Luciferase detection during stationary phase in Lactococcus lactis

Abstract

The luminescence signal of luxAB-encoded bacterial luciferase strongly depends on the metabolic state of the host cell, which restricts the use of this reporter system to metabolically active bacteria. Here we show that in stationary-phase cells of Lactococcus lactis, detection of luciferase is significantly improved by the addition of riboflavin or flavin mononucleotide to whole-cell assay systems.

FIG. 1.

Effect of riboflavin on luminescence of luciferase-expressing L. lactis MG5267. Growth was analyzed by monitoring OD₅₉₅. Filled symbols show luminescence per OD₅₉₅ value, and open symbols show the corresponding OD₅₉₅ measurements. Symbols: ▪, MG5267(pNZ5519) grown in LM17 measured in buffer plus riboflavin; •, MG5267(pNZ5519) grown in LM17 measured in buffer only; ▴, MG5267(pNZ5519) grown in LM17 plus riboflavin (10 mg/liter) measured in buffer only; ⧫, CB010(pNZ5519) grown in LM17 measured in buffer only. Each data point represents the average of 12 biological replicates (error bars show standard deviations). a.u., arbitrary units.

FIG. 2.

Effect of addition of riboflavin or FMN on luminescence signals of stationary-phase cells of L. lactis MG5267 with various luciferase expression levels. Luciferase activity was measured 3.5 h after cultures entered the stationary phase. The black bars show measurements performed in buffer supplemented with 10 mg/liter riboflavin. White bars show measurements in buffer supplemented with 10 mg/liter FMN. Gray bars show measurements in buffer only. Each bar represents the average value of four biological replicates (error bars show standard deviations). *, P < 0.001; **, P < 0.002 (pairwise t test). The experiment was repeated four times with similar results. The names of the samples refer to the promoter sequences upstream of the luciferase genes as annotated in L. lactis MG1363 (18). a.u., arbitrary units.