Clostridium botulinum produces highly potent neurotoxin, which can cause a potentially life- threatening neuroparalytic disease, botulism. Foodborne botulism is caused by the ingestion of preformed toxin in food. Harsh thermal treatments are used in canned food production to destroy the heat-resistant C. botulinum spores, but in fresh food products, the growth and toxin production are mainly prevented by mild treatments and adequate cold storage. The ability of C. botulinum to tolerate various stresses and grow at broad temperature ranges is a challenge for food industry, and increased understanding is needed to continuously improve the control of this potentially lethal pathogen. The aim of this study was to investigate the genetic mechanisms behind the stress tolerance of C. botulinum. We observed the occurrence of cold shock protein encoding genes (csps) in different C. botulinum strains and investigated the role of csp genes and DEAD-box RNA helicase csdA in the stress response of C. Botulinum ATCC 3502. Cold shock proteins and DEAD-box RNA helicases help bacteria to adapt to low temperature and other stress conditions by destabilizing the nucleotide secondary structures and enabling continuous transcription and translation. Group I C. botulinum ATCC 3502, the first sequenced C. botulinum strain, contains three csp genes: cspA, cspB, and cspC, as well as one DEAD-box RNA helicase, cbo2802 (csdA). To gain understanding on the role of these genes in C. botulinum, we first investigated the occurrence of the csp genes in sixteen C. botulinum genomes. Genome database searches and sequence comparisons revealed that all the studied Group I strains contained homologues for the cspA and cspB genes, and the homolog for cspC was present in all but four Group I strains. Only one of the Group II C. botulinum strains contained a csp homologue, and no homologues for csp genes were found in any of the three studied type E neurotoxin-producing C. botulinum genomes. Considering their highly conserved nature and abundance of these genes in organisms from all taxa, the occurrence of csp homologues in Group I genomes was expected. However, it was surprising to see that the psychrotrophic Group II strains did not contain any csp homologues. The finding indicates that some other, yet undefined, mechanisms are behind the cold tolerance of these strains. The activity of the csp and cbo2802 genes was studied in C. Botulinum ATCC 3502 by quantitative real-time reverse transcription PCR. The relative expression of cspB was increased 30 minutes after a cold shock treatment, where the bacterial culture was exposed to a sudden temperature drop from 37 °C to 15 °C, and the upregulation continued for five hours after the cold shock. The expression of cspC was increased at the time of cold shock and 30 minutes later, but decreased at later time points, 2 h and 5 h after the treatment. The cspA was upregulated at the 30-minute and 2-hour timepoints. The increase in relative expression was significant but modest (1.5 to 2-fold) for all csp genes. The relative expression of the cbo2802 gene, however, was strongly increased (up to 7.6-fold) at 30 mins, 2 h, and 5 h after cold sock. These observations indicated that the csp genes and cbo2802 play a role in the cold tolerance of the strain. The role of the csp genes in C. botulinum ATCC 3502 was further studied by the insertional inactivation of the studied genes and assessing the growth of the mutant strains at low temperature and under NaCl, pH and ethanol stresses. The inactivation of the cspB caused growth arrest of the mutant strain at 15 °C, and its growth rate was 70% lower than that of the wild-type strain at 20 °C. At the optimal growth temperature 37 °C, the growth of the ΔcspB mutant strain did not differ from the wild-type strain. The growth of the ΔcspB mutant strain was also affected by other tested stress conditions; increased NaCl and modified pH lowered the growth rate of the mutant, and added ethanol increased the lag phase of the