title: Marker-free chromosomal integration of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri.
abstract: A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-beta-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli [Russell, W.M. and Klaenhammer, T.R. 2001 Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361-4364]. L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild-type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25 degrees C. This is the first report showing the successful utilization of the pORI plasmids system to generate marker-free gene integration in L. gasseri strains.
authors.affiliation: Department of Microbiology, North Carolina State University, P.O. Box 7615, Raleigh, NC 27695-7615, USA.
authors.forename: José M
authors.initials: JM
authors.lastname: Bruno-Bárcena
authors.forename: M Andrea
authors.initials: MA
authors.lastname: Azcárate-Peril
authors.forename: Todd R
authors.initials: TR
authors.lastname: Klaenhammer
authors.forename: Hosni M
authors.initials: HM
authors.lastname: Hassan
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis; D002876:Chromosomes, Bacterial / Q000235:genetics*; D015870:Gene Expression; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D048249:Streptococcus thermophilus / Q000201:enzymology / Q000235:genetics*; D013482:Superoxide Dismutase / Q000235:genetics*; D014169:Transformation, Bacterial
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase