College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, China.
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Appl Environ Microbiol. 2024 Nov 20;90(11):e0126724. doi: 10.1128/aem.01267-24. Epub 2024 Oct 15.
The F1 genome and those of many other pseudomonads contain two tandem genes encoding acyl-CoA ligases Pput_1340 () and Pput_1339 () with Pput_1339 () being the upstream gene. The designation was assigned when both genes were found to complement the growth of an acyl-CoA synthetase deletion strain with oleic acid as sole carbon source. Site-directed mutagenesis showed that residues of the ATP/AMP domain required for function of FadD were also essential for full function of FadD1 and FadD2. Growth of the constructed ∆, ∆ and ∆∆ 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 ∆∆ unsaturated fatty acid auxotrophic strain carrying a deletion of either ∆ or ∆ were constructed. Growth of the ∆∆∆ strain was very weak, whereas the ∆∆∆ strain grew as well as the ∆∆ parent strain. Overexpression of either or restored growth of the ∆∆∆ strain with overexpression having a greater effect than overexpression. The ∆ or ∆ genes are cotranscribed although the expression level of is much higher than that of . This is attributed to a promoter located within the upstream FadD2 coding sequence.
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 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.
F1 基因组和许多其他假单胞菌的基因组包含两个串联基因，分别编码酰基辅酶 A 连接酶 Pput_1340（）和 Pput_1339（），其中 Pput_1339（）是上游基因。当这两个基因都被发现可以用油酸作为唯一碳源来补充酰基辅酶 A 合成酶缺失菌株的生长时，就被赋予了这个命名。定点突变显示，FadD 的 ATP/AMP 结构域的必需残基对于 FadD1 和 FadD2 的完全功能也是必需的。在含有不同链长脂肪酸作为唯一碳源的最小培养基中测试了构建的 ∆、∆和 ∆∆ 菌株的生长。缺乏 FadD1 会显著减缓不同链长脂肪酸的生长，而缺乏 FadD1 和 FadD2 则会进一步减缓生长。构建了携带缺失 ∆ 或 ∆ 的不饱和脂肪酸营养缺陷型衍生菌株。∆∆∆ 菌株的生长非常微弱，而 ∆∆∆ 菌株的生长与 ∆∆ 亲本菌株一样好。FadD1 和 FadD2 的过表达都可以恢复 ∆∆∆ 菌株的生长，而过表达 FadD1 的效果大于过表达 FadD2。虽然 的表达水平远高于 ，但 或 基因是共转录的。这归因于位于上游 FadD2 编码序列内的 启动子。
细菌表现出很大的代谢多样性，因此可以在广泛的生态位中定植。这些细菌的一个特征是一对串联的基因，注释为参与脂肪酸降解的酰基辅酶 A 连接酶。F1 基因组被注释为至少有九个编码酰基辅酶 A 连接酶的基因，这些基因散布在染色体周围，除了串联对。由于在其他假单胞菌中也发现了类似的串联对，我们构建并表征了串联连接酶的缺失突变体。我们报告说，编码的蛋白是参与脂肪酸降解的真正酰基辅酶 A 连接酶。
