Expression of the bglH Gene of Lactobacillus plantarum Is Controlled by Carbon Catabolite Repression

Abstract

A newly identified bglH gene coding for a phospho-β-glucosidase of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The bglH gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in L. plantarum cells grown on glucose compared to the β-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from −3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression ofbglH expression.

Mutational analysis of the bglH catabolite-responsive element (cre) in Lactobacillus plantarum

“…Gel retardation experiments were performed as described previously [20]. Cell extracts were obtained by resuspending exponential cultures in a bu¡er containing 20 mM Tris^HCl (pH 8.0), 100 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl £uoride, followed by grinding with glass beads.…”

“…Cell extracts were obtained by resuspending exponential cultures in a bu¡er containing 20 mM Tris^HCl (pH 8.0), 100 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl £uoride, followed by grinding with glass beads. A synthetic oligonucleotide (5P-GGAGGCGACTTGTTGTAAGGGC-TATCATTATTAGCGGACG-3P) and its complement were annealed to form a 40-bp fragment (CREa) containing the L. plantarum bglHcre sequence (in bold) and the surrounding sequence [20]. Fragment CREf was obtained by annealing two complementary oligonucleotides with a sequence identical to CREa except for one mismatch falling in position 2 of the 14-bp cre sequence (G to A).…”

“…We had previously shown that the G residue in position 2 (G 2 ) of the 14-bp bglHcre sequence was protected in vivo during growth on glucose, and not during growth on ribose [20]. In order to investigate further the function of the G 2 residue in CcpA binding, a gel retardation experiment was performed.…”

“…To monitor in vivo the function of the G 2 residue, transcriptional fusions of the wild-type and mutated bglH promoter with the cat reporter gene of the promoter probe vector pGKV210 were constructed (see Section 2). The analysis was extended to the C residue in position 13 (C 13 ) of bglHcre, previously shown to be protected in vivo during growth on glucose [20]. Oligonucleotides carrying zero, one or two mismatches with respect to the wild-type bglHcre sequence were used for PCR ampli¢cation of chromosomal L. plantarum DNA (Fig.…”

“…The positional distribution of cre sites has been reported to be within þ 200 bp of the translational start site [13], with cre sites downstream of the transcriptional start site that function as transcriptional roadblocks [18]. CcpA contacts guanine residues at the outer bounds of the cre rather than near the dyad axis as shown by in vitro studies of B. subtilis amyO [19] and in vivo studies of Lactobacillus plantarum bglHcre [20].…”