Abstract
Summary Acinetobacter oleivorans DR1 can utilize C12–C30 alkanes as a sole carbon source but not short‐chain alkanes (C6, C10). Two copies of each alkB‐, almA‐ and ladA‐type alkane hydroxylase (AH) are present in the genome of DR1 cells. Expression and mutational analyses of AHs showed that alkB1 and alkB2 are the major AH‐encoding genes under C12–C30, and the roles of other almA‐ and ladA genes are negligible. Our data suggested that AlkB1 is responsible for long‐chain alkane utilization (C24–C26), and AlkB2 is important for medium‐chain alkane (C12–C16) metabolism. Phylogenetic analyses revealed large incongruities between phylogenies of 16S rRNA and each AH gene, which implies that A. oleivorans DR1 has acquired multiple alkane hydroxylases through horizontal gene transfer. Transcriptomic and qRT‐PCR analyses suggested that genes participating in the synthesis of siderophore, trehalose and poly 3‐hydroxybutyrate (PHB) were expressed at much higher levels when cells used C30 than when used succinate as a carbon source. The following biochemical assays supported our gene expression analyses: (i) quantification of siderophore, (ii) measurement of trehalose and (iii) observation of PHB storage. Interestingly, highly induced both ackA gene encoding an acetate kinase A and pta gene encoding a phosphotransacetylase suggested unusual ATP synthesis during C30 alkane degradation, which was demonstrated by ATP measurement using the ΔackA mutant. Impaired growth of the ΔaceA mutant indicated that the glyoxylate shunt pathway is important when C30 alkane is utilized. Our data provide insight into long‐chain alkane degradation in soil microorganisms.
Highlights
Substrate ranges of alkane degradation in A. oleivorans DR1
Growth tests on various substrates indicated that DR1 cells could utilize both mediumand long-chain alkanes (C12–C30) but not short-chain alkanes (C6–C10) (Fig. 1A, Figs S1A and S1B)
Because of the extremely low water solubility of tested alkanes, the maximum growth rate could be monitored through simple growth measurements using each alkane at 0.1% concentration
Summary
Acinetobacter oleivorans DR1 can utilize C12–C30 alkanes as a sole carbon source but not short-chain alkanes (C6, C10). A large number of hydrocarbonoclastic- and alkanedegrading bacteria are widely distributed in nature (Liu et al, 2015) Their degrading mechanisms have been investigated owing to their ecological importance and the versatile applications of alkane-degrading enzymes with economic benefits (Rojo, 2010). From many different bacteria, have been characterized, revealing that they have different ranges of carbon-chain length preferences with respect to alkane substrates (Van Beilen and Funhoff, 2007). Genomic data do not reveal the substrate ranges of each alkane hydroxylase, and the regulation of gene expression involved in alkane degradation is not well understood in many known alkane degraders. Expression analysis of the alkane hydroxylase genes provides insight into how bacteria can utilize alkanes and which enzymes should be investigated for biotechnological applications. Our analyses provide considerable insight into metabolic and stress responses during bacterial long-chain alkane metabolism
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