Abstract: Strain YL-1 was isolated from soybean root tips and identified as Pseudomonas chlororaphis. This strain showed broad-spectrum antibacterial and antifungal activities against plant phytopathogens that are economically important in agriculture. In order to explore the function of its biocontrol-associated genes, an efficient genetic manipulation system was established in this study. The Bacillus subtilis sacB gene, whose product encodes levansucrase that is toxic to Gram-negative bacteria in the presence of sucrose, was considered as a counter- selection marker in the system. We selected a well-characterized pvdS as a representative example to generate an in-frame deletion mutant, because the product of pvdS encodes a sigma factor that is known to control pyoverdine biosynthesis at transcription level. A sacB-containing suicide-vector, pEX18 was used, in which both the upstream and downstream homolog fragment of pvdS was cloned, creating a recombinant plasmid, pEX18-pvdS. The recombinant plasmid was transformed into wild type strain YL-1 via an optimized bacterial conjugal approach. Via a double-crossover homologous recombinant approach, an in-frame deletion mutant of pvdS, named as ΔpvdS was generated and validated. Compared to the wild type, this mutant exhibited equal swimming motility and growth capacity, but a significant decrease in swarming motility and pyoverdine production.The function of pyoverdine production in the ΔpvdS could be rescued by introducing a plasmid-borne pvdS into this mutant. Together, our studies established an effective system for both in-frame gene deletion and complementation in YL-1, which facilitates to uncover the biocontrol mechanisms of YL-1 in the future.

Key words: Pseudomonas chlororaphis, sacB, pvdS, pyoverdine