ClpP participates in the degradation of misfolded protein in Lactococcus lactis

Abstract

SummaryClpP proteins constitute a family of homologous proteins found in both prokaryotic and eukaryotic organisms. In Escherichia coli, ClpP is the proteolytic component of a large complex also containing either the ClpA or the ClpX ATPases. We show here that the clpP gene from the Gram-positive bacterium Lactococcus lactis encodes a 22-kDa protein that is induced by low pH and by the t-RNA analogue puromycin, which interferes with translation, resulting in the production of misfolded puromycyl-containing peptides. Northern blot and primer extension analysis showed that clpP expression is also induced by heat shock and that stress induction occurs at the transcriptional level independent of the CIRCE regulatory element often implicated in stress regulation in Gram-positive bacteria. When we disrupted the L. lactis clpP gene by insertional inactivation, the resulting mutant was more sensitive to both heat and puromycin than wild-type cells. Furthermore, cells lacking ClpP had a reduced ability to degrade puromycyl-containing peptides, and they synthesized heat shock proteins constitutively in the absence of stress. Thus, our data suggest that ClpP plays a major role in the degradation of misfolded proteins.

Construction and characterization of a Lactococcus lactis strain deficient in intracellular ClpP and extracellular HtrA proteases

“…This phenotype has not been studied previously, and the original clpP mutant strain was only described to grow at similar rates on plates at 30 u C (colonies of equal size and frequency; Frees & Ingmer, 1999). Surprisingly, the growth of the clpP-htrA double mutant strain was improved compared to the clpP-NZ9000 single mutant (Fig.…”

“…ClpP is an ATP-dependent enzyme reported as the major intracellular housekeeping protease in L. lactis (Frees & Ingmer, 1999) whereas HtrA is a trypsin-like serine protease essential for growth at high temperatures (39 uC for L. lactis) and which degrades misfolded proteins at the cell surface (Poquet et al, 2000;Foucaud-Scheunemann & Poquet, 2003). In this study, we constructed a L. lactis NZ9000 strain deficient in its both major proteases, ClpP and HtrA (hereafter called clpP-htrA), and evaluated its physiology and heterologous protein production potential under the control of the NICE (nisin-controlled expression) system (Kuipers et al, 1998).…”

“…One transconjugant was grown in GM17 medium containing antibiotics at 30 uC until saturation. The culture was then diluted and plated without Cm R at 35 uC: this temperature was chosen to allow loss of the plasmid and lactis clpP-NZ9000 (for more details about the construction see Frees & Ingmer, 1999). (b, c) PCR identification of the clpP-NZ9000 and clpP-htrA strains using primers 5C and 3C for identification of clpP disruption (b) and 5E and 3B for confirmation of integrated plasmid (c).…”

“…to limit thermal stress, as both ClpP and HtrA are heat-shock proteins (Frees & Ingmer, 1999;Poquet et al, 2000;FoucaudScheunemann & Poquet, 2003). After 48 h incubation, some colonies were screened on selective and non-selective GM17 media incubated for 24 h. Colonies of the clpP-htrA double mutant strain which had lost pGhost3 were Cm sensitive (Cm S ), but they were Em R , and Em addition was needed to maintain the clpP mutation (Fig.…”

Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of Shigella dysenteriae Type-1 in Lactococcus lactis Using Nisin Inducible Controlled Expression (NICE)

“…In addition to HtrA, proteases like ClpP and several other housekeeping proteases which are poorly understood [23], were inhibited by incorporation of protease inhibitor cocktail. Such findings in our study suggest the critical role of proteases in efficient protein expression.…”