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.

FIG. 1.

Correlation between the level of Alr and the level of d-cycloserine resistance in L. lactis. PH3960 harboring pGIP013 was cultured with different concentrations of nisin and d-cycloserine. After 2 h, the growth rate was determined by OD₆₀₀ measurement. Cultures were induced with 0, 0.01, 0.1, or 1 ng of nisin per ml (black, gray, hatched, and white bars, respectively). Note that growth without d-cycloserine was similar for all nisin concentrations and is taken as 100%.

FIG. 2.

Integration of pGIP014 into the chromosome of MD007 at the tRNA^Ser locus by site-specific integration. pGIP014 harbors the attP site, which is identical to the chromosomally localized attB site. The int gene product catalyzes the recombination event between the attB and attP sites. The indicated primers tRNA and 40930L1 were used to identify the recombination event. The final strain was designated MD007::pGIP014. In this strain, the alr gene is orientated opposite to all neighboring genes, preventing readthrough expression from surrounding genes.

FIG. 3.

Growth of strain MD007::pGIP014 depends on nisin addition. An overnight culture of MD007::pGIP014 in MRS containing d-Ala was used to inoculate MRS containing nisin concentrations of 0.1 (solid squares), 1 (solid circles), 3 (triangles), and 20 (diamond) ng/ml. Open circles represent a control culture grown with d-Ala. Growth was monitored for 24 h by OD₆₀₀ measurement. Data are representative of three independent experiments.