FrnE,a Cadmium-Inducible Protein in Deinococcus radiodurans,Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo

Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ∼15- and ∼3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ∼2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42°C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation.     

DrRecQ regulates guanine quadruplex DNA structure dynamics and its impact on radioresistance in Deinococcus radiodurans

The GC‐rich genome of Deinococcus radiodurans contains a very high density of putative guanine quadruplex (G4) DNA motifs and its RecQ (drRecQ) was earlier characterized as a 3′→5′ dsDNA helicase. We saw that N‐Methyl mesoporphyrin IX (NMM), a G4 DNA binding drug affected normal growth as well as the gamma radiation resistance of the wild‐type bacterium. Interestingly, NMM treatment and recQ deletion showed additive effect on normal growth but there was no effect of NMM on gamma radiation resistance of recQ mutant. The recombinant drRecQ showed ~400 times higher affinity to G4 DNA (K_d = 11.74 ± 1.77 nM) as compared to dsDNA (K_d = 4.88 ± 1.30 µM). drRecQ showed ATP independent helicase function on G4 DNA, which was higher than ATP‐dependent helicase activity on dsDNA. Unlike wild‐type cells that sparingly stained for G4 structure with Thioflavin T (ThT), recQ mutant showed very high‐density of ThT fluorescence foci on DNA indicating an important role of drRecQ in regulation of G4 DNA structure dynamics in vivo. These results together suggested that drRecQ is an ATP independent G4 DNA helicase that plays an important role in the regulation of G4 DNA structure dynamics and its impact on radioresistance in D. radiodurans.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

RecBC enzyme overproduction affects UV and gamma radiation survival of Deinococcus radiodurans

Deinococcus radiodurans recovering from the effect of acute dose of gamma (gamma) radiation shows a biphasic mechanism of DNA double strands breaks repair that involves an efficient homologous recombination. However, it shows higher sensitivity to near-UV (NUV) than Escherichia coli and lacks RecBC, a DNA strand break (DSB) repair enzyme in some bacteria. Recombinant Deinococcus expressing the recBC genes of E. coli showed nearly three-fold improvements in near-UV tolerance and nearly 2 log cycle reductions in wild type gamma radiation resistance. RecBC over expression effect on radiation response of D. radiodurans was independent of indigenous RecD. Loss of gamma radiation tolerance was attributed to the enhanced rate of in vivo degradation of radiation damaged DNA and delayed kinetics of DSB repair during post-irradiation recovery. RecBC expressing cells of Deinococcus showed wild type response to Far-UV. These results suggest that the overproduction of RecBC competes with the indigenous mechanism of gamma radiation damaged DNA repair while it supports near-UV tolerance in D. radiodurans.     

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 gamma-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.