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Archives of Microbiology (v.191, #11)
Lysine-91 of the tetraheme c-type cytochrome CymA is essential for quinone interaction and arsenate respiration in Shewanella sp. strain ANA-3 by Kamrun Zargar; Chad W. Saltikov (pp. 797-806).
The tetraheme c-type cytochrome, CymA, is essential for arsenate respiratory reduction in Shewanella sp. ANA-3, a model arsenate reducer. CymA is predicted to mediate electron transfer from quinols to the arsenate respiratory reductase (ArrAB). Here, we present biochemical and physiological evidence that CymA interacts with menaquinol (MQH2) substrates. Fluorescence quench titration with the MQH2 analog, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), was used to demonstrate quinol binding of E. coli cytoplasmic membranes enriched with various forms of CymA. Wild-type CymA bound HOQNO with a K d of 0.1–1 μM. It was also shown that the redox active MQH2 analog, 2,3-dimethoxy-1,4-naphthoquinone (DMNH2), could reduce CymA in cytoplasmic membrane preparations. Based on a CymA homology model made from the NrfH tetraheme cytochrome structure, it was predicted that Lys91 would be involved in CymA-quinol interactions. CymA with a K91Q substitution showed little interaction with HOQNO. In addition, DMNH2-dependent reduction of CymA-K91Q was diminished by 45% compared to wild-type CymA. A ΔcymA ANA-3 strain containing a plasmid copy of cymA-K91Q failed to grow with arsenate as an electron acceptor. These results suggest that Lys91 is physiologically important for arsenate respiration and support the hypothesis that CymA interacts with menaquinol resulting in the reduction of the cytochrome.
Keywords: Tetraheme cytochrome; Shewanella; Quinone; Arsenate reduction
NifH-based studies on azotobacterial diversity in cotton soils of India by Ranjana Bhatia; Silke Ruppel; Neeru Narula (pp. 807-813).
In order to promote the use of Azotobacter inoculants for cotton crop, a complete characterization of soil isolates of Azotobacter, isolated and screened on the basis of physiological properties, from four different cotton–wheat cropping regions of India was carried out, and their genetic diversity determined by RFLP (restriction fragment length polymorphism) analysis of the functional gene nifH. Genetic analysis of these isolates depicted a similarity coefficient of ≥80% among them, suggesting that though the isolates were obtained from different cotton soils of India, still they have large commonality in the nifH gene and constituted a homogeneous nifH population.
Keywords: Azotobacter ; Cotton; Diversity; nifH gene; Nitrogen fixation
Characterization of the mineral phosphate solubilizing activity of Serratia marcescens CTM 50650 isolated from the phosphate mine of Gafsa by Mounira Ben Farhat; Ameny Farhat; Wacim Bejar; Radhouan Kammoun; Kameleddine Bouchaala; Amin Fourati; Hani Antoun; Samir Bejar; Hichem Chouayekh (pp. 815-824).
The mineral phosphate solubilizing (MPS) ability of a Serratia marcescens strain, namely CTM 50650, isolated from the phosphate mine of Gafsa, was characterized on a chemically defined medium (NBRIP broth). Various insoluble inorganic phosphates, including rock phosphate (RP), calcium phosphate (CaHPO4), tri-calcium phosphate (Ca3(PO4)2) and hydroxyapatite were tested as sole sources of phosphate for bacterial growth. Solubilization of these phosphates by S. marcescens CTM 50650 was very efficient. Indeed, under optimal conditions, the soluble phosphorus (P) concentration it produced reached 967, 500, 595 and 326 mg/l from CaHPO4, Ca3(PO4)2, hydroxyapatite and RP, respectively. Study of the mechanisms involved in the MPS activity of CTM 50650, showed that phosphate solubilization was concomitant with significant drop in pH. HPLC-analysis of culture supernatants revealed the secretion of gluconic acid (GA) resulting from direct oxidation pathway of glucose when the CTM 50650 cells were grown on NBRIP containing glucose as unique carbon source. This was correlated with the simultaneous detection by PCR for the first time in a S. marcescens strain producing GA, of a gene encoding glucose dehydrogenase responsible for GA production, as well as the genes pqqA, B, C and E involved in biosynthesis of its PQQ cofactor. This study is expected to lead to the development of an environmental-friendly process for fertilizer production considering the capacity of S. marcescens CTM 50650 to achieve yields of P extraction up to 75% from the Gafsa RP.
