BACKGROUND The pOV plasmid isolated from the Pasteurella multocida strain PMOV is a new plasmid, and its molecular characterization is important for determining its gene content and its replicative properties in Pasteurellaceae family bacteria. METHODS Antimicrobial resistance mediated by the pOV plasmid was tested in bacteria. Purified pOV plasmid DNA was used to transform E. coli DH5α and Gallibacterium anatis 12656-12, including the pBluescript II KS(-) plasmid DNA as a control for genetic transformation. The pOV plasmid was digested with EcoRI for cloning fragments into the pBluescript II KS(-) vector to obtain constructs and to determine the full DNA sequence of pOV. RESULTS The pOV plasmid is 13.5 kb in size; confers sulfonamide, streptomycin and ampicillin resistance to P. multocida PMOV; and can transform E. coli DH5α and G. anatis 12656-12. The pOV plasmid was digested for the preparation of chimeric constructs and used to transform E. coli DH5α, conferring resistance to streptomycin (plasmid pSEP3), ampicillin (pSEP4) and sulfonamide (pSEP5) on the bacteria; however, similar to pBluescript II KS(-), the chimeric plasmids did not transform G. anatis 12656-12. A 1.4 kb fragment of the streptomycin cassette from pSEP3 was amplified by PCR and used to construct pSEP7, which in turn was used to interrupt a chromosomal DNA locus of G. anatis by double homologous recombination, introducing strA-strB into the G. anatis chromosome. CONCLUSION The pOV plasmid is a wide-range, low-copy-number plasmid that is able to replicate in some gamma-proteobacteria. Part of this plasmid was integrated into the G. anatis 12656-12 chromosome. This construct may prove to be a useful tool for genetic studies of G. anatis.

中文翻译：

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多杀性巴斯德氏菌pOV质粒的分子和遗传表征以及Analitic Gallibacterium整合载体的构建。

背景技术从多杀巴斯德氏菌PMOV株中分离出的pOV质粒是一种新质粒，其分子特征对于确定其基因含量及其在巴斯德氏菌科细菌中的复制特性很重要。方法在细菌中检测pOV质粒介导的抗药性。纯化的pOV质粒DNA用于转化大肠杆菌DH5α和anatis 12656-12，包括pBluescript II KS（-）质粒DNA作为基因转化的对照。用EcoRI消化pOV质粒，以将片段克隆到pBluescript II KS（-）载体中，以获得构建体并确定pOV的完整DNA序列。结果pOV质粒大小为13.5 kb。赋予磺胺类，链霉素和氨苄青霉素对多杀性巴氏杆菌PMOV的抗性；并可以转化大肠杆菌DH5α和G。anatis 12656-12。消化pOV质粒以制备嵌合构建体，并用于转化大肠杆菌DH5α，赋予细菌对链霉素（质粒pSEP3），氨苄青霉素（pSEP4）和磺酰胺（pSEP5）的抗性；然而，类似于pBluescript II KS（-），嵌合质粒不能转化Anatiss G. anatis 12656-12。通过PCR扩增来自pSEP3的链霉素盒的1.4kb片段，并用于构建pSEP7，其随后被用于通过双重同源重组来中断Anatis的染色体DNA基因座，将strA-strB引入Anatis染色体中。 。结论pOV质粒是一种广谱，低拷贝数的质粒，能够在某些γ-变形杆菌中复制。该质粒的一部分被整合到Anatiss 12656-12染色体中。