title: Quantification of the Genetic Expression of bgl-A, bgl, and CspA and Enzymatic Characterization of β-Glucosidases from Shewanella sp. G5.
abstract: Shewanella sp. G5, a psychrotolerant marine bacterium, has a cold-shock protein (CspA) and three β-glucosidases, two of which were classified in the glycosyl hydrolase families 1 and 3 and are encoded by bgl-A and bgl genes, respectively. Shewanella sp. G5 was cultured on Luria-Bertani (LB) and Mineral Medium Brunner (MMB) media with glucose and cellobiose at various temperatures and pH 6 and 8. Relative quantification of the expression levels of all three genes was studied by real-time PCR with the comparative Ct method (2(-ΔΔCt)) using the gyrB housekeeping gene as a normalizer. Results showed that the genes had remarkably different genetic expression levels under the conditions evaluated, with increased expression of all genes obtained on MMB with cellobiose at 30 °C. Specific growth rate and specific β-glucosidase activity were also determined for all the culture conditions. Shewanella sp. G5 was able to grow on both media at 4 °C, showing the maximum specific growth rate on LB with cellobiose at 37 °C. The specific β-glucosidase activity obtained on MMB with cellobiose at 30 °C was 25 to 50 % higher than for all other conditions. At pH 8, relative activity was 34, 60, and 63 % higher at 30 °C than at 10 °C, with three peaks at 10, 25, and 37 °C on both media. Enzyme activity increased by 61 and 47 % in the presence of Ca(2+) and by 24 and 31 % in the presence of Mg(2+) on LB and MMB at 30 °C, respectively, but it was totally inhibited by Hg(2+), Cu(2+), and EDTA. Moreover, this activity was slightly decreased by SDS, Zn(2+), and DTT, all at 5 mM. Ethanol (14 % v/v) and glucose (100 mM) also reduced the activity by 63 and 60 %, respectively.
authors.affiliation: Instituto de Investigaciones para la Industria Química, Universidad Nacional de Salta (INIQUI - CONICET-UNSa), Av. Bolivia 5150, 4400, Salta, Argentina. hacristobal@gmail.com.|Planta Piloto de Procesos Industriales y Microbiológicos (PROIMI - CONICET), Av. Belgrano y Pje. Caseros, 4000, Tucumán, Argentina. hacristobal@gmail.com.
authors.forename: Héctor Antonio
authors.initials: HA
authors.lastname: Cristóbal
authors.affiliation: Instituto de Investigaciones para la Industria Química, Universidad Nacional de Salta (INIQUI - CONICET-UNSa), Av. Bolivia 5150, 4400, Salta, Argentina.
authors.forename: Hugo Ramiro
authors.initials: HR
authors.lastname: Poma
authors.affiliation: Instituto de Investigaciones para la Industria Química, Universidad Nacional de Salta (INIQUI - CONICET-UNSa), Av. Bolivia 5150, 4400, Salta, Argentina.
authors.forename: Carlos Mauricio
authors.initials: CM
authors.lastname: Abate
authors.affiliation: Instituto de Investigaciones para la Industria Química, Universidad Nacional de Salta (INIQUI - CONICET-UNSa), Av. Bolivia 5150, 4400, Salta, Argentina.|Facultad de Ingeniería, Universidad Nacional de Salta, Avda. Bolivia 5150, 4400, Salta, Argentina.
authors.forename: Verónica Beatriz
authors.initials: VB
authors.lastname: Rajal
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics; D059001:Aquatic Organisms; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent; D002475:Cellobiose / Q000378:metabolism* / Q000494:pharmacology; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D054712:Salinity; D020592:Shewanella / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D007527:Isoenzymes; C062260:cold shock protein CS7.4, Bacteria; D002475:Cellobiose; D001617:beta-Glucosidase; D027081:DNA Gyrase; D008274:Magnesium; D005947:Glucose
keywords: Biotechnology industry; Marine bacteria; Real-time PCR; Shewanella; β-glucosidases