Rao Qingyan, Li Yongfang, Sun Mengwei, Wei Fengdan, Shi Fulong, Ma Yanling
College of Life Science, Northwest University, 229 Tai bai North Rd, Xi'an, Shaanxi 710069, China.
Shaanxi Provincial Key Laboratory of Biotechnology, Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, 229 Tai bai North Rd, Xi'an, Shaanxi 710069, China.
J Appl Microbiol. 2025 Jun 2;136(6). doi: 10.1093/jambio/lxaf135.
The removal of n-alkanes by bacteria is a promising strategy for bioremediation. Especially, the transmembrane transport of n-alkanes is a critical intermediate process for transfer and adsorption. Herein, the structure and function of two FadL outer membrane transporters in Pseudomonas aeruginosa TJM4 were explored to elucidate the impact on efficient removal of C18 n-alkanes.
Phylogenetic analysis revealed considerable distinction in FadL sequences among strains involved in alkane catabolism. RT-qPCR analysis revealed that the expression of the two outer membrane transporter-encoding genes in strain TJM4, fadL1 and fadL2, was significantly upregulated in response to C18 n-alkane induction. To further investigate whether FadL was involved in the uptake and transport of C18 n-alkane, the fadL1 and fadL2 genes were deleted to generate mutants. Growth and degradation assays demonstrated that loss of the fadL gene reduced the ability of strain TJM4 to utilize and degrade C18 n-alkane, and FadL1 played a key role in the removal of C18 n-alkane. Moreover, the cell surface hydrophobicity (CSH) of the mutants was significantly lower than that of the wild-type (WT) strain TJM4. However, the complementation of fadL gene restored alkane degradation capacity and CSH of those mutant strains. These findings supported the role of FadL in enhancement of n-alkane catabolism. Finally, molecular docking analysis revealed hydrophobic interactions between the two FadL transporters and C18 n-alkane, in which Val, Leu, Ile, and Ala played a role in both complexes.
Those results indicated that FadL was a pivotal step for removal of C18 n-alkane in strain TJM4. The pronounced differences in sequence between the two FadL transporters and their interaction sites with C18 n-alkane suggested that they might be substrate-specific involved in the n-alkane transport pathway of strain TJM4.
利用细菌去除正构烷烃是一种很有前景的生物修复策略。特别是，正构烷烃的跨膜运输是转移和吸附的关键中间过程。在此，研究了铜绿假单胞菌TJM4中两种FadL外膜转运蛋白的结构和功能，以阐明其对高效去除C18正构烷烃的影响。
系统发育分析表明，参与烷烃分解代谢的菌株间FadL序列存在显著差异。RT-qPCR分析表明，菌株TJM4中两个外膜转运蛋白编码基因fadL1和fadL2的表达在C18正构烷烃诱导下显著上调。为进一步研究FadL是否参与C18正构烷烃的摄取和运输，缺失fadL1和fadL2基因以产生突变体。生长和降解试验表明，fadL基因的缺失降低了菌株TJM4利用和降解C18正构烷烃的能力，且FadL1在去除C18正构烷烃中起关键作用。此外，突变体的细胞表面疏水性（CSH）显著低于野生型（WT）菌株TJM4。然而，fadL基因的互补恢复了这些突变菌株的烷烃降解能力和CSH。这些发现支持了FadL在增强正构烷烃分解代谢中的作用。最后，分子对接分析揭示了两种FadL转运蛋白与C18正构烷烃之间的疏水相互作用，其中Val、Leu、Ile和Ala在两种复合物中均起作用。
这些结果表明，FadL是菌株TJM4中去除C18正构烷烃的关键步骤。两种FadL转运蛋白的序列及其与C18正构烷烃的相互作用位点存在明显差异，表明它们可能是参与菌株TJM4正构烷烃运输途径的底物特异性蛋白。
