Construction Of A Temperature-sensitive Plasmid In Pasteurellaceae

Pasteurellaceae consists of Pasteurella, Actinobacillus and Haemophilus. This bacteriaceae contains a large number of animal pathogens which can infect a lot of domestic animals and even lead to deadth. These pathogens bring huge economic losses to livestock breeding industry. More and more attentions have been paid to the pathogens belonging to Pasteurellaceae worldwide.In order to study the pathogenic mechanism of pathogenic bacteria, the investigations on their gene functions are required. The usual technique to study gene function is to build isogenic mutant strain. The current methods of mutation including natural transformation, conjugation and electroporation using suicide plasmid are defective in the efficiency of transforming and double exchange of homologous recombination. So it is necessary to establish a set of effective mutation system. Because temperature sensitive plasmid can be lost under certain temperature, so it can be used to rapidly identify the mutant with double exchange of homologous recombination. In addition, it can be used for PCR directed mutation system as expression vector and the cloning vector for double exchange. With the help of recombinases, the probability of double exchange will be greatly enhanced. Therefore, temperature sensitive plasmid can greatly improve the efficiency of mutation system.In this study, the plasmid pD70KanR genetically modified from pD70 isolated from Pasteurellaceae bacteria was sequenced and analyzed. The sequences contained a kanamycin resistance gene, a streptomycin resistance gene, a sulfonamide resistance gene and a mobA gene essential in conjugation. The restriction sites in this plasmid were analyzed using DANMAN software. Some unique restriction sites which could be used in gene clone were identified. By PCR site-directed mutagenesis, two temperature sensitive plasmids in Pm and APP, pSK-GA318 and pGA318, were constructed. The replication features of the two plasmids in Pm and APP at different cultural times and temperatures were analyzed. The two plasmids could replicate normally at 30℃, but lost under the temperature higher than 41℃after passage. pGA318 and pSK-GA318 in Pm could be lost at 42℃after 3 generations; while pGA318 and pSK-GA318 in APP could be lost at 42℃after 9 generations. These results built bases for construction of efficient mutation system in Pasteurellaceae bacteria such as Pm and APP. Finally, the temperature sensitive plasmid pGA318 was applied to delete the APP ureC gene. The upstream and downstream arms of ureC were cloned into pGA318 to try to delete ureC using the principle of homologous recombination. But we failed to obtain the mutant. The possible reason could be that the kanamycin resistance gene did not express under 42℃in APP, thus inhibited the identification of mutant. Additionally, we verified that the plasmid pJN105 from Bordelella bronchiseptica and pD70kanR could co-exist in APP. This provided genetic manipulation tools for expressing two different proteins in APP at the same time.