title: Phage-mediated transfer of a dextranase gene in Lactobacillus sanfranciscensis and characterization of the enzyme.
abstract: While phages of lactobacilli are extensively studied with respect to their structure and role in the dairy environment, knowledge about phages in bacteria residing in sourdough fermentation is limited. Based on the previous finding that the Lactobacillus sanfranciscensis phage EV3 carries a putative dextranase gene (dex), we have investigated the distribution of similar dex(+) phages in L. sanfranciscensis, the chance of gene transfer and the properties of the dextranase encoded by phage EV3. L. sanfranciscensis H2A (dex(-)), originally isolated from a wheat sourdough, expressed a Dex(+) phenotype upon infection with EV3. The dextranase gene was isolated from the transductant and heterologously expressed in Escherichia coli. The gene encoded a protein of 801 amino acids with a calculated molecular weight (Mw) of 89.09 kDa and a calculated pI of 5.62. Upon purification aided by a 6-His tag, enzyme kinetic parameters were determined. The Km value was 370 mM, and the Vmax was calculated in about 16 μmol of glucose released from dextran by 1 mg of enzyme in 1 min in a buffer solution at pH 5.0. The optimum conditions were 60 °C and pH 4.5. The enzyme retained its activity for >3h at 60 °C and exhibited only 40% activity at 30 °C; the highest homology of 72% was found to a dextranase gene from Lactobacillus fermentum phage φPYB5. Within 25 L. sanfransiscensis isolates tested, the strain 4B5 carried a similar prophage encoding a dextranase gene. Our data suggest a phage-mediated transfer of dextranase genes in the sourdough environment resulting in superinfection-resistant L. sanfranciscensis Dex(+) strains with a possible ecological advantage in dextran-containing sourdoughs.
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione, l'Ambiente (DeFENS), Università degli Studi di Milano, Milano, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Picozzi
authors.affiliation: Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Freising, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Meissner
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione, l'Ambiente (DeFENS), Università degli Studi di Milano, Milano, Italy.
authors.forename: Margherita
authors.initials: M
authors.lastname: Chierici
authors.affiliation: Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Freising, Germany.
authors.forename: Matthias A
authors.initials: MA
authors.lastname: Ehrmann
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione, l'Ambiente (DeFENS), Università degli Studi di Milano, Milano, Italy.
authors.forename: Ileana
authors.initials: I
authors.lastname: Vigentini
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione, l'Ambiente (DeFENS), Università degli Studi di Milano, Milano, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Foschino
authors.affiliation: Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Freising, Germany. Electronic address: rudi.vogel@wzw.tum.de.
authors.forename: Rudi F
authors.initials: RF
authors.lastname: Vogel
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D003910:Dextranase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics* / Q000821:virology; D008970:Molecular Weight; D013696:Temperature
chemical_list: D003910:Dextranase
keywords: Bacteriophage; Dextranase; Lactobacillus sanfranciscensis; Lysogenic conversion; Sourdough