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Metabolic and stress responses of Acinetobacter oleivorans DR1 during long-chain alkane degradation.

ABSTRACT: 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 ?ackA mutant. Impaired growth of the ?aceA 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.

SUBMITTER: Park C

PROVIDER: S-EPMC5658608 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Metabolic and stress responses of Acinetobacter oleivorans DR1 during long-chain alkane degradation.

Park Chulwoo C Shin Bora B Jung Jaejoon J Lee Yunho Y Park Woojun W

Microbial biotechnology 20170831 6

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 tha ...[more]

PMID: 28857443

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Project description:The glyoxylate shunt (GS), involving isocitrate lyase (encoded by aceA) and malate synthase G (encoded by glcB), is known to play important roles under several conditions including oxidative stress, antibiotic defense, or certain carbon source metabolism (acetate and fatty acids). Comparative growth analyses of wild type (WT), aceA, and glcB null-strains revealed that aceA, but not glcB, is essential for cells to grow on either acetate (1%) or hexadecane (1%) in Acinetobacter oleivorans DR1. Interestingly. the aceA knockout strain was able to grow slower in 0.1% acetate than the parent strain. Northern Blot analysis showed that the expression of aceA was dependent on the concentration of acetate or H2O2, while glcB was constitutively expressed. Up-regulation of stress response-related genes and down-regulation of main carbon metabolism-participating genes in a ΔaceA mutant, compared to that in the parent strain, suggested that an ΔaceA mutant is susceptible to acetate toxicity, but grows slowly in 0.1% acetate. However, a ΔglcB mutant showed no growth defect in acetate or hexadecane and no susceptibility to H2O2, suggesting the presence of an alternative pathway to eliminate glyoxylate toxicity. A lactate dehydrogenase (LDH, encoded by a ldh) could possibly mediate the conversion from glyoxylate to oxalate based on our RNA-seq profiles. Oxalate production during hexadecane degradation and impaired growth of a ΔldhΔglcB double mutant in both acetate and hexadecane-supplemented media suggested that LDH is a potential detoxifying enzyme for glyoxylate. Our constructed LDH-overexpressing Escherichia coli strain also showed an important role of LDH under lactate, acetate, and glyoxylate metabolisms. The LDH-overexpressing E. coli strain, but not wild type strain, produced oxalate under glyoxylate condition. In conclusion, the GS is a main player, but alternative glyoxylate pathways exist during acetate and hexadecane metabolism in A. oleivorans DR1.

Dataset Information

Metabolic and stress responses of Acinetobacter oleivorans DR1 during long-chain alkane degradation.

Publications

Metabolic and stress responses of Acinetobacter oleivorans DR1 during long-chain alkane degradation.

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