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. 2004 Apr;186(7):2200-5.
doi: 10.1128/JB.186.7.2200-2205.2003.

Evidence for multiple levels of regulation of Oenococcus oeni clpP-clpL locus expression in response to stress

Charlotte Beltramo  1 , Cosette Grandvalet, Fabrice Pierre, Jean Guzzo
Affiliations

Evidence for multiple levels of regulation of Oenococcus oeni clpP-clpL locus expression in response to stress

Charlotte Beltramo et al. J Bacteriol. 2004 Apr.

Abstract

A locus containing the clpP and clpL genes in the lactic acid bacterium Oenococcus oeni was studied. Real-time reverse transcription-PCR analysis revealed different induction factors involved in expression of these genes during stress. According to the conditions, clpP and clpL genes could be transcripted as two distinct transcripts or cotranscripted. The clpP promoter depended on the CtsR regulator, but surprisingly the clpL promoter did not. The amount of the clpL transcript depended on mRNA stability. This clp ATPase gene is at least controlled at the posttranscriptional level.

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Figures

FIG. 1.
FIG. 1.
Schematic diagram of the clpP-clpL locus and positions of probes (black lines) and primers (arrows) used in this study. Positions relative to ATG are indicated in parentheses. pb, base pairs.
FIG. 2.
FIG. 2.
Expression of clpP and clpL as a function of growth phase. (A) Growth curve of O. oeni. Arrows indicate cells samples for RNA extraction. (B) Northern blot analysis of RNA samples 1 to 8 using the clpP- and clpL-specific probes P and L (at each of the eight sampling times indicated in panel A). In all cases, 20 μg of RNA was applied per lane. This experiment was carried out two times. Note that with longer time exposure, a 2.4-kb signal was weakly detected during the mid-exponential phase. The sizes of the transcripts detected are indicated, with 0.7 kb corresponding to clpP and 2.4 kb corresponding to clpL. An asterisk indicates transcript detected with the clpL probe, the size of which did not correlate with the entire clpL gene.
FIG. 3.
FIG. 3.
Expression of clpP and clpL genes under stress conditions. Total RNAs were isolated from O. oeni cells grown at 30°C (lane 1) and submitted to heat shock (30 min at 42°C) (lane 2) or ethanolic shock (30 min at 10% [vol/vol]) (lane 3). Northern blot analyses of RNAs were performed with clpP probe (A); clpL probe (B); or a mixture of clpP, clpL, and intergenic region-specific probes (C). The sizes of the transcripts detected are indicated. In all cases, 15 μg of RNA was applied per lane.
FIG. 4.
FIG. 4.
Northern blotting performed to determine the relative rates of degradation of transcripts produced from the clp locus. The decay was measured in the exponential phase (A) at an OD600 of 0.7, with a heat stress at 42°C during 30 min (B) at an OD600 of 0.7, and in the stationary phase (C) at an OD600 of 1.8. The RNAs were prepared after 0, 3, 4.5, or 6.5 min after rifampin addition (250 μg ml−1). Northern blot analyses of RNAs were performed with a mixture of the three clpP, clpL, and intergenic region-specific probes. In all cases, 15 μg of RNA was applied per lane. This experiment was carried out two times. (D) Semilogarithmic plot of (i) clpP mRNA decay at OD600 of 0.7 (solid squares) or after heat-shock (open squares), (ii) clpL mRNA decay at an OD600 of 0.7 (solid circles) or after heat shock (open circles), and (iii) cotranscript mRNA decay at an OD600 of 0.7 after heat shock (open diamonds). The correlation coefficients (R2) and half-life (T1/2) were determined for each regression analysis.
FIG. 5.
FIG. 5.
Determination of the 5′ end of clp transcripts. The RT reaction was performed with 5 μg of total RNA isolated from O. oeni grown at 30°C or heat shocked for 1 h at 42°C. Lanes A, C, G, and T show the dideoxy sequencing ladder obtained with the same primer as that used for the reverse transcriptase reaction. The asterisk indicates the base representing the 5′ end of clpP mRNA or clpL mRNA in the sequence shown to the left. The RBS is double underlined. The putative −10 and −35 sequences are underlined and in boldface. An asterisk indicates the transcription start site. The ATG start codon was boxed. Arrows indicate the probable operator sites for CtsR. Positions relative to the translation initiation site are indicated to the left. (A) Determination of the 5′ end of the clpP transcript with oligonucleotide Pi2. (B) Determination of the 5′ end of the clpL transcript with oligonucleotide L2.
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