Hari S. Misra

Silver nanoparticle-loaded PVA/gum acacia hydrogel: synthesis, characterization and antibacterial study.

A simple one-pot method for in situ synthesis of silver nanoparticles (AgNPs), within polyvinyl alcohol/gum acacia (PVA-GA) hydrogel matrix, by gamma radiation-induced cross-linking is reported here. The synthesized hydrogels were characterized by FT-IR, thermogravimetry, dynamic light scattering and inductively coupled mass spectrometry method. The thermal stability was found to be more for the hydrogel loaded with silver nanoparticles and also the percentage silver loading was found to increase with increase in cross-linking density. The influence of gum acacia (GA) concentration on the equilibrium degree of swelling of the synthesized hydrogels, and also on the silver release from hydrogel matrix, was investigated. The size of the silver nanoparticles formed in the hydrogel matrix was in the range of 10-40 nm. The rheological gel point was found to be at 25.34 kGy of radiation dose, for a typical hydrogel synthesized, using 5% GA, 3% PVA and 1mM AgNO3. The antibacterial studies of the synthesized nanosilver-containing hydrogels showed good antibacterial activity against gram-negative bacterium, Escherichia coli.

Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria

Transgenic Escherichia coli expressing pyrroloquinoline‐quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non‐transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non‐reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by γ‐irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.

Pyrroloquinoline-quinone and its versatile roles in biological processes.

Pyrroloquinoline-quinine (PQQ) was initially characterized as a redox cofactor for membrane-bound dehydrogenases in the bacterial system. Subsequently, PQQ was shown to be an antioxidant protecting the living cells from oxidative damage in vivo and the biomolecules from artificially produced reaction oxygen species in vitro. The presence of PQQ has been documented from different biological samples. It functions as a nutrient and vitamin for supporting the growth and protection of living cells under stress. Recently, the role of PQQ has also been shown as a bio-control agent for plant fungal pathogens, an inducer for proteins kinases involved in cellular differentiation of mammalian cells and as a redox sensor leading to development of biosensor. Recent reviews published on PQQ and enzymes requiring this cofactor have brought forth the case specific roles of PQQ. This review covers the comprehensive information on various aspects of PQQ known till date. These include the roles of PQQ in the regulation of cellular growth and differentiation in mammalian system, as a nutrient and vitamin in stress tolerance, in crop productivity through increasing the availability of insoluble phosphate and as a bio-control agent, and as a redox agent leading to the biosensor development. Most recent findings correlating the exceptionally high redox recycling ability of PQQ to its potential as anti-neurodegenerative, anticancer and pharmacological agents, and as a signalling molecule have been distinctly brought out. This review discusses different findings suggesting the versatility in PQQ functions and provides the most plausible intellectual basis to the ubiquitous roles of this compound in a large number of biological processes, as a nutrient and a perspective vitamin.

An exonuclease I-sensitive DNA repair pathway in Deinococcus radiodurans : a major determinant of radiation resistance

Deinococcus radiodurans R1 recovering from acute dose of γ radiation shows a biphasic mechanism of DNA double‐strand break repair. The possible involvement of microsequence homology‐dependent, or non‐homologous end joining type mechanisms during initial period followed by RecA‐dependent homologous recombination pathways has been suggested for the reconstruction of complete genomes in this microbe. We have exploited the known roles of exonuclease I in DNA recombination to elucidate the nature of recombination involved in DNA double‐strand break repair during post‐irradiation recovery of D. radiodurans. Transgenic Deinococcus cells expressing exonuclease I functions of Escherichia coli showed significant reduction in γ radiation radioresistance, while the resistance to far‐UV and hydrogen peroxide remained unaffected. The overexpression of E. coli exonuclease I in Deinococcus inhibited DNA double‐strand break repair. Such cells exhibited normal post‐irradiation expression kinetics of RecA, PprA and single‐stranded DNA‐binding proteins but lacked the divalent cation manganese [(Mn(II)]‐dependent protection from γ radiation. The results strongly suggest that 3′ (ρ) 5′ single‐stranded DNA ends constitute an important component in recombination pathway involved in DNA double‐strand break repair and that absence of sbcB from deinococcal genome may significantly aid its extreme radioresistance phenotype.

Genome-wide study predicts promoter-G4 DNA motifs regulate selective functions in bacteria: radioresistance of D. radiodurans involves G4 DNA-mediated regulation

A remarkable number of guanine-rich sequences with potential to adopt non-canonical secondary structures called G-quadruplexes (or G4 DNA) are found within gene promoters. Despite growing interest, regulatory role of quadruplex DNA motifs in intrinsic cellular function remains poorly understood. Herein, we asked whether occurrence of potential G4 (PG4) DNA in promoters is associated with specific function(s) in bacteria. Using a normalized promoter-PG4-content (PG4P) index we analysed >60 000 promoters in 19 well-annotated species for (a) function class(es) and (b) gene(s) with enriched PG4P. Unexpectedly, PG4-associated functional classes were organism specific, suggesting that PG4 motifs may impart specific function to organisms. As a case study, we analysed radioresistance. Interestingly, unsupervised clustering using PG4P of 21 genes, crucial for radioresistance, grouped three radioresistant microorganisms including Deinococcus radiodurans. Based on these predictions we tested and found that in presence of nanomolar amounts of the intracellular quadruplex-binding ligand N-methyl mesoporphyrin (NMM), radioresistance of D. radiodurans was attenuated by ∼60%. In addition, important components of the RecF recombinational repair pathway recA, recF, recO, recR and recQ genes were found to harbour promoter-PG4 motifs and were also down-regulated in presence of NMM. Together these results provide first evidence that radioresistance may involve G4 DNA-mediated regulation and support the rationale that promoter-PG4s influence selective functions.

