Cyanobacterial KdpD modulates in vivo and in vitro activities of a membrane-anchored histidine kinase.

The prokaryotic KdpATPAse complex, encoded by the kdpABC operon, is an inducible, high-affinity K transporter. In E. coli, the operon is transcriptionally regulated by a two-component sensor-kinase response-regulator system, constituted by the KdpD and KdpE proteins.

Metal removal by metallothionein and an acid phosphatase PhoN, surface-displayed on the cells of the extremophile, Deinococcus radiodurans.

The utility of surface layer proteins (Hpi and SlpA) of the radiation resistant bacterium, Deinococcus radiodurans, was investigated for surface display and bioremediation of cadmium and uranium. The smtA gene, from Synechococcus elongatus (encoding the metal binding metallothionein protein), was cloned and over-expressed in D. radiodurans, either as such or as a chimeric gene fused with hpi ORF (Hpi-SmtA), or fused to the nucleotide sequence encoding the SLH domain of the SlpA protein (SLH-SmtA).

Effect of Co-Gamma Ionizing Radiation and Desiccation Stress on Protein Profile of Anabaena 7120.

Physiological stress can bring major molecular and cellular change to a living cell which further decide its survival or tolerance to the stress exposure. Cyanobacteria like Anabaena has been shown to tolerate high levels of different stresses like oxidative, desiccation, UV, and gamma radiation. They are able to withstand and recover remarkably without any lethal mutation when exposed to high doses of gamma radiation or prolonged duration of desiccation.

Low concentrations of ethanol during irradiation drastically reduce DNA damage caused by very high doses of ionizing radiation.

Presence of low concentrations (1-2%) of ethanol during irradiation exhibited significant protection against DNA damage caused by very high doses (2-12 kGy) of 60 Co-gamma-rays in vitro. Radiation-induced DNA damage was substantially reduced in different types of DNA molecules (chromosomal DNA from Anabaena 7120 or Deinococcus radiodurans or bacteriophage Lambda, and plasmid pBluescript DNA) when irradiated in the presence of ethanol, thus indicating the generic nature of ethanol protection. The radioprotection appeared to be a consequence of the well known ability of ethanol to scavenge hydroxyl radicals.

Regulation of potassium dependent ATPase (kdp) operon of Deinococcus radiodurans.

The genome of D. radiodurans harbors genes for structural and regulatory proteins of Kdp ATPase, in an operon pattern, on Mega plasmid 1. Organization of its two-component regulatory genes is unique.

Transcriptional Modulation of Transport- and Metabolism-Associated Gene Clusters Leading to Utilization of Benzoate in Preference to Glucose in Pseudomonas putida CSV86.

The effective elimination of xenobiotic pollutants from the environment can be achieved by efficient degradation by microorganisms even in the presence of sugars or organic acids. Soil isolate CSV86 displays a unique ability to utilize aromatic compounds prior to glucose. The draft genome and transcription analyses revealed that glucose uptake and benzoate transport and metabolism genes are clustered at the and loci, respectively, as two distinct operons.

Proximity of Radiation Desiccation Response Motif to the core promoter is essential for basal repression as well as gamma radiation-induced gyrB gene expression in Deinococcus radiodurans.

The radioresistant D. radiodurans regulates its DNA damage regulon (DDR) through interaction between a 17bp palindromic cis-regulatory element called the Radiation Desiccation Response Motif (RDRM), the DdrO repressor and a protease IrrE. The role of RDRM in regulation of DDR was dissected by constructing RDRM sequence-, position- or deletion-variants of Deinococcal gyrB gene (DR0906) promoter and by RDRM insertion in the non-RDRM groESL gene (DR0606) promoter, and monitoring the effect of such modifications on the basal as well as gamma radiation inducible promoter activity by quantifying fluorescence of a GFP reporter.

Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase.

Background: Control of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans.

Methods: C-5 methyl cytosines (mC) were detected by immuno-blotting with mC-specific antibody.

Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium.

Unlabelled: Two strains of the nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, displayed differential sensitivity to exposure to uranyl carbonate at neutral pH. Anabaena sp. strain PCC 7120 and Anabaena sp.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation.

Unlabelled: Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase.

In situ real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe Deinococcus radiodurans.

A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG.

Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins.

The radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi.

GroEL of the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31 exhibits GroES and ATP-independent refolding activity.

The nitrogen-fixing cyanobacterium, Anabaena L-31 has two Hsp60 proteins, 59 kDa GroEL coded by the second gene of groESL operon and 61 kDa Cpn60 coded by cpn60 gene. Anabaena GroEL formed stable higher oligomer (>12-mer) in the presence of K(+) and prevented thermal aggregation of malate dehydrogenase (MDH). Using three protein substrates (MDH, All1541 and green fluorescent protein), it was found that the refolding activity of Anabaena GroEL was lower than that of Escherichia coli GroEL, but independent of both GroES and ATP.

Involvement of phosphoesterases in tributyl phosphate degradation in Sphingobium sp. strain RSMS.

