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

Abstract

The role of the alternative sigma factor SigK in cold and osmotic stress tolerance of Clostridium botulinum ATCC 3502 was demonstrated by induction of sigK after temperature downshift and exposure to hyperosmotic conditions and by impaired growth of the sigK mutants under the respective conditions. T he ability of the anaerobic, Gram-positive, spore-forming, food-borne pathogen Clostridium botulinum to survive, grow, and subsequently produce botulinum neurotoxin in foods (1) raises substantial concern over food safety (2, 3). In minimally processed foods, the extrinsic and intrinsic hurdles used to control the outgrowth and toxin production by C. botulinum include refrigeration, heat treatment, increased osmolarity or low water activity, extreme pH, and preservatives (2, 4). Understanding the mechanisms by which food-borne pathogenic bacteria cope with these stress conditions is of key importance in designing modern food safety measures.Sigma factors are dissociable RNA polymerase subunits that alter the promoter specificity of the RNA polymerase complex under different environmental and growth phase-dependent conditions. A stress-responsive alternative sigma factor, SigB, has been identified (5-8) and has been shown to play a role in cold adaptation of Listeria monocytogenes (9) and Bacillus subtilis (10). However, the genome of C. botulinum ATCC 3502 does not harbor a homolog for sigB (11), suggesting mechanisms of general stress response different from those in the Gram-positive model organism B. subtilis. A gene encoding a homolog for the sporulation sigma factor SigK of bacilli is present in the genome of C. botulinum ATCC 3502 (open reading frame [ORF] CBO2541) (11) and was recently shown to be essential in early stage sporulation in C. botulinum (12) and in Clostridium perfringens (13,14) and putatively in transcriptional activation of the sporulation master switch Spo0A of C. botulinum (12). In B. subtilis, recent findings suggest an interesting interconnection between the decision to sporulate and adaptation to stress as nongrowing "vegetative dormant" cells (15,16). These observations propose a role for the stress sigma factor SigB in regulating the activity of Spo0A of B. subtilis. The lack of sigB in the C. botulinum ATCC 3502 genome suggests that the network for decision making between sporulation initiation and stress adaptation of this organism is regulated differently from the one proposed for B. subtilis (15,16). We thus sought to investigate the behavior and role of the alternative sigma factor SigK in response to stress in C. botulinum ATCC 3502.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=]) (17-19) and the Pfaffl method (20) for quantitation, with 16S rrn expression as a normalization re...

Transcriptomic Analysis of (Group I) Clostridium botulinum ATCC 3502 Cold Shock Response

“…Further characterization showed these mechanisms to have a role in cold tolerance of C. botulinum [9]–[11]. Significant induction of the cbo0364-cbo0366 operon was also observed in the present DNA microarray experiment, albeit the 5-h log 2 fold changes fell slightly outside of the defined cut-off value of 2.…”

“…A modest induction was also observed for cbo2306 , while no significant induction was observed for cbo2307 or cbo2541 . In our previous RT-qPCR studies, however, the transcription of cbo2307 or cbo2541 reached a 3.0 to 4.4-fold induction 5 h after cold shock [10], [11]. The RT-qPCR verification of the DNA microarray data suggests that differences in transcript levels are more modestly interpreted by the microarray experiment than by RT-qPCR.…”

Diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes

“…would provide critical insight into these questions. Lastly, it should be noted that σ K regulates additional cellular processes beyond sporulation in these species: in C. botulinum it controls stress responses [62], and in C. acetobutylicum it controls solventogenesis [56]. …”

Alternative Sigma Factors SigF, SigE, and SigG Are Essential for Sporulation in Clostridium botulinum ATCC 3502

“…A notable exception among the clostridia is C. difficile, which possesses a skin element, but this element has been proposed to play a different role than that of the skin element of B. subtilis (27,28). Apart from sporulation, SigK of C. botulinum also has been associated with stress response (29). In addition to SigK, SigG also appeared to possess various roles, as related mutants in the clostridia were reported to advance further in sporulation than those in B. subtilis (28,(30)(31)(32).…”