Keywords: Serratia marcescens CTM 50650; Mineral phosphate solubilization; Rock phosphate; Gluconic acid; PQQ cofactor
A new β-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization by Aneta Monika Białkowska; Hubert Cieśliński; Karolina Maria Nowakowska; Józef Kur; Marianna Turkiewicz (pp. 825-835).
A psychrotrophic bacterium producing a cold-adapted β-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F′ and screening on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-containing agar plates led to the isolation of β-galactosidase gene. The β-galactosidase gene (bgaS) encoding a protein of 1,053 amino acids, with a calculated molecular mass of 113,695 kDa. Analysis of the amino acid sequence of BgaS protein, deduced from the bgaS ORF, suggested that it is a member of the glycosyl hydrolase family 2. A native cold-adapted β-galactosidase was purified to homogeneity and characterized. It is a homotetrameric enzyme, each subunit being approximately 116 kDa polypeptide as deduced from native and SDS–PAGE, respectively. The β-galactosidase was optimally active at pH 6.0–8.0 and 25°C. P-nitrophenyl-β-d-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG—o-nitrophenyl-β-d-galactopyranoside). The Arthrobacter sp. 20B β-galactosidase is activated by thiol compounds (53% rise in activity in the presence of 10 mM 2-mercaptoethanol), some metal ions (activity increased by 50% for Na+, K+ and by 11% for Mn2+) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb2+, Zn2+, Cu2+).
Keywords: Cold-adapted enzymes; β-Galactosidase; Arthrobacter sp.; Lactose hydrolysis; Psychrotrophic microorganisms
Response to different oxidants of Saccharomyces cerevisiae ure2Δ mutant by Tatina T. Todorova; Ventsislava Y. Petrova; Stéphane Vuilleumier; Anna V. Kujumdzieva (pp. 837-845).
Growth of Saccharomyces cerevisiae ure2Δ mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium supplemented with H2O2 was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established. Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed. It was suggested that the susceptibility of ure2Δ mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of the γ-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level.
Keywords: Ure2p; Glutathione transferase; SOD; Catalase; Oxidative stress; Yeasts
Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test by Luciana A. Gonçalves; Zélia I. P. Lobato; Rodrigo O. S. Silva; Felipe M. Salvarani; Prhiscylla S. Pires; Ronnie A. Assis; Francisco C. F. Lobato (pp. 847-851).
Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.
Keywords: Clostridium perfringens ; Epsilon; Dot-immunoblotting; etx ; Vaccine
Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species by Hui Li; Sara Jubelirer; Amaya M. Garcia Costas; Niels-Ulrik Frigaard; Donald A. Bryant (pp. 853-867).
The genome of the green sulfur bacterium Chlorobaculum (Cba.) tepidum, a strictly anaerobic photolithoautotroph, is predicted to encode more than ten genes whose products are potentially involved in protection from reactive oxygen species and an oxidative stress response. The encoded proteins include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were insertionally inactivated. Wild-type Cba. tepidum cells were very sensitive to oxygen in the light but were remarkably resistant to oxygen in the dark. When wild-type and mutant cells were subjected to air for various times under dark or light condition, significant decreases in viability were detected in most of the mutants relative to wild type. Treatments with hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BOOH) and methyl viologen resulted in more severe effects in most of the mutants than in the wild type. The results demonstrated that these putative antioxidant proteins combine to form an effective defense against oxygen and reactive oxygen species. Reverse-transcriptase polymerase chain reaction studies showed that the genes with functions in oxidative stress protection were constitutively transcribed under anoxic growth conditions.
Keywords: Green sulfur bacteria; Oxidative stress; Chlorobaculum tepidum ; Photosynthesis