title: Engineered strains of Streptococcus macedonicus towards an osmotic stress resistant phenotype retain their ability to produce the bacteriocin macedocin under hyperosmotic conditions.
abstract: Streptococcus macedonicus ACA-DC 198 produces the bacteriocin macedocin in milk only under low NaCl concentrations (<1.0%w/v). The thermosensitive plasmid pGh9:ISS1 was employed to generate osmotic stress resistant (osmr) mutants of S. macedonicus. Three osmr mutants showing integration of the vector in unique chromosomal sites were identified and the disrupted loci were characterized. Interestingly, the mutants were able to grow and to produce macedocin at considerably higher concentrations of NaCl compared to the wild-type (up to 4.0%w/v). The production of macedocin under hyperosmotic conditions solely by the osmr mutants was validated by the well diffusion assay and by mass spectrometry analysis. RT-PCR experiments demonstrated that the macedocin biosynthetic regulon was transcribed at high salt concentrations only in the mutants. Mutant osmr3, the most robust mutant, was converted in its markerless derivative (osmr3f). Co-culture of S. macedonicus with spores of Clostridium tyrobutyricum in milk demonstrated that only the osmr3f mutant and not the wild-type inhibited the growth of the spores under hyperosmotic conditions (i.e., 2.5%w/v NaCl) due to the production of macedocin. Our study shows how genetic manipulation of a strain towards a stress resistant phenotype could improve bacteriocin production under conditions of the same stress.
authors: Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.
authors: Rania
authors: R
authors: Anastasiou
authors: Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.
authors: Gonzalez Van
authors: GV
authors: Driessche
authors: Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimioupolis-Zographou, 157 84 Athens, Greece.
authors: Effrossyni
authors: E
authors: Boutou
authors: Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.
authors: Maria
authors: M
authors: Kazou
authors: Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.
authors: Voula
authors: V
authors: Alexandraki
authors: Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimioupolis-Zographou, 157 84 Athens, Greece.
authors: Constantinos E
authors: CE
authors: Vorgias
authors: Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.
authors: Bart
authors: B
authors: Devreese
authors: Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.
authors: Effie
authors: E
authors: Tsakalidou
authors: Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece; Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimioupolis-Zographou, 157 84 Athens, Greece. Electronic address: kpapadimitriou@aua.gr.
authors: Konstantinos
authors: K
authors: Papadimitriou
mesh_terms: D000818:Animals; D001430:Bacteriocins / Q000096:biosynthesis*; D003016:Clostridium perfringens; D018920:Coculture Techniques; D008892:Milk / Q000382:microbiology; D009154:Mutation; D009997:Osmotic Pressure / Q000502:physiology*; D010641:Phenotype; D012965:Sodium Chloride / Q000494:pharmacology; D013171:Spores, Bacterial; D013291:Streptococcus / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D013312:Stress, Physiological / Q000502:physiology*
chemical_list: D001430:Bacteriocins; C471066:macedocin, Streptococcus macedonicus; D012965:Sodium Chloride
keywords: Bacteriocin; Clostridium; Mutants; Osmotic; Streptococcus; Stress