Metabolic characterization of alkane monooxygenases and the growth phenotypes of <i>Pseudomonas aeruginosa</i> ATCC 33988 on hydrocarbons

There is a demand and widespread interest in evaluating microbial community structures and metabolic processes in hydrocarbon environments. The current work aims to detect microbial subgroups (phenotypic subsets) and their metabolic processes, such as substrate specificity and expression of niche-associated genes. In this study, we were able to discriminate different cell types in real time from a complex sample matrix to allow the detection of live, dead, and injured cell populations in jet fuels. We found that the expression of <i>alkB1</i> and <i>alkB2</i> genes is induced in a growth-dependent manner and <i>alkB2</i> induction started before <i>alkB1</i>. This indicates that as an early response of <i>Pseudomonas aeruginosa</i> cells' exposure to alkanes, cells activate <i>alkB2</i> gene induction. Deletion of <i>alkB1</i> and <i>alkB2</i> genes completely inhibited <i>P. aeruginosa</i> ATCC 33988 growth in jet fuel, suggesting that two alkane monooxygenases are responsible for the degradation of alkanes and jet fuel. Interestingly, the AlkB2 has a broader (<i>n</i>-C8<i>-n</i>-C16) substrate range compared to AlkB1 (<i>n</i>-C12-<i>n</i>-C16). The data indicate that two alkane utilization pathways can coexist in <i>P. aeruginosa</i> ATCC 33988, and they are differentially expressed in response to <i>n</i>-C6<i>-n-</i>C16 alkanes found in jet fuel. This study provided additional information on the heterogeneity and phenotypic diversity within the same species after exposure to hydrocarbons. This work advances our understanding of microbial community structures and provides new insight into the alkane metabolism of <i>P. aeruginosa</i>.IMPORTANCEAlkane degradation allows for the natural breakdown of hydrocarbons found in crude oil, which can significantly contribute to environmental remediation. The metabolic process of microbes to hydrocarbons and the expression of niche-associated genes are not well understood. <i>Pseudomonas aeruginosa</i> ATCC 33988, originally isolated from a jet fuel tank, degrades hydrocarbons effectively and outcompetes the type strain <i>Pseudomonas aeruginosa</i> PAO1. In this study, we found differential expression of <i>alkB1</i> and <i>alkB2</i> alkane monooxygenase genes and the relative importance of these genes in alkane degradation. We found different phenotypic subsets within the same genotype, which are influenced by hydrocarbon stress. Overall, the research conducted in this study significantly contributes to our knowledge about microbial processes and community structure in hydrocarbon environments.