AUTHOR=Feng Saixiang , Xu Chenggang , Yang Kaijie , Wang Haihong , Fan Huiying , Liao Ming 

TITLE=Either fadD1 or fadD2, Which Encode acyl-CoA Synthetase, Is Essential for the Survival of Haemophilus parasuis SC096

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=7

YEAR=2017

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2017.00072

DOI=10.3389/fcimb.2017.00072

ISSN=2235-2988

ABSTRACT=<p>In <italic>Haemophilus parasuis</italic>, the genes <italic>HAPS_0217</italic> and <italic>HAPS_1695</italic> are 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 in <italic>Escherichia coli</italic> FadD (EcFadD). In this study, we demonstrate that <italic>HAPS_0217</italic> and <italic>HAPS_1695</italic> can functionally replace EcFadD in the <italic>E. coli fadD</italic> mutant JW1794, and were thus designated <italic>fadD1</italic> and <italic>fadD2</italic>, 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 the <italic>fadD</italic> genes were easily constructed, whereas double mutants were not. These results were further confirmed using genomic site-directed mutagenesis, which supported the idea that <italic>H. parasuis</italic> requires either <italic>fadD1</italic> or <italic>fadD2</italic> for survival. The <italic>fadD1</italic> mutant 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 disrupted <italic>fadD1</italic> gene also exhibited increased sensitivity to quinolone antibiotics, including levofloxacin, enrofloxacin, ciprofloxacin and nalidixic acid.</p>