Wolfson Centre for Age-Related Diseases, King's College London , London SE1 9RT, United Kingdom.

The diacylglycerol lipases (DAGLα and DAGLβ) hydrolyze DAG to generate 2-arachidonoylglycerol (2-AG), the principal endocannabinoid and main precursor of arachidonic acid (AA). The DAGLs make distinct tissue specific contributions toward 2-AG and AA levels, and therefore, selective modulators for these enzymes could play crucial roles toward harnessing their therapeutic potential. Relatively high-throughput assays have recently been reported for DAGLα and have proven useful toward the characterization of inhibitors of this enzyme. Similar assays are also warranted for DAGLβ which was the aim of this study. We first adapted previously reported DAGLα membrane assays (using PNPB and DiFMUO as substrates) to measure recombinant DAGLβ activity in membranes. In contrast to results with DAGLα, both substrates provided a relatively limited signal window for measuring DAGLβ activity, however, an improved window was obtained when employing a third commercially available substrate, EnzChek. In order to further improve on the assay parameters, we successfully purified the glutathione S-transferase (GST) tagged catalytic domain of DAGLβ. Activity of the enzyme was confirmed using EnzChek as well as two DAGL inhibitors (THL and OMDM-188). The purified DAGLβ catalytic domain assay described here provides the basis for a relatively clean and convenient assay with the potential to be adapted for high-throughput drug discovery efforts.
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http://dx.doi.org/10.1021/acs.biochem.6b00221DOI Listing
May 2016

A novel live cell assay to measure diacylglycerol lipase α activity.

Biosci Rep 2016 06 6;36(3). Epub 2016 May 6.

Wolfson Centre for Age-Related Diseases, King's College London, SE1 9RT, U.K.

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.
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http://dx.doi.org/10.1042/BSR20160073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859088PMC
June 2016

Quantitative analysis of heavy metals in medicinal plants collected from environmentally diverse locations in India for use in a novel phytopharmaceutical product.

Environ Monit Assess 2015 Aug 30;187(8):542. Epub 2015 Jul 30.

Collaborative Program, Institute of Medical Science, BHU, Varanasi, India,

It is important to monitor the quality of the phytopharmaceutical product as its therapeutic potential depends on standardized delivery of active ingredients present in the botanical source. Minimal presence of toxic impurities like heavy metals (HMs) is warranted to ensure product safety and prevent hazardous health impacts. In the present study, conducted as part of the development of a novel phytopharmaceutical product, the chemical profile of 13 heavy metals (Fe, Cu, Mn, Zn, Ni, Co, Mo, V, Cr, As, Pb, Hg, and Cd) was studied in the whole plant, fruit, and rhizome of Bacopa monnieri, Hippophae rhamnoides, and Dioscorea bulbifera, respectively, from environmentally diverse regions in India. Most samples had HM profiles within permissible limits as established by regulatory authorities, with the exception of Cd and Hg in low-altitude regions. This study indicates geographical regions in India suitable for procuring raw materials to develop and manufacture phytopharmaceutical products.
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http://dx.doi.org/10.1007/s10661-015-4764-3DOI Listing
August 2015

Age dependent levels of plasma homocysteine and cognitive performance.

Behav Brain Res 2015 Apr 16;283:139-44. Epub 2015 Jan 16.

Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.

Elevated plasma homocysteine (hcy) levels, also known as hyperhomocysteinemia (hhcy), have been associated with cognitive impairment and neurodegenerative disorders. Hhcy has been attributed to deficiency of B vitamins which can adversely affect the brain and result in memory loss and poor attention power. Monitoring hcy levels and the use of vitamin supplementation to treat hhcy may therefore prove advantageous for the prevention and management of cognitive impairment. With this in consideration, we measured plasma hcy, folate and vitamin B12 levels in 639 subjects from different age groups in two sub-regions of India. Cognitive function was also measured using attention span and immediate and delayed memory recall tests. Depression scores were obtained using the Beck Depression Inventory-II and functional impairment was assessed using the functional activities questionnaire (FAQ) score. As hhcy has also been linked to inflammation, plasma levels of high sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) were also measured. The results demonstrated significant negative correlations between hcy levels and folic acid levels, vitamin B12 levels and cognitive performance (attention span and delayed but not immediate memory recall) along with significant positive correlations between hcy levels and depression scores and hsCRP (but not IL-6) levels. A positive correlation was also observed between hcy levels and FAQ scores, however this was not found to be significant. Based on these results, folic acid and vitamin B12 intervention in people with elevated hcy levels in India could prove to be effective in lowering hcy levels and help maintain or improve cognitive function.
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http://dx.doi.org/10.1016/j.bbr.2015.01.016DOI Listing
April 2015

A complex genetic switch involving overlapping divergent promoters and DNA looping regulates expression of conjugation genes of a gram-positive plasmid.

