title: EitherfadD1orfadD2, Which Encode acyl-CoA Synthetase, Is Essential for the Survival ofHaemophilus parasuisSC096.
abstract: InHaemophilus parasuis, the genesHAPS_0217andHAPS_1695are predicted to encode long-chain fatty acid-CoA ligases (FACSs). These proteins contain ATP/AMP signature motifs and FACS conserved motifs that are homologous to those inEscherichia coliFadD (EcFadD). In this study, we demonstrate thatHAPS_0217andHAPS_1695can functionally replace EcFadD in theE. coli fadDmutant JW1794, and were thus designatedfadD1andfadD2, respectively. An evaluation of kinetic parameters indicated that FadD1 and FadD2 have a substrate preference for long-chain fatty acids. Moreover, FadD2 exhibited substrate inhibition in the presence of high concentrations of oleic acid. Single mutants of each of thefadDgenes were easily constructed, whereas double mutants were not. These results were further confirmed using genomic site-directed mutagenesis, which supported the idea thatH. parasuisrequires eitherfadD1orfadD2for survival. ThefadD1mutant exhibited slower growth than the wild-type strain SC096, and its complementation resulted in a restored phenotype. The wild-type strain did not grow on chemically defined medium without the addition of oleic acid, indicating that lipids are a vital nutrient for this bacterium. Additionally, strains with a disruptedfadD1gene also exhibited increased sensitivity to quinolone antibiotics, including levofloxacin, enrofloxacin, ciprofloxacin and nalidixic acid.
authors.affiliation: Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University Guangzhou, China.
authors.forename: Saixiang
authors.initials: S
authors.lastname: Feng
authors.affiliation: Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University Guangzhou, China.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Xu
authors.affiliation: Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University Guangzhou, China.
authors.forename: Kaijie
authors.initials: K
authors.lastname: Yang
authors.affiliation: Key Laboratory of Protein Function and Regulation in Agricultural Organisms of Guangdong province, College of Life Science, South China Agricultural University Guangzhou, China.
authors.forename: Haihong
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University Guangzhou, China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Fan
authors.affiliation: Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University Guangzhou, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Liao
mesh_terms: D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D020043:Genes, Essential; D005816:Genetic Complementation Test; D044137:Haemophilus parasuis / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D050296:Microbial Viability; D013379:Substrate Specificity
chemical_list: D003066:Coenzyme A Ligases
keywords: FadD; Haemophilus parasuis; acyl-CoA synthetase; fatty acid; quinolones