Involvement of a Protein Kinase Activity Inducer in DNA Double Strand Break Repair and Radioresistance of Deinococcus radiodurans

Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wild-type bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in gamma radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of gamma resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to gamma radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of (32)P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.

PprA: a protein implicated in radioresistance of Deinococcus radiodurans stimulates catalase activity in Escherichia coli

PprA: a pleiotropic protein promoting DNA repair, role in radiation resistance of Deinococcus radiodurans was demonstrated. In this study, the effect of radiation and oxidative stress on transgenic Escherichia coli expressing pprA has been studied. The pprA gene from D. radiodurans KR1 was cloned and expressed in E. coli. Transgenic E. coli cells expressing PprA showed twofold to threefold higher tolerance to hydrogen peroxide as compared to control. The 2.8-fold in vivo stimulation of catalase activity largely contributed by KatE was observed as compared to nonrecombinant control. Furthermore, the purified PprA could stimulate the E. coli catalase activity by 1.7-fold in solution. The effect of PprA on catalase activity observed both in vivo and in vitro was reverted to normal levels in the presence of PprA antibodies. The results suggest that enhanced oxidative stress tolerance in E. coli expressing PprA was due to the PprA stimulation of catalase activity, perhaps through the interaction of these proteins.

Genetic transformation of chickpea (Cicer arietinum L.) with insecticidal crystal protein gene using particle gun bombardment

Here, we report the establishment of an efficient particle gun bombardment mediated genetic transformation in chickpea (Cicer arietinum L.) using cryIAc gene of Bacillus thuringiensis. Explants were bombarded with recombinant plasmids engineered for the expression of cryIAc transgene in plants and stable transformants regenerated in presence of benzyladenine, kinetin and kanamycin. Transformation frequency showed dependence on explant type, cultivars, plasmids, helium pressure and microcarrier type used. Integration of transgenes was demonstrated using polymerase chain reaction and Southern blot hybridization approaches in T0 plants. The expression of CryIA(c) delta-endotoxin and GUS enzyme was ascertained by enzyme linked immunosorbent assay and histochemical assays, respectively. These transgenic plants (T0) showed more protection and high mortality for Heliothis armigera and Spodoptera litura larvae as compared to control plants. The results of the present study indicate that highest transformation frequency (18%) could be achieved by use of gold as a microcarrier in combination with helium pressure of 900 psi. Among the other factors tested, plasmid pHS 102 was the most efficient plasmid, while epicotyl explant was the best explant source for particle gun bombardment. Among the different cultivars of chickpea tested, cultivar ICCC37 and PG-12 produced higher frequency of transformation frequency compared to others.

Characterization of a DNA damage‐inducible membrane protein kinase from Deinococcus radiodurans and its role in bacterial radioresistance and DNA strand break repair

Deinococcus radiodurans mutant lacking pyrroloquinoline–quinone (PQQ) synthesis shows sensitivity to γ‐rays and impairment of DNA double strand break repair. The genome of this bacterium encodes five putative proteins having multiple PQQ binding motifs. The deletion mutants of corresponding genes were generated, and their response to DNA damage was monitored. Only the Δdr2518 mutant exhibited higher sensitivity to DNA damage. Survival of these cells decreased by 3‐log cycle both at 6 kGy γ‐rays and 1200 J m−2 UV (254 nm) radiation, and 2.5‐log cycle upon 14 days desiccation at 5% humidity. The Δdr2518 mutant showed complete inhibition of DSB repair until 24 h PIR and disappearance of a few phosphoproteins. The Δdr2518pqqE:cat double mutant showed γ‐ray sensitivity similar to Δdr2518 indicating functional interaction of these genes in D. radiodurans. DR2518 contains a eukaryotic type Ser/Thr kinase domain and structural topology suggesting stress responsive transmembrane protein. Its autokinase activity in solution was stimulated by nearly threefold with PQQ and twofold with linear DNA, but not with circular plasmid DNA. More than 15‐fold increase in dr2518 transcription and several‐fold enhanced in vivo phosphorylation of DR2518 were observed in response to γ irradiation. These results suggest that DR2518 as a DNA damage‐responsive protein kinase plays an important role in radiation resistance and DNA strand break repair in D. radiodurans.

Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli

The involvement of signal transduction in the repair of radiation‐induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline‐quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline‐quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline‐quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10‐fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline‐quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild‐type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild‐type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation‐induced DNA strand break repair and homologous recombination in E. coli.