PLoS Genet 2014 Oct 23;10(10):e1004733. Epub 2014 Oct 23.

Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.

Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed.
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http://dx.doi.org/10.1371/journal.pgen.1004733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207663PMC
October 2014

Diverse regulatory circuits for transfer of conjugative elements.

FEMS Microbiol Lett 2014 Sep 17;358(2):119-28. Epub 2014 Jul 17.

Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), C. Nicolás Cabrera 1, Universidad Autónoma, Canto Blanco, Madrid, Spain.

Conjugation systems are present on many plasmids as well as on chromosomally integrated elements. Conjugation, which is a major route by which bacteria exchange genetic material, is a complex and energy-consuming process. Hence, a shared feature of conjugation systems is that expression of the genes involved is strictly controlled in such a way that conjugation is kept in a default 'OFF' state and that the process is switched on only under conditions that favor the transfer of the conjugative element into a recipient cell. However, there is a remarkable diversity in the way by which conjugation genes present on different transferable elements are regulated. Here, we review these diverse regulatory circuits on the basis of several prototypes with a special focus on the recently discovered regulation of the conjugation genes present on the native Bacillus subtilis plasmid pLS20.
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http://dx.doi.org/10.1111/1574-6968.12526DOI Listing
September 2014

Comparative evolutionary analysis of cell cycle proteins networks in fission and budding yeast.

Cell Biochem Biophys 2014 Nov;70(2):1167-75

Department of Bioinformatics, Maulana Azad National Institute of Technology, Bhopal, India,

Fission yeast and budding yeast are the two distantly related species with common ancestors. Various studies have shown significant differences in metabolic networks and regulatory networks. Cell cycle regulatory proteins in both species have differences in structural as well as in functional organization. Orthologous proteins in cell cycle regulatory protein networks seem to play contemporary role in both species during the evolution but little is known about non-orthologous proteins. Here, we used system biology approach to compare topological parameters of orthologous and non-orthologous proteins to find their contributions during the evolution to make an efficient cell cycle regulation. Observed results have shown a significant role of non-orthologous proteins in fission yeast in maintaining the efficiency of cell cycle regulation with less number of proteins as compared to budding yeast.
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http://dx.doi.org/10.1007/s12013-014-0037-yDOI Listing
November 2014

Semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1 protects against gamma radiation-induced oxidative stress.

Environ Toxicol Pharmacol 2014 Mar 25;37(2):553-62. Epub 2014 Jan 25.

Radiation Biotechnology Laboratory, Department of Radiation Biosciences, Institute of Nuclear and Allied Sciences, Delhi 110054, India; Department of Biochemistry, Faculty of Science, Jamia Hamdard, Delhi 110062, India. Electronic address:

In the present study, radioprotective potential of Semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 was evaluated. γ-Radiation induced protein carbonylation, plasmid DNA damage, enzyme functional impairment, lipid peroxidation, HO radicals generation and their protection by SQGD was assessed. As a result of SQGD treatment, significant inhibition (p<0.05) in protein carbonylation was observed with BSA. SQGD treatment was found to restore supercoiled (~70±3.21%) form of irradiated plasmid DNA against γ-irradiation. SQGD protects enzymes (EcoR1 and BamH1) against radiation-induced dysfunctioning. SQGD significantly inhibited (p<0.05) lipid peroxidation in liposomes, brain and liver homogenate. Higher HO(•) radicals-averting activity of SQGD was observed in the serum and liver homogenate of C57BL/6 mice against H2O2-induced oxidative stress. In conclusion, SQGD demonstrates excellent radical-scavenging activity towards bio-macromolecules in irradiated environment and can be developed as an ideal radioprotector against radiation-induced oxidative stress in future.
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http://dx.doi.org/10.1016/j.etap.2014.01.003DOI Listing
March 2014