Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheS m as selection markers.

Autor:	Jiang J; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China., Zhao Y; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China., Chen A; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China., Sun J; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China., Zhou M; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China., Hu J; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China., Cao X; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China., Dai N; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China., Liang Z; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.; Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, Guangzhou, China.; Key Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs, Guangzhou, China.; Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture and Rural Affairs, Guangzhou, China.; National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangzhou, China., Feng S; College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.; Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, Guangzhou, China.; Key Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs, Guangzhou, China.; Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture and Rural Affairs, Guangzhou, China.; National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangzhou, China.
Jazyk:	angličtina
Zdroj:	Applied and environmental microbiology [Appl Environ Microbiol] 2024 Apr 17; Vol. 90 (4), pp. e0204323. Date of Electronic Publication: 2024 Mar 28.
DOI:	10.1128/aem.02043-23
Abstrakt:	Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida . Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB , opa , and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheS m screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p -chloro-phenylalanine, demonstrating the feasibility of pheS m as a counter-selective marker. The pheS m system was used for markerless deletions of glgB , opa , and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheS m counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida .IMPORTANCE Pasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheS m screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheS m as counterselection markers to improve counterselection efficiency in P. multocida . This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida . Competing Interests: The authors declare no conflict of interest.
Databáze:	MEDLINE
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