ClpL is essential for induction of thermotolerance and is potentially part of the HrcA regulon in Lactobacillus gasseri

Abstract

Stress-inducible proteins are likely to contribute to the survival and activity of probiotic bacteria during industrial processes and in the gastrointestinal tract. The recently published genome sequence of probiotic Lactobacillus gasseri ATCC 33323 suggests the presence of ClpC, ClpE, ClpL, and ClpX from the Clp ATPase family of stress proteins. The heat-shock response of L. gasseri was studied using 2-D DIGE. A total of 20 protein spots showing significant (p<0.05) increase in abundance after 30 min heat-shock were identified, including DnaK, GroEL, ClpC, ClpE, and ClpL. To study the physiological role of ClpL, one of the most highly induced proteins during heat-shock, its corresponding gene was inactivated. The DeltaclpL mutant strain had growth characteristics that were indistinguishable from wild-type under several stress conditions. However, in the absence of functional ClpL, L. gasseri exhibited drastically reduced survival at a lethal temperature and was unable to induce thermotolerance. Genome sequences indicate that the expression of clp genes in several Lactobacillus species is regulated by HrcA, instead of CtsR, the conserved clp gene regulator of low G+C Gram-positive bacteria. Electrophoretic mobility shift assays using L. gasseri HrcA protein and clpL upstream fragments revealed, for the first time, a direct interaction between HrcA and the promoter of a clp gene from a Lactobacillus.

CtsR Regulation in mcsAB -Deficient Gram-Positive Bacteria

“…ClpL is a heat shock protein found mainly in Gram-positive bacteria (16). Although the detailed functional studies on ClpL have been limited, several previous reports have shown that the protein plays an important role as a chaperone during heat shock (32,41,42,44). The protein was previously reported to be part of the CtsR regulon in several Gram-positive bacteria, such as Streptococcus thermophilus and Oenococcus oeni (18,44).…”

“…ClpL has never been reported to be involved in a specific protein degradation process. ClpL belongs to the HSP100/Clp ATPase family and has been shown to be important for the stress tolerance response in some organisms (15,41,42,45). Apart from its putative chaperone function and involvement in stress tolerance, very little is known about ClpL ATPase activity.…”

“…In staphylococci, clpL is solely under the control of a secondary sigma factor, B (15). In lactobacilli, the clpL promoter region contains a CIRCE sequence where HcrA, a transcriptional repressor, binds and regulates expression (41). In lactococci and some streptococci, clpL expression is regulated primarily by CtsR by binding to a consensus heptanucleotide repeat, A/GGTCAA/T, known as the CtsR box and located in the promoter region of the target genes.…”

ClpL Is Required for Folding of CtsR in Streptococcus mutans

“…This protein is found strictly in Gram-positive bacteria, including streptococci, and is involved in various cellular processes, such as the stress tolerance response, virulence, long-term cell survival, and antibiotic resistance (7,(30)(31)(32). Despite its importance, very little is known about the molecular functions of this protein.…”

“…In Streptococcus pneumoniae, a respiratory pathogen, ClpL has been shown to be involved in thermotolerance, acid tolerance, virulence regulation, and resistance to various antibiotics that target the cell wall (29)(30)(31)(32).…”