A CONJUGATION PROCEDURE FOR BDELLOVIBRIO-BACTERIOVORUS AND ITS USE TO IDENTIFY DNA-SEQUENCES THAT ENHANCE THE PLAQUE-FORMING ABILITY OF A SPONTANEOUS HOST-INDEPENDENT MUTANT
A CONJUGATION PROCEDURE FOR BDELLOVIBRIO-BACTERIOVORUS AND ITS USE TO IDENTIFY DNA-SEQUENCES THAT ENHANCE THE PLAQUE-FORMING ABILITY OF A SPONTANEOUS HOST-INDEPENDENT MUTANT
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DOI：
10.1128/jb.174.19.6011-6017.1992
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发表时间：
1992-10-01
影响因子：
3.2
通讯作者：
THOMASHOW, MF
中科院分区：
文献类型：
--
作者：
COTTER, TW;THOMASHOW, MF
野生型蛭弧菌是其他革兰氏阴性细菌的专性周质内寄生虫。然而，可以在没有宿主细胞的情况下培养的自发突变体以10（-6）至10（-7）的频率发生。这种宿主非依赖性（H-I）突变体通常显示降低的周质内生长能力，并且形成比野生型菌株在宿主细胞菌苔上形成的斑块更小且更浑浊的斑块。对负责H-I表型的基因的分析应该提供对蛭弧菌宿主依赖性的本质的重要洞察。为此，开发了将IncQ和IncP载体从大肠杆菌转移到噬菌蛭弧菌的接合程序。发现IncQ型质粒能够在B中自主复制。而IncP衍生物则没有。然而，IncP质粒可以在B中保持。通过克隆的B经同源重组获得噬菌菌。噬菌DNA序列。同时发现野生型B.构建在IncP粘粒pVK 100中的噬菌109 J DNA在E.在IncQ粘粒pBM 33中构建的质粒不稳定。最后，我们使用共轭程序和B。噬菌菌体文库以鉴定野生型B的5.6-kb BamHI片段。噬菌DNA，显着提高噬菌斑形成能力的H-I突变体，B。bacteriovorus BB5.
Wild-type bdellovibrios are obligate intraperiplasmic parasites of other gram-negative bacteria. However, spontaneous mutants that can be cultured in the absence of host cells occur at a frequency of 10(-6) to 10(-7). Such host-independent (H-I) mutants generally display diminished intraperiplasmic-growth capabilities and form plaques that are smaller and more turbid than those formed by wild-type strains on lawns of host cells. An analysis of the gene(s) responsible for the H-I phenotype should provide significant insight into the nature of Bdellovibrio host dependence. Toward this end, a conjugation procedure to transfer both IncQ and IncP vectors from Escherichia coli to Bdellovibrio bacteriovorus was developed. It was found that IncQ-type plasmids were capable of autonomous replication in B. bacteriovorus, while IncP derivatives were not. However, IncP plasmids could be maintained in B. bacteriovorus via homologous recombination through cloned B. bacteriovorus DNA sequences. It was also found that genomic libraries of wild-type B. bacteriovorus 109J DNA constructed in the IncP cosmid pVK100 were stably maintained in E. coli; those constructed in the IncQ cosmid pBM33 were unstable. Finally, we used the conjugation procedure and the B. bacteriovorus libraries to identify a 5.6-kb BamHI fragment of wild-type B. bacteriovorus DNA that significantly enhanced the plaque-forming ability of an H-I mutant, B. bacteriovorus BB5.