苏云金芽孢杆菌两株溶原性噬菌体的生物学特性

Biology of two lysogenic phages from Bacillus thuringiensis MZ1

摘要:

研究苏云金芽孢杆菌（Bacillus thuringiensis）的溶原性及其噬菌体的生物学特性，从生产菌株MZ1中分离了两株溶原性噬菌体。MZ1经诱导后产生直径约为3mm和1mm的噬斑，分离获得属长尾噬菌体科的噬菌体MZTP01和MZTP02两株；分别对6株和7株不同亚种的Bt菌株具有侵染力；免疫血清与相应噬菌体的中和反应K值分别为45和326，且两者无相关抗原性。MZTP01抵抗酸、碱、紫外线和热的能力比MZTP02强，但抵抗有机溶剂的程度比MZTP02弱。MZTP01的潜伏期为80min，裂解量为55；MZTP02的潜伏期为40min，裂解量为175。核酸结构分析均表明为线性dsDNA分子。两基因组DNA的凝胶电泳表明分子量均在9.4～23kb之间，并被HindⅢ酶切分别产生8条和9条清晰条带。该菌株被证明为二元溶原菌，可能是造成生产损失的主要原因；为防治溶原性噬菌体提供了生物学信息。

Abstract:

A Bacillus thuringiensis (Bt) fermentative strain MZ1 (subsp.kurstaki), from a company in Meixian County of Guangdong Province, produce toxins during sporulation and are extensively used in the field to control pest insects (Lepidoptera) in China. But some unknown or random factors that inhibited or stopped B.t growth in the fermentation can be regarded as reflecting the exist of lysogenic phage. Therefore, strain MZ1 and its lysogenic phages were studied in this paper. With indicator strain ZK1, two kind of phage plaques, one with about 3mm diameter and the other with about 1mm diameter, can be observed after strain MZ1 cultured in plates or flasks was induced by mitomycin C. Then, two lysogenic phages, namely MZTP01 and MZTP02, were isolated and characterized in biology. They belonged to family Siphoviridae, which had icosahedral heads (MZTP01:82nm×85nm; MZTP02: 75nm×55nm) and long tails (MZTP01: 220nm×18nm; MZTP02:183nm×12nm) without flexibility. Host range examination showed that six and seven (including indicator strain ZK1) out of 113 B.t strains saved in our laboratory were sensitive to MZTP01 and MZTP02, respectively. MZTP01 was more stable than MZTP02 against pH value, ultraviolet and heat treatment, but contrary against organic solvents. For MZTP01 and MZTP02, K values in the neutralization reactions were 45 and 326, respectively. Both phages had no relationship in their antigenicity. Burst size of phage MZTP02 was 175, two times more than that of MZTP01. Latent time of MZTP02 was 40min, one times shorter than that of MZTP01. It was suggested that both phages DNA be linear dsDNA through the typical absorption curves, reaction with diphenylamine, DNase sensitivity and acridine orange staining. And this was in good accord with the previous findings that all tailed phages being dsDNA moleculars. The genomic DNA and their restriction maps showed that both molecular weights should be between 9.4～23kb. Both phage genomic DNAs were digested by HindⅢ, producing eight and nine bands, respectively. The results above show that strain MZ1 is proved a double lysogen. It is considered to be the main reason of great losses in the production. The biological information of the two lysogenic phages belonging to strain MZ1 is provided to help solve the problem and the two lysogenic phages is prepared for complete DNA sequencing in the next step.