clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor σ^H

Abstract

SummaryThe ATP-dependent protease Clp plays important roles in the cell's protein quality control system and in the regulation of cellular processes. In Corynebacterium glutamicum , the levels of the proteolytic subunits ClpP1 and ClpP2 as well as of the corresponding mRNAs were drastically increased upon deletion of the clpC gene, coding for a Clp ATPase subunit. We identified a regulatory protein, designated ClgR, binding to a common palindromic sequence motif in front of clpP1P2 as well as of clpC . Deletion of clgR in the D D D D clpC background completely abolished the increased transcription of both operons, indicating that ClgR activates transcription of these genes. ClgR activity itself is probably controlled via ClpC-dependent regulation of its stability, as ClgR is only present in D D D D clpC and not in wild-type cells, whereas the levels of clgR mRNA are comparable in both strains. clpC , clpP1P2 and clgR expression is induced upon severe heat stress, however, independently of ClgR. Identification of the heat-responsive transcriptional start sites in front of these genes revealed the presence of sequence motifs typical for s s s s ECF -dependent promoters. The ECF sigma factor s s s s H could be identified as being required for transcriptional activation of clpC , clpP1P2 and clgR in response to severe heat stress. A second heat-responsive but s s s s H -independent promoter in front of clgR could be identified that is subject to negative regulation by the transcriptional repressor HspR. Taken together, these results show that clpC and clpP1P2 expression in C. glutamicum is subject to complex regulation via both independent and hierarchically organized pathways, allowing for the integration of multiple environmental stimuli. Both the ClgR-and s s s s H -dependent regulation of clpC and clpP1P2 expression appears to be conserved in other actinomycetes. IntroductionProteolysis in bacterial cells is mainly performed by ATPdependent proteases. Together with several chaperones, i.e. DnaKJ-GrpE and ClpB, these proteases are integral parts of the cell's protein quality control system, which is responsible for clearing the cell of non-functional proteins (for recent reviews, see Wickner et al ., 1999;Dougan et al ., 2002). Most of these proteases also perform important regulatory functions by controlling the availability of transcriptional regulators, enzymes and other proteins via conditional degradation (for a recent review, see Jenal and Hengge-Aronis, 2003). Of these proteases, Clp has been most extensively studied both mechanistically and functionally. The Clp holoenzyme consists of two separate and functionally distinct subunits. The proteolytic subunits, ClpP, perform the actual hydrolysis of substrates. However, their active sites are buried within the cavity of the so-called proteolytic core formed by 14 ClpP subunits. Therefore, hexamers of ATPase subunits (ClpA, ClpC or ClpX), which are members of the Clp/Hsp100 superfamily (Schirmer et al ., 1996) and associate with the core, are required in orde...

ClgR regulation of chaperone and protease systems is essential for Mycobacterium tuberculosis parasitism of the macrophage

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“…Low-GC Gram-positive bacteria such as B. subtilis have evolved a negative control strategy using the transcriptional repressor CtsR. In contrast, the high-GC actinomycetes appear to have adopted a highly conserved transcriptional activator ClgR (clp gene regulator), which has been shown to control clp genes in Corynebacterium glutamicum (Engels et al, 2004(Engels et al, , 2005, Streptomyces lividans (Bellier & Mazodier, 2004;Bellier et al, 2006) and Bifidobacterium breve (Ventura et al, 2005). A homologue of ClgR exists in M. tuberculosis and its expression is highly upregulated in stress conditions such as heat shock (Stewart et al, 2002) and during macrophage infection (Schnappinger et al, 2003).…”

Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum

“…A DNA-affinity approach (Gabrielsen et al, 1989;Rey et al, 2003;Engels et al, 2004) was used to purify such a protein, with the ldh-cg3220 intergenic region as a target. Protein purification was performed with dialysed crude extracts from cells grown on glucose or acetate, harvested after 7.5 h cultivation when lactate reached its maximum concentration on glucose.…”

Post-translational control of the Streptomyces lividans ClgR regulon by ClpP

“…glutamicum ClgR does not end with two alanines. Moreover, the motif is probably not located at the C terminus since the addition of 10 amino acid residues did not interfere with its degradation (Engels et al, 2004).…”