A tri- and dibutyl phosphate (TBP/DBP) non-degrading spontaneous mutant, Sphingobium SS22, was derived from the Sphingobium sp. strain RSMS (wild type). Unlike the wild type strain, Sphingobium SS22 could not grow in a minimal medium supplemented with TBP or DBP as the sole source of carbon or phosphorous.

Engineered Deinococcus radiodurans R1 with NiCoT genes for bioremoval of trace cobalt from spent decontamination solutions of nuclear power reactors.

The aim of the present work was to engineer bacteria for the removal of Co in contaminated effluents. Radioactive cobalt ((60)Co) is known as a major contributor for person-sievert budgetary because of its long half-life and high γ-energy values. Some bacterial Ni/Co transporter (NiCoT) genes were described to have preferential uptake for cobalt.

Membrane targeting of MnSOD is essential for oxidative stress tolerance of nitrogen-fixing cultures of Anabaena sp. strain PCC7120.

The nitrogen-fixing cyanobacterium, Anabaena PCC7120 encodes for a membrane-targeted 30 kDa Mn-superoxide dismutase (MnSOD) and a cytosolic FeSOD. The MnSOD is post-translationally processed to 27 and 24 kDa forms in the cytosol and periplasm/thylakoid lumen. The extent of cleavage of signal and linker peptides at the N-terminus is dependent on the availability of combined nitrogen during growth.

DNA adenine hypomethylation leads to metabolic rewiring in Deinococcus radiodurans.

The protein encoded by DR_0643 gene from Deinococcus radiodurans was shown to be an active N-6 adenine-specific DNA methyltransferase (Dam). Deletion of corresponding protein reduced adenine methylation in the genome by 60% and resulted in slow-growth phenotype. Proteomic changes induced by DNA adenine hypomethylation were mapped by two-dimensional protein electrophoresis coupled with mass spectrometry.

Comparative proteomics of oxidative stress response in three cyanobacterial strains native to Indian paddy fields.

Three strains of photoautotrophic, heterocystous, nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, were examined for their tolerance and proteomic response to the frequently used weedicide paraquat (methyl viologen). Anabaena 7120 (LD50 dose: 2μM for 6h) and Anabaena L-31 (LD50 dose: 2μM for 5h) showed distinctly better tolerance than Anabaena doliolum (LD50 dose: 2μM for 3h), to methyl viologen induced oxidative stress. The proteomic response, at respective LD50 dose, was mapped by 2D gel protein electrophoresis followed by protein identification by MALDI-ToF mass spectrometry.

Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans.

Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N-terminal without connector), SsbNC (N-terminal with connector) and SsbC (C-terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (SsbFL).

Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans.

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD50 = 40 μM tellurite, 40 min exposure). The organism reduced 70% of 40 μM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status.

Harnessing a radiation inducible promoter of Deinococcus radiodurans for enhanced precipitation of uranium.

Bioremediation is an attractive option for the treatment of radioactive waste. We provide a proof of principle for augmentation of uranium bioprecipitation using the radiation inducible promoter, Pssb from Deinococcus radiodurans. Recombinant cells of D.

The hypothetical protein 'All4779', and not the annotated 'Alr0088' and 'Alr7579' proteins, is the major typical single-stranded DNA binding protein of the cyanobacterium, Anabaena sp. PCC7120.

Single-stranded DNA binding (SSB) proteins are essential for all DNA-dependent cellular processes. Typical SSB proteins have an N-terminal Oligonucleotide-Binding (OB) fold, a Proline/Glycine rich region, followed by a C-terminal acidic tail. In the genome of the heterocystous nitrogen-fixing cyanobacterium, Anabaena sp.

Cyanobacterial heat-shock response: role and regulation of molecular chaperones.

Cyanobacteria constitute a morphologically diverse group of oxygenic photoautotrophic microbes which range from unicellular to multicellular, and non-nitrogen-fixing to nitrogen-fixing types. Sustained long-term exposure to changing environmental conditions, during their three billion years of evolution, has presumably led to their adaptation to diverse ecological niches. The ability to maintain protein conformational homeostasis (folding-misfolding-refolding or aggregation-degradation) by molecular chaperones holds the key to the stress adaptability of cyanobacteria.

Bioprecipitation of uranium from alkaline waste solutions using recombinant Deinococcus radiodurans.

Bioremediation of uranium (U) from alkaline waste solutions remains inadequately explored. We engineered the phoK gene (encoding a novel alkaline phosphatase, PhoK) from Sphingomonas sp. for overexpression in the radioresistant bacterium Deinococcus radiodurans.

Engineering bacteria for bioremediation of persistent organochlorine pesticide lindane (γ-hexachlorocyclohexane).

Strategies were designed for bioremediation of the highly persistent toxic pesticide γ-hexachlorocyclohexane (γ-HCH) or lindane from the environment. Lindane caused the loss of stress-protective chaperone GroEL, and inhibited photosynthesis, respiration and nitrogen-fixation in Anabaena, resulting in growth arrest. To alleviate lindane toxicity, the linA2 gene, encoding HCH dehydrochlorinase from Sphingomonas paucimobilis B90, was knocked-in at an innocuous locus in Anabaena genome and over-expressed from an eco-friendly light-inducible PpsbA1 promoter.