Identification and function research of the fatty acyl-CoA synthetase in Ralstonia solanacearum

Abstract:

[Objective] Ralstonia solanacearum, a devastating, soil-borne plant pathogen, causes a bacterial wilt disease in diverse plants. Studies on the metabolic mechanisms of fatty acids will facilitate the discovery of novel methods or biopesticides to efficiently control the bacterial wilt disease. [Methods] RSc2857 (RsfadD) gene was found in the genome of Ralstonia solanacearum GMI1000 through sequence alignment with Escherichia coli FadD, which was annotated to encode a fatty acyl-CoA synthetase (FACS). For complementation analysis, RsfadD gene was amplified by PCR, and was ligated into an expression vector pBAD24M, which was subsequently transferred into an E. coli fadD mutant JW1794. The growth of transformant was analyzed. RsfadD was also fused in-frame to pET-28b, and expressed in E. coli BL21(DE3). The hexahistidine-tagged RsFadD was purified by Ni-NTA, and the activity was analyzed in vitro. RsfadD deletion mutant was obtained by homologous recombination, and the mutant growth was also analyzed. [Results] RsfadD conferred the E. coli fadD mutant to grow on the minimal medium with fatty acids as the sole carbon source. In vitro enzymatic analysis demonstrated that RsFadD has FACS activity, and could utilize fatty acids of different chain lengths as substrates to form fatty acyl-CoAs. While the activity of RsFadD was lower than that of E. coli FadD. RsfadD deletion mutant grew well on nutritional medium, but grew weak on the minimal medium with fatty acids as the sole carbon source. [Conclusion] All of above suggested that RsfadD encodes a FACS, which plays an important role in fatty acids utilization. The weak growth of RsfadD deletion mutant on the minimal medium indicated other genes encoding FACS may exist in the genome. This study will contribute to further research about FACS and fatty acids utilizing mechanism in R. solanacearum.