title: Use of the alr gene as a food-grade selection marker in lactic acid bacteria.
abstract: Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.
authors.affiliation: Wageningen Centre for Food Sciences, 6700 AN Wageningen, The Netherlands.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Bron
authors.forename: Marcos G
authors.initials: MG
authors.lastname: Benchimol
authors.forename: Jolanda
authors.initials: J
authors.lastname: Lambert
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Palumbo
authors.forename: Marie
authors.initials: M
authors.lastname: Deghorain
authors.forename: Jean
authors.initials: J
authors.lastname: Delcour
authors.forename: Willem M
authors.initials: WM
authors.lastname: De Vos
authors.forename: Michiel
authors.initials: M
authors.lastname: Kleerebezem
authors.forename: Pascal
authors.initials: P
authors.lastname: Hols
mesh_terms: D000900:Anti-Bacterial Agents; D018407:Bacterial Physiological Phenomena; D001430:Bacteriocins / Q000737:chemistry* / Q000378:metabolism; D003523:Cycloserine / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000032:analysis*; D005518:Food Preferences; D005816:Genetic Complementation Test; D013294:Lactococcus lactis / Q000737:chemistry* / Q000378:metabolism; D009363:Neoplasm Proteins; D009561:Nisin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D004268:DNA-Binding Proteins; C120898:KMT2D protein, human; D009363:Neoplasm Proteins; C417164:plantaricin UG1; D009561:Nisin; D003523:Cycloserine