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Si M.,Northwest University, China | Zhang L.,Northwest University, China | Chaudhry M.T.,Environmental Analytical Laboratory | Ding W.,Northwest University, China | And 6 more authors.
Applied and Environmental Microbiology | Year: 2015

Oxidation of methionine leads to the formation of the S and R diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in Actinomycetes. Here, we report that a Corynebacterium glutamicum methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the msrA gene in C. glutamicum resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an S-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditions in vivo, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed. © 2015, American Society for Microbiology.

Si M.,Northwest University, China | Wang J.,Northwest University, China | Xiao X.,Northwest University, China | Guan J.,Northwest University, China | And 5 more authors.
PLoS ONE | Year: 2015

Ohr, a bacterial protein encoded by the Organic Hydroperoxide Resistance (ohr) gene, plays a critical role in resistance to organic hydroperoxides. In the present study, we show that the Cys-based thiol-dependent Ohr of Corynebacterium glutamicum decomposes organic hydroperoxides more efficiently than hydrogen peroxide. Replacement of either of the two Cys residues of Ohr by a Ser residue resulted in drastic loss of activity. The electron donors supporting regeneration of the peroxidase activity of the oxidized Ohr of C. glutamicum were principally lipoylated proteins (LpdA and Lpd/SucB). A Δohr mutant exhibited significantly decreased resistance to organic hydroperoxides and marked accumulation of reactive oxygen species (ROS) in vivo; protein carbonylation was also enhanced notably. The resistance to hydrogen peroxide also decreased, but protein carbonylation did not rise to any great extent. Together, the results unequivocally show that Ohr is essential for mediation of organic hydroperoxide resistance by C. glutamicum. © 2015 Si et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Si M.,Northwest University, China | Long M.,Northwest University, China | Chaudhry M.T.,Environmental Analytical Laboratory | Xu Y.,Northwest University, China | And 3 more authors.
PLoS ONE | Year: 2014

The present study focuses on the genetic and biochemical characterization of mycothiol S-conjugate amidase (Mca) of Corynebacterium glutamicum. Recombinant C. glutamicum Mca was heterologously expressed in Escherichia coli and purified to apparent homogeneity. The molecular weight of native Mca protein determined by gel filtration chromatography was 35 kDa, indicating that Mca exists as monomers in the purification condition. Mca showed amidase activity with mycothiol S-conjugate of monobromobimane (MSmB) in vivo while mca mutant lost the ability to cleave MSmB. In addition, Mca showed limited deacetylase activity with N-acetyl-D-glucosamine (GlcNAc) as substrate. Optimum pH for amidase activity was between 7.5 and 8.5, while the highest activity in the presence of Zn2+ confirmed Mca as a zinc metalloprotein. Amino acid residues conserved among Mca family members were located in C. glutamicum Mca and site-directed mutagenesis of these residues indicated that Asp14, Tyr137, His139 and Asp141 were important for activity. The mca deletion mutant showed decreased resistance to antibiotics, alkylating agents, oxidants and heavy metals, and these sensitive phenotypes were recovered in the complementary strain to a great extent. The physiological roles of Mca in resistance to various toxins were further supported by the induced expression of Mca in C. glutamicum under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. © 2014 Si et al.

Liu Y.-B.,Northwest University, China | Chen C.,Northwest University, China | Chaudhry M.T.,Environmental Analytical Laboratory | Si M.-R.,Northwest University, China | And 3 more authors.
Biotechnology Letters | Year: 2014

Over-expression of the gene, mshA, coding for mycothiol glycosyl transferase improved the robustness of Corynebacterium glutamicum to various stresses. Intracellular mycothiol (MSH) content was increased by 114 % in WT(pXMJ19-mshA) compared to WT(pXMJ19). Survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to WT(pXMJ19) under stress by H2O2 (40 mM), methylglyoxal (5.8 mM), erythromycin (0.08 mg ml-1), streptomycin (0.005 mg ml-1), Cd2+ (0.01 mM), Mn2+ (2 mM), formic acid (0.05 %), acetic acid (0.15 %), levulinic acid (0.25 %), furfural (7.2 mM), and ethanol (10 % v/v), respectively. Increased MSH content also decreased the concentration of reactive oxygen species in the presence of the above stresses. Our results may open a new avenue for enhancing robustness of industrial bacteria for production of commodity chemicals. © 2014 Springer Science+Business Media Dordrecht.

Hussain F.,Environmental Analytical Laboratory | Tahir S.S.,Environmental Analytical Laboratory | Rauf N.,Environmental Analytical Laboratory | Batool A.,Environmental Analytical Laboratory
Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences | Year: 2012

This study was conducted to determine the health risks in the indoor air of the four government hospitals of the Islamabad city, Pakistan. Four different main wards, i.e., general male/female surgical wards, children's ward and microbiology laboratory were included. The sampling was done in the summer season due to the possibility of maximum recovery of microorganisms. Results showed presence of bacterial and fungal pathogens in the air of hospitals especially in surgical wards of all hospitals. Lowest bacterial counts were recorded in microbiology laboratory. Among bacterial isolates Micrococcus and Staphylococcus aureus were abundantly found in all hospitals as 22.09 % & 21.2 %, respectively followed by gram negative group of bacteria i.e, Enterobacteriaceae spp. (Escherichia coli), Pseudomonas spp. were found to be lowest as 6.5 % of the total bacterial load in all hospitals. Among the fungi Aspergillus (fumigatus, niger, flavus) recovery was the most in the environment of all hospitals with the value of 27.7 % and Tricosporon was observed lowest with the value of 1.15 %. p-value for total microbial load among the hospitals sampled was not significant.

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