The puzzle of two tandem acyl-CoA ligases of Pseudomonas putida F1.

Abstract

The Pseudomonas putida F1 genome and those of many other pseudomonads contain two tandem genes encoding acyl-CoA ligases Pput_1340 (fadD1) and Pput_1339 (fadD2) with Pput_1339 (fadD2) being the upstream gene. The fadD designation was assigned when both genes were found to complement the growth of an Escherichia coli acyl-CoA synthetase fadD deletion strain with oleic acid as sole carbon source. Site-directed mutagenesis showed that residues of the ATP/AMP domain required for function of E. coli FadD were also essential for full function of FadD1 and FadD2. Growth of the constructed ∆fadD1, ∆fadD2, and ∆fadD1∆fadD2 strains was tested in minimal medium with different chain length fatty acids as sole carbon sources. Lack of FadD1 significantly retarded growth with different chain length fatty acids and lack of both FadD1 and FadD2 further retarded growth. Derivatives of the ∆fabA∆desA unsaturated fatty acid auxotrophic strain carrying a deletion of either ∆fadD1 or ∆fadD2 were constructed. Growth of the ∆fabA∆desA∆fadD1 strain was very weak, whereas the ∆fabA∆desA∆fadD2 strain grew as well as the ∆fabA∆desA parent strain. Overexpression of either fadD1 or fadD2 restored growth of the ∆fabA∆desA∆fadD1 strain with fadD2 overexpression having a greater effect than fadD1 overexpression. The ∆fadD1 or ∆fadD2 genes are cotranscribed although the expression level of fadD1 is much higher than that of fadD2. This is attributed to a fadD1 promoter located within the upstream FadD2 coding sequence.

Importance: Pseudomonas bacteria demonstrate a great deal of metabolic diversity and consequently colonize a wide range of ecological niches. A characteristic of these bacteria is a pair of genes in tandem annotated as acyl-CoA ligases involved in fatty acid degradation. The Pseudomonas putida F1 genome is annotated as having at least nine genes encoding acyl-CoA ligases which are scattered around the chromosome excepting the tandem pair. Since similar tandem pairs are found in other pseudomonads, we have constructed and characterized deletion mutants of the tandem ligases. We report that the encoded proteins are authentic acyl-CoA ligases involved in fatty acid degradation.