Xanthomonas campestris FabH is required for branched-chain fatty acid and DSF-family quorum sensing signal biosynthesis

Abstract

Xanthomonas campestris pv. campestris (Xcc), a Gram-negative phytopathogenic bacterium, causes black rot disease of cruciferous vegetables. Although Xcc has a complex fatty acid profile comprised of straight-chain fatty acids and branched-chain fatty acids (BCFAs), and encodes a complete set of genes required for fatty acid synthesis, there is still little known about the mechanism of BCFA synthesis. We reported that expression of Xcc fabH restores the growth of Ralstonia solanacearum fabH mutant, and this allows the R. solanacearum fabH mutant to produce BCFAs. Using in vitro assays, we demonstrated that Xcc FabH is able to condense branched-chain acyl-CoAs with malonyl-ACP to initiate BCFA synthesis. Moreover, although the fabH gene is essential for growth of Xcc, it can be replaced with Escherichia coli fabH, and Xcc mutants failed to produce BCFAs. These results suggest that Xcc does not have an obligatory requirement for BCFAs. Furthermore, Xcc mutants lost the ability to produce cis-11-methyl-2-dodecenoic acid, a diffusible signal factor (DSF) required for quorum sensing of Xcc, which confirms that the fatty acid synthetic pathway supplies the intermediates for DSF signal biosynthesis. Our study also showed that replacing Xcc fabH with E. coli fabH affected Xcc pathogenesis in host plants.

Biological Functions of ilvC in Branched-Chain Fatty Acid Synthesis and Diffusible Signal Factor Family Production in Xanthomonas campestris

“…Although supplementing with valine and isoleucine mixtures increased the percentage of BCFAs, especially anteiso -C 15:0 , and decreased the amount of UFAs in Xcc membrane lipids, Xcc significantly increased UFAs at low temperature, even when supplemented with high concentrations of valine and isoleucine mixtures at low temperature, which indicated that BCFAs did not have a major effect on response to a decrease in ambient temperature. These results were consistent with previous studies that replacement of Xcc fabH with E. coli fabH caused Xcc to lose the ability to synthesize BCFAs, but the mutant strain grew well at low temperature ( Yu et al, 2016 ). This confirms that the adaptive mechanism to low temperature in Xcc differs from that in L. monocytogenes , which mainly stimulates anteiso -BCFA biosynthesis to increase membrane disorder in response to low temperatures ( Zhu et al, 2005a , b ).…”

“…The precursors of the DSF-family signal come from the fatty acid synthesis pathway ( Zhou et al, 2015b ; Yu et al, 2016 ). However, though ilvC deletion mutant produced fatty acid normally, the total amount of DSF-family signal production was reduced during its growth.…”

“…campestris ( Xcc ), the causal agent of crucifer black rot, is possibly the most important disease of crucifers worldwide ( He and Zhang, 2008 ; Ryan and Dow, 2011 ). Xcc is a rod-shaped, aerobic Gram-negative, non-spore-forming bacterium that produces BCFAs, which account for approximately 50% of the total cellular fatty acids ( Kaneda, 1991 ; Yu et al, 2016 ). In addition, at least three branched-chain diffusible signal factor (DSF)-family signals are known in Xcc : DSF ( cis -11-methyl-2-dodecenoic acid [11-Me-C 12 :Δ 2 ]), CDSF ( cis -11-methyldodeca-2,5-dienoic acid [11-Me-C 12 : Δ 2,5 ]), and IDSF ( cis -10-methyl-2-dodecenoic acid [10-Me-C 12 :Δ 2 ]) ( Deng et al, 2015 ; Zhou et al, 2015a , b ).…”

“…These are involved in quorum sensing (QS) mechanisms to regulate the production of a range of virulence determinants, including extracellular polysaccharides (EPS), extracellular enzymes (proteases, pectinases, and cellulases), and a type III secretion system ( He and Zhang, 2008 ; Ryan and Dow, 2011 ). Xcc FabH, like FabHs from Gram-positive bacteria, prefers to use branched-chain acyl-CoAs as primers to initiate BCFAs synthesis ( Yu et al, 2016 ). However, little is known about how Xcc produces branch-chain acyl-CoAs.…”

Novel Xanthomonas campestris Long-Chain-Specific 3-Oxoacyl-Acyl Carrier Protein Reductase Involved in Diffusible Signal Factor Synthesis

“…In this second scenario, X. campestris pv. campestris FabH condensed octanoyl coenzyme A (octanoyl-CoA) with malonyl-ACP to produce 3-oxodecanoyl-ACP ( 24 ), which FabG2 reduced to 3-hydroxydecanoyl-ACP, as described above. Fatty acid synthesis was thus primed, and growth at 42°C proceeded.…”