Swathi Kota

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.

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.

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.

PprA, a pleiotropic protein for radioresistance, works through DNA gyrase and shows cellular dynamics during postirradiation recovery in Deinococcus radiodurans

PprA, a pleiotropic protein involved in radioresistance of Deinococcus radiodurans was detected in multiprotein DNA processing complex identified from this bacterium. pprA mutant expressing GFP-PprA could restore its wild type resistance of γ radiation. Under normal conditions, GFP-PprA expressing cells showed PprA localization on both septum trapped nucleoids (STN) and nucleoids located elsewhere (MCN). Cell exposed to 4 kGy γ radiation showed nearly 2 h growth lag and during this growth arrest phase, the majority of the cells had GFP-PprA located on MCN. While in late phase (∼120 min) PIR cells, when cells are nearly out of growth arrest, PprA was maximally found with STN. These cells when treated with nalidixic acid showed diffused localization of PprA across the septum. gyrA disruption mutant of D. radiodurans showed growth inhibition, which increased further in gyrA pprA mutant. Interestingly, gyrA mutant showed ∼20-fold less resistance to γ radiation as compared to wild type, which did increase further in gyrA pprA mutant. These results suggested that PprA localization undergoes a dynamic change during PIR, and its localization on nucleoid near septum and functional interaction with gyrase A might suggest a mechanism that could explain PprA role in genome segregation possibly through topoisomerase II.

G-quadruplex forming structural motifs in the genome of Deinococcus radiodurans and their regulatory roles in promoter functions

Deinococcus radiodurans displays compromised radioresistance in the presence of guanine quadruplex (G4)-binding drugs (G4 drugs). Genome-wide scanning showed islands of guanine runs (G-motif) in the upstream regions of coding sequences as well as in the structural regions of many genes, indicating a role for G4 DNA in the regulation of genome functions in this bacterium. G-motifs present upstream to some of the DNA damage-responsive genes like lexA, pprI, recF, recQ, mutL and radA were synthesized, and the formation of G4 DNA structures was probed in vitro. The G-motifs present at the 67th position upstream to recQ and at the 121st position upstream to mutL produced parallel and mixed G4 DNA structures, respectively. Expression of β-galactosidase under recQ and mutL promoters containing respective G-motifs was inhibited by G4 drugs under normal growth conditions in D. radiodurans. However, when such cells were exposed to γ radiation, mutL promoter activity was stimulated while recQ promoter activity was inhibited in the presence of G4 drugs. Deletion of the G-motif from the recQ promoter could relax it from G4 drug repression. D. radiodurans cells treated with G4 drug showed reduction in recQ expression and γ radiation resistance, indicating an involvement of G4 DNA in the radioresistance of this bacterium. These results suggest that G-motifs from D. radiodurans genome form different types of G4 DNA structures at least in vitro, and the recQ and mutL promoters seem to be differentially regulated at the levels of G4 DNA structures.

Maintenance of multipartite genome system and its functional significance in bacteria

Bacteria are unicellular organisms that do not show compartmentalization of the genetic material and other cellular organelles as seen in higher organisms. Earlier, bacterial genomes were defined as single circular chromosome and extrachromosomal plasmids. Recently, many bacteria were found harbouring multipartite genome system and the numbers of copies of genome elements including chromosomes vary from one to several per cell. Interestingly, it is noticed that majority of multipartite genome-harbouring bacteria are either stress tolerant or pathogens. Further, it is observed that the secondary genomes in these bacteria encode proteins that are involved in bacterial genome maintenance and also contribute to higher stress tolerance, and pathogenicity in pathogenic bacteria. Surprisingly, in some bacteria the genes encoding the proteins of classical homologous recombination pathways are present only on the secondary chromosomes, and some do not have either of the classical homologous recombination pathways. This review highlights the presence of ploidy and multipartite genomes in bacterial system, the underlying mechanisms of genome maintenance and the possibilities of these features contributing to higher abiotic and biotic stress tolerance in these bacteria.

Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro

Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase Ib of D. radiodurans (DraTopoIB) could change the electrophoretic mobility of fast migrating intramolecular recF-G4 DNA into the slow migrating species. DraTopoIB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular recF-G4 DNA structures stabilized by K+. On the contrary, when DraTopoIB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopoIB. This implies that DraTopoIB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopoIB activity on intramolecular G4 DNA structures. These results suggested that DraTopoIB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.