Cold shock induction of the cspL gene in Lactobacillus plantarum involves transcriptional regulation

Abstract

Fragments of the cspL promoter region were fused to the gusA reporter and reintroduced into Lactobacillus plantarum cells, either on multicopy plasmids or through single-copy chromosomal integration. beta-Glucuronidase activity and primer extension data demonstrate that the cspL promoter is induced in response to cold shock and that multicopy constructs quench the induction of the resident cspL gene.

FIG. 1.

Molecular structure of the genetic constructs. (A) Schematic map of the predicted mRNA secondary structure of the 5′ untranslated cspL region, showing the MluI and PstI restriction sites used in constructing the fusions, the Shine-Dalgarno sequence (SD, in bold), and the putative DB and UB (in bold). The cspL ORF (AUG codon, boxed) starts 89 nt downstream of the transcription start site (boxed +1). The predicted RNA secondary structure was obtained by free-energy minimization with the MFOLD program, version 2.0 (19, 20, 35). (B) Schematic drawing of the various genetic fusions used to study cspL regulation. The top line shows the gusA reporter carried by pGIS105. The next lines show schematic maps of the intact cspL and cbh genes (lanes 2 and 6, respectively), as well as those of the cspL-gusA, cbh-gusA, and cbh-cspL fusions (lanes 3 to 5, 7, and 8, respectively). Hairpins, hexagons, circles, and boxes represent, respectively, the terminators, promoters, Shine-Dalgarno sequences, and coding regions of gusA, cspL, or cbh. The cspL, gusA, and cbh fragments are indicated in white, black, and grey, respectively. The putative 77-aa ORF (orfX) downstream of cspL is also indicated by a white box (lanes 2 and 8). The fusion points are indicated by black straight lines and are numbered. Nucleotide numbers start from the transcription initiation site (+1), as determined previously (9). Arrows locate the specific cspL and gusA primers used to perform primer extension analyses.

FIG. 2.

Relative Gus activities of the various recombinant strains after a cold shock of 3 h at 8°C (values are averages of results of the numbers of experiments indicated below the bars). The reference level of 1 corresponds to the Gus activity measured before the cold shock in cells grown at 27°C and collected at an optical density (OD) (600 nm) of 0.8. Error bars represent 1 standard error of the mean. Statistical analysis was performed with the Student t test. ∗, significant difference (P < 0.05); ∗∗, highly significant difference (P < 0.01).

FIG. 3.

Primer extension analysis of cold shock induction (multicopy constructs). (A) Primer extension analysis of cold shock induction of the various autoreplicative constructs. Cells were grown at 27°C to an OD (600 nm) of 0.8 and shifted to 8°C for 3 h. Samples for RNA extraction were taken before (lane 1) and after (lane 2) the shift. Graphical presentation of the relative mRNA abundances of the various fusions are shown, with the lowest mRNA level taken as 1. (B) Similar analysis performed on mRNAs transcribed from the resident cspL gene in wild-type cells (NC8) and in cells carrying the various multicopy plasmids.

FIG. 4.

Primer extension analysis of cold shock induction (integrated constructs). (Upper panel) Primer extension analysis of cold shock induction of the various single-copy constructs integrated in the tRNA^Ser gene. Cells were grown at 27°C to an OD (600 nm) of 0.8 and shifted to 8°C for 3 h. Samples for RNA extraction were taken before (lane 1) and after (lane 2) the shift. ND, not detected. The asterisks indicate the position of the extension products showing the expected size. (Lower panel) Similar analysis performed on mRNAs transcribed from the resident cspL gene in wild-type cells (NC8) and in cells carrying the various single-copy constructs.