cspB encodes a major cold shock protein in Clostridium botulinum ATCC 3502

Söderholm, Henna; Lindström, Miia; Somervuo, Panu; Heap, John; Minton, Nigel P.; Lindén, Jere; Korkeala, Hannu

doi:10.1016/j.ijfoodmicro.2011.01.033

Cited by 27 publications

(24 citation statements)

References 39 publications

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“…The significantly reduced growth rate of both sigK mutants at 17°C, compared to the wildtype strain, supports an important role for SigK in adaptation to low temperature. A previous study has shown that ClosTron manipulation (22,23) does not affect the overall fitness of ATCC 3502 at optimal and cold temperatures (18). The similar behavior of the two sigK-427s and sigK-298as mutant strains, with intron insertions at different sites and orientations in sigK, further supports disruption of sigK as the sole cause behind the cold-sensitive phenotype.…”

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confidence: 54%

“…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.…”

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confidence: 99%

“…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.…”

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confidence: 99%

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

Dahlsten

Kirk

Lindström

et al. 2013

Appl Environ Microbiol

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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...

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confidence: 54%

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confidence: 99%

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confidence: 99%

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

Dahlsten

Kirk

Lindström

et al. 2013

Appl Environ Microbiol

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“…By way of example, it has been used to establish the role of bacterial factors (toxins, flagella and adhesins) in infection, 23,[26][27][28][29] to garner a greater understanding of the role and activities of enzymes in primary metabolism, [30][31][32][33] to begin to elucidate mechanistic details of the developmental process of spore formation/ germination 21,34,35 and provide fundamental information on regulatory processes important in virulence factor and metabolite production. [36][37][38][39] Its availability has provided the research community with a sequence of the re-targeted region used to generate the insertion should be given. So, for example, when creating a toxin A mutant in C. difficile strain R20291, the implementation of the Perutka algorithm 13 at www.clostron.com suggests a possible insertion site in the sense orientation after nucleotide 1584.…”

Section: Discussionmentioning

confidence: 99%

ClosTron-Mediated Engineering of Clostridium

Kuehne

Heap

Cooksley

et al. 2011

Methods in Molecular Biology

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“…While group I C. botulinum strains carry Csp genes in their genome (20), none were found in the genomes of C. botulinum type E strains (21), which raises the question of alternative strategies for coping with cold by C. botulinum type E. It is known that twocomponent signal transduction systems (TCSs) play a role in the cold tolerance and cold shock response of various bacteria (22)(23)(24)(25)(26), and recent studies have shown the importance of two TCSs in the cold adaption of the mesophilic group I C. botulinum (27,28). Typical TCSs consist of a sensory histidine kinase and a response regulator.…”

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confidence: 99%

The CLO3403/CLO3404 Two-Component System of Clostridium botulinum E1 Beluga Is Important for Cold Shock Response and Growth at Low Temperatures

Mascher

Derman

Kirk

et al. 2014

Appl Environ Microbiol

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In order to survive a temperature downshift, bacteria have to sense the changing environment and adjust their metabolism and structure. Two-component signal transduction systems (TCSs) play a central role in sensing and responding to many different environmental stimuli. Although the nonproteolytic (group II) Clostridium botulinum represents a major hazard in chilled foods, the cold adaption mechanisms of group II C. botulinum organisms are not known. Here, we show that the CLO3403/ CLO3404 TCS of C. botulinum E1 Beluga is involved in the cold shock response and growth at 12°C. Cold shock induced the expression of the genes encoding the histidine kinase (clo3403) and the response regulator (clo3404) by more than 100-fold after 5 h relative to their expression in a nonshocked culture at the corresponding time point. The involvement of CLO3403/CLO3404 in growth at low temperature was demonstrated by impaired growth of the insertional clo3403 and clo3404 knockout mutants at 12°C compared to the growth of the wild-type culture. Additionally, the inactivation of clo3403 had a negative effect on motility. The growth efficiency at 12°C of the TCS mutants and the motility of the kinase mutants were restored by introducing a plasmid harboring the operon of the CLO3403/CLO3404 TCS. The results suggest that the CLO3403/CLO3404 TCS is important for the cold tolerance of C. botulinum E1 Beluga. G roup II (nonproteolytic)Clostridium botulinum type E is a notorious food-borne pathogen which is mainly found in aquatic environments in cold regions of the world (e.g., Northern Europe, Japan, Canada, Alaska, and Greenland) (1, 2), with a high prevalence of spores in the Baltic Sea (3). Type E botulism is usually caused through consumption of contaminated fish or seafood products such as vacuum-packed smoked fish, salted fish, and fermented marine mammals (4-11). Concerning modern food processing, C. botulinum type E is the principal food safety hazard in refrigerated, minimally processed packaged foods of aquatic origin (12). The mild heat treatments used in the production of these foods are not sufficient to eliminate all spores, and the limited use of salt and preservatives, as well as vacuum packaging, may support growth and toxin production by C. botulinum. Most importantly, thermal control is often not sufficient to prevent food poisoning, as some strains of group II C. botulinum are able to grow and produce toxin at temperatures as low as 3°C (13-15).The mechanisms of cold shock response and growth at low temperature have been thoroughly studied in the model organisms Escherichia coli and Bacillus subtilis (16-18) but are still unknown in the psychrotrophic C. botulinum type E. The cold shock proteins (Csps) are a universal group of cold-induced proteins occurring in a wide range of bacteria and other organisms (17,19). While group I C. botulinum strains carry Csp genes in their genome (20), none were found in the genomes of C. botulinum type E strains (21), which raises the question of alternative strategies for coping with...

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cspB encodes a major cold shock protein in Clostridium botulinum ATCC 3502

Cited by 27 publications

References 39 publications

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

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

ClosTron-Mediated Engineering of Clostridium

The CLO3403/CLO3404 Two-Component System of Clostridium botulinum E1 Beluga Is Important for Cold Shock Response and Growth at Low Temperatures

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