Alvarez B, Secades P, McBride M J, Guijarro J A
Area de Microbiologia, Departamento de Biología Funcional, IUBA, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain.
Appl Environ Microbiol. 2004 Jan;70(1):581-7. doi: 10.1128/AEM.70.1.581-587.2004.
Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.
嗜冷黄杆菌是噬纤维菌-黄杆菌-拟杆菌群的成员，是鲑科鱼类的一种重要病原体。此前针对这种苛求的嗜冷细菌开发遗传技术的尝试均以失败告终。在此，我们描述了嗜冷黄杆菌遗传操作技术的开发，以及在该鱼类病原体的多个分离株中发挥作用的质粒、选择标记、报告系统和转座子的鉴定。抗生素抗性基因ermF、cfxA和tetQ在嗜冷黄杆菌中发挥作用。基于携带这些选择标记的嗜冷黄杆菌隐蔽质粒pCP1构建的克隆载体通过从大肠杆菌进行接合转移引入，从而产生了嗜冷黄杆菌的抗生素抗性菌落。DNA向嗜冷黄杆菌的接合转移具有菌株依赖性。在所测试的7个菌株中的2个（THC02-90和THC04-90）中观察到了高效转移。当从pCP1启动子表达时，大肠杆菌lacZY在嗜冷黄杆菌中发挥作用，使其能够作为基因表达研究的报告基因得以开发。通过电穿孔将从嗜冷黄杆菌中分离的质粒高效引入嗜冷黄杆菌，但从大肠杆菌中分离的质粒不适合通过这种途径转移，这表明存在限制屏障。从嗜冷黄杆菌中分离的DNA对Sau3AI和BamHI消化具有抗性，这表明类似Sau3AI的限制修饰系统可能构成了这一屏障的一部分。Tn4351从大肠杆菌引入嗜冷黄杆菌并以明显的随机性进行转座，从而产生了红霉素抗性菌落。本研究中开发的技术使得对这种重要的鱼类病原体进行遗传操作和分析成为可能。
