Bradshaw Marite, Dineen Sean S, Maks Nicole D, Johnson Eric A
Department of Food Microbiology and Toxicology and Bacteriology, Food Research Institute, University of Wisconsin, 1925 Willow Drive, Madison, WI 53706, USA.
Anaerobe. 2004 Dec;10(6):321-33. doi: 10.1016/j.anaerobe.2004.07.001.
The kinetics of botulinum toxin gene expression have been investigated in Clostridium botulinum type A strains 62A, Hall A-hyper, and type A(B) strain NCTC 2916 during the growth cycle. The analyses were performed in TPGY and type A Toxin Production Media (TPM). The mRNA transcript levels encoding the proteins of the neurotoxin complex were determined using Northern analyses. Neurotoxin concentrations in culture supernatants and lysed cell pellets were assayed using ELISA, Western blots, and mouse bioassay. Proteolytic activation of botulinum neurotoxin during the growth cycle was evaluated by Western blots. For all three strains, mRNA transcripts for the toxin complex genes were initially detected in early log phase, reached peak levels in early stationary phase, and rapidly decreased in mid-to-late stationary phase and during lysis. Toxin expression varied depending on the strain and growth medium. Toxin production was highest in strain Hall A-hyper, followed by NCTC 2916 and 62A. For C. botulinum strain Hall A-hyper, cell lysis and toxin release into the supernatant occurred rapidly for cells grown in TPM, while cells grown in TPGY remained in stationary phase with minimal lysis and toxin release through 96 h of growth. In contrast, strains 62A and NCTC 2916 lysed more extensively than Hall A-hyper in TPGY. TPM supported higher toxin production and activation than TPGY in strains 62A and Hall A-hyper. These data support that the genes of the botulinum neurotoxin complex are temporally expressed during late-log and early stationary phase and that toxin complex formation depends on the strain and growth medium. Botulinum toxin synthesis and activation appears to be a complex process that is highly regulated by nutritional and environmental conditions. Further research is needed to elucidate the sensing mechanisms and genetic regulatory factors controlling these processes.
研究了A型肉毒杆菌菌株62A、Hall A-hyper以及A(B)型菌株NCTC 2916在生长周期中肉毒杆菌毒素基因表达的动力学。分析在TPGY和A型毒素生产培养基（TPM）中进行。使用Northern分析确定编码神经毒素复合物蛋白的mRNA转录水平。使用ELISA、Western印迹和小鼠生物测定法检测培养上清液和裂解细胞沉淀中的神经毒素浓度。通过Western印迹评估生长周期中肉毒杆菌神经毒素的蛋白水解激活情况。对于所有这三种菌株，毒素复合物基因的mRNA转录本最初在对数早期被检测到，在稳定早期达到峰值水平，并在稳定中后期和裂解期间迅速下降。毒素表达因菌株和生长培养基而异。毒素产量在Hall A-hyper菌株中最高，其次是NCTC 2916和62A。对于肉毒杆菌菌株Hall A-hyper，在TPM中生长的细胞迅速发生细胞裂解并将毒素释放到上清液中，而在TPGY中生长的细胞在96小时的生长过程中保持在稳定期，裂解和毒素释放最少。相比之下，在TPGY中，62A和NCTC 2916菌株的裂解比Hall A-hyper更广泛。在62A和Hall A-hyper菌株中，TPM比TPGY支持更高的毒素产生和激活。这些数据支持肉毒杆菌神经毒素复合物的基因在对数后期和稳定早期进行时序表达，并且毒素复合物的形成取决于菌株和生长培养基。肉毒杆菌毒素的合成和激活似乎是一个受营养和环境条件高度调控的复杂过程。需要进一步研究以阐明控制这些过程的传感机制和遗传调控因子。
