Selection and characterization of conditionally active promoters in Lactobacillus plantarum, using alanine racemase as a promoter probe

Abstract

This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum. Random fragments of the L. plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an L. plantarum Deltaalr strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the D-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 micro g of the competitive Alr inhibitor D-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of D-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen. Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.

FIG. 1.

Plasmid map of the promoter-probe vector pNZ7120. The vector is a derivative of the low-copy-number vector pIL252 (34) and carries an erythromycin resistance gene (ery) for selection in L. plantarum. pNZ7120 harbors a promoterless copy of the L. lactis MG1363 alr gene, which is transcribed in the orientation opposite to that of all other genes encoded on pNZ7120. The DNA sequence visualizes the multiple-cloning site and the lactococcal prtP ribosome binding site (18). Furthermore, stop codons in all three reading frames (three black arrows) upstream of alr prevent translational fusions when chromosomal fragments are cloned in the BglII or SalI site of pNZ7120. The gray box indicates the 5′ end of the alr gene.

FIG. 2.

Western blot analysis of Alr expression levels in colonies sensitive to different concentrations of d-cycloserine. Randomly selected individual clones that originally were selected on MRS plates containing 10 (lanes 1 through 3), 100 (lanes 4 through 6), or 300 (lanes 7 and 8) μg of d-cycloserine/ml.

FIG. 3.

Western blot analysis of 0.8 M NaCl-induced Alr expression in 8 clones. Alr production levels were clearly higher in all eight clones (lanes 1 to 8) when cells were grown in media to which 0.8 M NaCl was added (lower panel) than when cells were grown in standard MRS media (upper panel). It should be noted that, when larger amounts of cell extract were used, a more obvious signal for clones pNZ7120-con2 and -con5 was detected (data not shown).