Important Role of Class I Heat Shock Genes hrcA and dnaK in the Heat Shock Response and the Response to pH and NaCl Stress of Group I Clostridium botulinum Strain ATCC 3502

Abstract

Class I heat shock genes (HSGs) code for molecular chaperones which play a major role in the bacterial response to sudden increases of environmental temperature by assisting protein folding. Quantitative reverse transcriptase real-time PCR gene expression analysis of the food-borne pathogen Clostridium botulinum grown at 37°C showed that the class I HSGs grpE, dnaK, dnaJ, groEL, and groES and their repressor, hrcA, were expressed at constant levels in the exponential and transitional growth phases, whereas strong downregulation of all six genes was observed during stationary phase. After heat shock from 37 to 45°C, all HSGs were transiently upregulated. A mutant with insertionally inactivated hrcA expressed higher levels of class I HSGs during exponential growth than the wild type, followed by upregulation of only groES and groES after heat shock. Inactivation of hrcA or of dnaK encoding a major chaperone resulted in lower maximum growth temperatures than for the wild type and reduced growth rates under optimal conditions compared to the wild type. The dnaK mutant showed growth inhibition under all tested temperature, pH, and NaCl stress conditions. In contrast, the growth of an hrcA mutant was unaffected by mild temperature or acid stress compared to the wild-type strain, indicating that induced class I HSGs support growth under moderately nonoptimal conditions. We show that the expression of class I HSGs plays a major role for survival and growth of C. botulinum under the stressful environmental conditions that may be encountered during food processing or growth in food products, in the mammalian intestine, or in wounds.

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

“…The ClosTron system has proven effective for genetic studies in C. botulinum (1,4,30,31). The sigK locus (CBO2541) was disrupted by inserting a mobile group II intron from Lactococcus lactis (Ll.ltrB) in sigK using the ClosTron system developed by Heap et al (10).…”

Alternative Sigma Factor SigK Has a Role in Stress Tolerance of Group I Clostridium botulinum Strain ATCC 3502

“…Insertional inactivation mutants of sigK with the ClosTron insertion (22,23) in a sense (sigK-427s) or antisense (sigK-296as) orientation (12) were evaluated for their growth characteristics at 17 and 37°C under hyperosmotic conditions (4.5% [wt/vol] NaCl) and at pH 6.0, 5.3, and 5.0 (17)(18)(19)24). The growth rate and maximum optical density of the wild-type strain at 17°C were significantly higher than those of the sigK-427s and sigK-298as mutants (Fig.…”

“…The C. botulinum ATCC 3502 wild-type strain was evaluated for relative sigK expression levels after cold shock, exposure to hyperosmotic conditions, exposure to acidity, or under optimal growth conditions, using quantitative reverse transcription-PCR (primers sigK-qPCR-F [5=-ACTTATGGGATGTACTAGGAAGT G-3=] and sigK-qPCR-R [5=-TTCTTCTTCATCACTTAGAGGCT TG-3=]) (17)(18)(19) and the Pfaffl method (20) for quantitation, with 16S rrn expression as a normalization reference (primers 16Srrn-qPCR-F [5=-AGCGGTGAAATGCGTAGAGA-3=] and 16Srrn-qPCR-R [5=-GGCACAGGGGGAGTTGATAC-3=]). In temperature downshift, cultures grown to the early logarithmic phase at 37°C were rapidly cooled to 15°C and thereafter anaerobically incubated at 15°C for 5 h. The actual temperature of the cultures varied between 14 and 18°C.…”

Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures

“…To study the involvement of the TCS CBO0366/CBO0365 in the cold shock response, the relative mRNA levels of cbo0365 and cbo0366 in ATCC 3502 cultures (Table 1) were measured via quantitative reverse transcription-PCR(qRT-PCR) (31,35) immediately before (T0) and 1 min, 30 min, 2 h, and 5 h after a Derived from pMTL007 by retargeting to cbo0365 This study pMTL007-cbo0366-267s…”