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, 187 (16), 5614-23

CtsR Is the Master Regulator of Stress Response Gene Expression in Oenococcus Oeni

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CtsR Is the Master Regulator of Stress Response Gene Expression in Oenococcus Oeni

Cosette Grandvalet et al. J Bacteriol.

Abstract

Although many stress response genes have been characterized in Oenococcus oeni, little is known about the regulation of stress response in this malolactic bacterium. The expression of eubacterial stress genes is controlled both positively and negatively at the transcriptional level. Overall, negative regulation of heat shock genes appears to be more widespread among gram-positive bacteria. We recently identified an ortholog of the ctsR gene in O. oeni. In Bacillus subtilis, CtsR negatively regulates expression of the clp genes, which belong to the class III family of heat shock genes. The ctsR gene of O. oeni is cotranscribed with the downstream clpC gene. Sequence analysis of the O. oeni IOB 8413 (ATCC BAA-1163) genome revealed the presence of potential CtsR operator sites upstream from most of the major molecular chaperone genes, including the clp genes and the groES and dnaK operons. Using B. subtilis as a heterologous host, CtsR-dependent regulation of O. oeni molecular chaperone genes was demonstrated with transcriptional fusions. No alternative sigma factors appear to be encoded by the O. oeni IOB 8413 (ATCC BAA-1163) genome. Moreover, apart from CtsR, no known genes encoding regulators of stress response, such as HrcA, could be identified in this genome. Unlike the multiple regulatory mechanisms of stress response described in many closely related gram-positive bacteria, this is the first example where dnaK and groESL are controlled by CtsR but not by HrcA.

Figures

FIG. 1.
FIG. 1.
(A) Determination of the transcription initiation site of the ctsR gene by primer extension analysis. Total RNA was extracted from O. oeni cells harvested in the exponential phase before (lane 1) or after (lane 2) a 30-min shift to 42°C. Primer extension products corresponding to the ctsR gene are shown alongside DNA-sequencing reaction products (lanes T, G, C, and A). The corresponding nucleotide sequence is shown on the left. The transcriptional start site is indicated by an asterisk, the −10 sequence is boxed, and arrows indicate the likely CtsR operator sites. (B) Organization of the ctsR-clpC operon and nucleotide sequence of the ctsR promoter region. The putative −10 and −35 sequences are underlined and boldface, arrows indicate the likely CtsR operator sites, and the initiation codon (ATG) is in boldface.
FIG. 2.
FIG. 2.
Alignment of CtsR-binding sites identified upstream from ctsR, hsp18, clpX, grpE, clpP, groES, and clpL2 genes of O. oeni IOB 8413 (ATCC BAA-1163). Identical nucleotides are shaded. The numbers indicate positions relative to the transcriptional start site. GenBank accession numbers for database sequences are as follows: clpL2 (AJ890337), groES (AJ890340), grpE (AJ890339), ctsR (AJ890338), hsp18 (AJ250422), clpX (Y15953), and clpP (AJ606044).
FIG. 3.
FIG. 3.
(A) Primer extension analysis of clpL2, groES, and grpE mRNAs. Total RNA was extracted from O. oeni cells harvested in the exponential phase (lane C) or after a 30-min shift to 42°C (lane HS). Primer extension products are shown alongside DNA-sequencing reaction products (lanes T, G, C, and A). (B) Nucleotide sequences of the clpL2, groESL, grpE, ctsR, hsp18, clpX, and clpP promoter regions. Potential −35 and −10 sequences are underlined and boldface, transcriptional start points are indicated by +1, and CtsR heptad direct-repeat operator sequences are indicated by arrows. Mutation of the groES promoter performed by insertion of a PstI site in the repeated sequence of the CtsR operator site is indicated.
FIG. 4.
FIG. 4.
CtsR negatively regulates hsp gene expression. Expression of the mleA-bgaB, ctsR-bgaB, clpX-bgaB, clpL2-bgaB, groES-bgaB, and groESmut-bgaB (A) and the hsp18-bgaB, clpP-bgaB, and grpE-bgaB (B) transcriptional fusions was measured in the wild-type strain (white and gray bars) or in the ΔctsR mutant (QB4991) (black bars). Cells were grown in LB medium at 37°C. Expression of bgaB fusions was compared in the mid-exponential phase before (white and black bars) or after (gray bars) transfer to 48°C. The numbers indicate induction factors, which were calculated relative to the expression level of each fusion measured in the wild-type strain grown at 37°C.
FIG. 5.
FIG. 5.
Dual regulation by CtsR and HrcA in different gram-positive bacteria. In many gram-positive bacteria, the CtsR and the HrcA regulons coexist. In B. subtilis and closely related bacilli (B. anthracis, B. stearothermophilus, B. halodurans, C. acetobutylicum, C. difficile, C. perfringens, L. monocytogenes, and L. innocua), the two regulons are distinct, whereas in the streptococcal group (S. pneumoniae, L. lactis, and S. salivarius), they partially overlap, and the HrcA regulon is entirely embedded within the CtsR regulon in S. aureus. O. oeni and L. bulgaricus have original heat shock gene regulation with a predominant control of molecular chaperone genes by either CtsR or HrcA, respectively (adapted from Chastanet et al. [11]).

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