ΔcspB mutant compared to the wild-type strain. Based on these findings, we proposed that CspB could have a role as a universal stress protein in C. botulinum ATCC 3502. The inactivation of cspC led to a lower growth rate at 15 °C and 20 °C, but also at 37 °C. Addition of ethanol and changes in pH decreased the growth rate of the ΔcspC mutant strain, but the growth rate of the mutant was higher than that of the wild-type strain under NaCl stress. The lag phases were longer for the mutant strain in all NaCl concentrations tested and most of the pH values. These observations indicate that the role of cspC could be related to other physiological events in addition to the cold response. In contrast to the other genes, the inactivation of cspA increased the growth rate of the mutant strain compared to the wild-type strain at all studied temperatures, 15 °C, 20 °C, and 37 °C. The growth rate of the ΔcspA mutant was higher than that of the wild-type strain also at all higher NaCl concentrations and all ethanol concentrations tested. The lag phase of the ΔcspA mutant was shorter compared to the wild-type strain at all pH conditions. cspA thus seems to have a role as a growth repressor. The role of cbo2802 was studied by insertional inactivation and observing the growth of the mutant strains at low temperature. At 20 °C, the growth rate of the cbo2802 mutants was lower compared to the wild-type strain and the initiation of the exponential growth was delayed. The mean minimum growth temperatures of the cbo2802 mutant strains were significantly higher compared to the wild-type strains. Inactivation of cbo2802 also decreased the motility of the strain at 20 °C. These observations indicate that cbo2802 has an important, yet unspecified, role in the cold tolerance of C. botulinum ATCC 3502. Kylmänsietogeenit vaikuttavat Clostridium botulinum -ruokamyrkytysbakteerin kykyyn selviytyä stressiolosuhteista. Terveellinen ruoka on paitsi ravitsemukselliselta koostumukseltaan hyvinvointia tukevaa, myös puhdasta ja turvallista, vailla terveydelle haitallisia tekijöitä. Haitallisten taudinaiheuttajien ja ruokaa pilaavien bakteerien esiintymistä elintarvikkeissa pyritään kontrolloimaan mm. kuumennuskäsittelyin, säilöntäainein, ruoan happamuutta säätelemällä sekä kylmäsäilytyksellä. Elintarvikkeissa esiintyvät bakteerit reagoivat elintarvikkeiden käsittelyistä johtuviin ympäristön muutoksiin stressivasteella, jonka avulla ne pyrkivät sopeutumaan epäedullisiin kasvuolosuhteisiin. Stressivasteessa tietyt stressinsietogeenit aktivoituvat, ja bakteeri alkaa tuottaa proteiineja, jotka auttavat sitä jatkamaan kasvua muuttuneissa elinolosuhteissa, kuten kylmässä. Tässä tutkimuksessa selvitettiin stressivasteen esiintymistä Clostridium botulinum –bakteerilla, joka aiheuttaa verrattain harvinaisen mutta seurauksiltaan hyvin vakavan ruokamyrkytyksen, botulismin. Työssä kartoitettiin geenejä, jotka kyseisellä bakteerilla aktivoituvat ympäristön olosuhteiden muuttuessa. Lisäksi tutkittiin näiden geenien merkitystä C. botulinumin selviytymiselle stressiolosuhteissa, kuten kylmässä, happamassa tai emäksisessä ympäristössä sekä korkeassa suolapitoisuudessa. Useimmilla tutkituilla C. botulinum -kannoilla osoittautui olevan yksi tai useampi erityisesti kylmänkestävyyteen liittyvä stressinsietogeeni. Yllättäen kyseisiä geenejä ei kuitenkaan havaittu erityisen hyvin kylmää sietävien C. botulinum -kantojen perimässä, eli näillä kylmänsieto perustunee johonkin muuhun, toistaiseksi tuntemattomaan stressinsietomekanismiin. Kylmänsietogeenien toiminnan estäminen pääosin heikensi tutkitun C. botulinum -kannan kykyä selviytyä muuttuvassa ympäristössä, osoittaen, että kyseisten geenien toiminta on tärkeä osa bakteerin stressivastetta. Bakteerien stressinsietomekanismien tutkimus on perustutkimusta. Bakteerien stressinsiedon ymmärtäminen auttaa elintarvikeketjun riskien arvioinnissa ja riskinhallinnassa. Kun mekanismit tunnetaan riittävän hyvin, voidaan kehittää uusia innovatiivisia riskinarviointi- ja hallintakeinoja.