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Quantification of the Genetic Expression of bgl-A, bgl, and CspA and Enzymatic Characterization of β-Glucosidases from Shewanella sp. G5

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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 Ca2+ and by 24 and 31 % in the presence of Mg2+ on LB and MMB at 30 °C, respectively, but it was totally inhibited by Hg2+, Cu2+, and EDTA. Moreover, this activity was slightly decreased by SDS, Zn2+, and DTT, all at 5 mM. Ethanol (14 % v/v) and glucose (100 mM) also reduced the activity by 63 and 60 %, respectively.

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Acknowledgments

This project was funded by Consejo de Investigaciones de la Universidad Nacional de Salta (Salta, Argentina) through the research projects N° 1669 and 1798. Héctor A. Cristóbal and Hugo R. Poma were holders of graduate fellowships awarded by Consejo Nacional de Investigaciones Científicas y Técnicas.

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Author notes

  1. Héctor Antonio Cristóbal

    Present address: Instituto de Investigaciones para la Industria Química, Universidad Nacional de Salta (INIQUI – CONICET-UNSa), Av. Bolivia 5150, 4400, Salta, Argentina

Authors and Affiliations

  1. Instituto de Investigaciones para la Industria Química, Universidad Nacional de Salta (INIQUI – CONICET-UNSa), Av. Bolivia 5150, 4400, Salta, Argentina

    Hugo Ramiro Poma, Carlos Mauricio Abate & Verónica Beatriz Rajal

  2. Planta Piloto de Procesos Industriales y Microbiológicos (PROIMI – CONICET), Av. Belgrano y Pje. Caseros, 4000, Tucumán, Argentina

    Héctor Antonio Cristóbal

  3. Facultad de Ingeniería, Universidad Nacional de Salta, Avda. Bolivia 5150, 4400, Salta, Argentina

    Verónica Beatriz Rajal

Authors
  1. Héctor Antonio Cristóbal
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  2. Hugo Ramiro Poma
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Correspondence to Héctor Antonio Cristóbal.

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Carlos Mauricio Abate passed away during the preparation of the study.

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Fig. S1

Primer analysis by conventional PCR and real-time PCR: (a) Agarose gel electrophoresis of the PCR products from the genes studied with designed primers. Lanes are M: 100 bp DNA Ladder, 1: CspA, 2: bgl-A, 3: bgl, 4: gyrB, 5: 16S rDNA, and 6: negative control. Dissociation curves generated with Applied Biosystems software: (b) 16S rDNA, (c) gyrB, (d) bgl-A, (e) bgl, and (f) CspA. (DOCX 1871 kb)

Fig. S2

Standard curves for the genes assayed determined that the amplification efficiency of all the systems was optimal: 99.9 % for 16S rDNA (slope −3.69), 99.3 % for gyrB (slope −3.24), 99.7 % for bgl (slope −3.43), 99.2 % for bgl-A and CspA (slopes −3.39). (DOCX 80 kb)

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Cristóbal, H.A., Poma, H.R., Abate, C.M. et al. Quantification of the Genetic Expression of bgl-A, bgl, and CspA and Enzymatic Characterization of β-Glucosidases from Shewanella sp. G5. Mar Biotechnol 18, 396–408 (2016). https://doi.org/10.1007/s10126-016-9702-z

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