Growth temperature variation and heat stress response of Clostridium botulinum

Clostridium botulinum, the causative agent of botulism in humans and animals, is frequently exposed to stressful environments during its growth in food or colonization of a host body. The wide genetic diversity of the strains of this foodborne pathogen has been thoroughly studied using different molecular biological methods; however, it is still largely unknown how this diversity reflects in the ability of different C. botulinum strains to tolerate environmental stresses. In contrast to cold tolerance, which has been the focus of intensive research in recent years, the molecular mechanisms C. botulinum utilizes in response to heat shock and during adaptation to high temperature stress are poorly understood. The aims of this study were to investigate the strain variation of Group I and II C. botulinum with regard to growth at low, high, and optimal temperature; the roles of hrcA, the negative regulator of Class I heat shock genes (HSG) and dnaK, a molecular chaperone coding Class I HSG, in the response of the Group I C. botulinum strain ATCC 3502 to heat and other environmental stresses; and the molecular mechanisms this strain employs in response to acute and prolonged heat stress. The maximum and minimum growth temperatures of 23 Group I and 24 Group II C. botulinum strains were studied. Further, maximum growth rates of the Group I strains at 20, 37, and 42°C and of the Group II strains at 10, 30, 37, and 42°C were determined. Within their groups, the C. botulinum strains showed significant variation in growth-limiting temperatures and their capability to grow at extreme temperature, especially at high temperature. Largest strain variation was found for Group I within type B and for Group II within type E strains, which further showed more mesophilic growth tendencies than the other Group II strains. However, the genetic background of the selected C. botulinum strains reflected only weakly in their growth characteristics. Group I strains showed larger physiological variation despite being genetically more closely related than Group II. A number of strains of both groups showed faster growth at temperatures above than at their commonly assumed optimal growth temperatures of 30°C for Group II and 37°C for Group I strains. In addition, they possessed higher maximum growth temperatures than the average of the studied strains. These strains can be expected to have higher than assumed optimal growth temperatures and pronounced high temperature stress tolerance. Good correlation was detected between maximum growth temperatures and growth rates at high temperature, although not for all strains. Therefore direct prediction from one studied growth trait to the other was impossible. These findings need to be taken into account when estimating the safety of food products with regard to C. botulinum by risk assessment and challenge studies. The role of Class I HSGs in C. botulinum Group I strain ATCC 3502 was studied by quantitative real-time reverse transcription PCR and insertional inactivation of the Class I HSGs hrcA and dnaK. During exponential and transitional growth, Class I HSGs were constantly expressed followed by down-regulation in the stationary phase. Exposure of mid-exponentially growing culture to heat shock led to strong, transient Class I HSG up-regulation. Inactivation of hrcA resulted in over-expression of all Class I HSGs, which confirmed its role as negative regulator of Class I HSGs in C. botulinum. Both inactivation mutants showed impaired high temperature tolerance as indicated by reduced growth rates at 45°C, a reduced maximum growth temperature, and increased log-reduction after exposure to lethal temperature. The growth of the dnaK mutant was more strongly affected than that of the hrcA mutant, emphasizing the importance of the molecular chaperone DnaK for C. botulinum. Reduced growth rates were evident for both mutants under optimal conditions and heat stress, but also under low pH, and high saline concentration. This suggests a probable role for Class I HSG in cross protection of C. botulinum against other environmental stresses. C. botulinum ATCC 3502 was grown in continuous culture and exposed to heat shock followed by prolonged high temperature stress at 45°C. Changes in the global gene expression pattern induced by heat stress were investigated using DNA microarray hybridization. Class I and III HSGs, as well as members of the SOS regulon, were employed in response to acute heat stress. High temperature led to suppression of the botulinum neurotoxin coding botA and the associated non-toxic protein-coding genes. During adaptation and in the heat-adapted culture, motility- and chemotaxis-related genes were found to be up-regulated, whereas sporulation related genes were suppressed. Thus, increase in motility appeared to be the long-term high-temperature stress-response mechanism preferred to sporulation. Prophage genes, including regulatory genes, were activated by high temperature and might therefore contribute to the high temperature tolerance of C. botulinum strain ATCC 3502. Further, remodeling of parts of the protein metabolism and changes in carbohydrate metabolism were observed.