Gene structures and regulation of the alkane hydroxylase complex in Acinetobacter sp. strain M-1.
Ontology highlight
ABSTRACT:
SUBMITTER: Tani A
PROVIDER: S-EPMC95074 | biostudies-literature | 2001 Mar
REPOSITORIES: biostudies-literature
Publications
Gene structures and regulation of the alkane hydroxylase complex in Acinetobacter sp. strain M-1.
Tani A A Ishige T T Sakai Y Y Kato N N
Journal of bacteriology 20010301 5
In the long-chain n-alkane degrader Acinetobacter sp. strain M-1, two alkane hydroxylase complexes are switched by controlling the expression of two n-alkane hydroxylase-encoding genes in response to the chain length of n-alkanes, while rubredoxin and rubredoxin ruductase are encoded by a single gene and expressed constitutively. ...[more]
Similar Datasets
Project description:CYP153, one of the most common medium-chain n-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed in n-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading various n-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrum n-alkane-degrading Gram-positive bacterium, Dietzia sp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the -10 and -35 regions in Actinobacteria. Further analysis of the β-galactosidase activity in the CYP153 gene promoter-lacZ fusion cell indicated that the CYP153 gene promoter was induced by n-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed a cypR mutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence of n-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements in Actinobacteria and Proteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by many Actinobacteria.
| S-EPMC4711121 | biostudies-literature
Project description:Degradation of long-chain alkanes by Acinetobacter sp. strain ADP1 involves rubredoxin and rubredoxin reductase. We complemented a mutant deficient in alkane utilization and sequenced four open reading frames (ORFs) on the complementing DNA. Each of these ORFs was disrupted by insertional mutagenesis on the chromosome. As determined from sequence comparisons, ORF1 and ORF4 seem to encode a rotamase of the PpiC type and an acyl coenzyme A dehydrogenase, respectively. Disruption of these ORFs does not affect alkane utilization. In contrast, the two other ORFs, alkR and alkM, are essential for growth on alkanes as sole carbon sources. alkR encodes a polypeptide with extensive homology to AraC-XyIS-like transcriptional regulators. It is located next to alkM, which encodes the terminal alkane hydroxylase, but is in the opposite orientation. Sequence homologies with other bacterial integral-membrane hydrocarbon hydroxylases suggest that AlkM may be the first member of a new protein family. The genes identified here are not linked to the rubredoxin- and rubredoxin reductase-encoding genes on the Acinetobacter sp. strain ADP1 chromosome.
| S-EPMC106126 | biostudies-literature
Project description:Enzymes involved in oxidation of long-chain n-alkanes are still not well known, especially those in gram-positive bacteria. This work describes the alkane degradation system of the n-alkane degrader actinobacterium Gordonia sp. strain SoCg, which is able to grow on n-alkanes from dodecane (C(12)) to hexatriacontane (C(36)) as the sole C source. SoCg harbors in its chromosome a single alk locus carrying six open reading frames (ORFs), which shows 78 to 79% identity with the alkane hydroxylase (AH)-encoding systems of other alkane-degrading actinobacteria. Quantitative reverse transcription-PCR showed that the genes encoding AlkB (alkane 1-monooxygenase), RubA3 (rubredoxin), RubA4 (rubredoxin), and RubB (rubredoxin reductase) were induced by both n-hexadecane and n-triacontane, which were chosen as representative long-chain liquid and solid n-alkane molecules, respectively. Biotransformation of n-hexadecane into the corresponding 1-hexadecanol was detected by solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME/GC-MS) analysis. The Gordonia SoCg alkB was heterologously expressed in Escherichia coli BL21 and in Streptomyces coelicolor M145, and both hosts acquired the ability to transform n-hexadecane into 1-hexadecanol, but the corresponding long-chain alcohol was never detected on n-triacontane. However, the recombinant S. coelicolor M145-AH, expressing the Gordonia alkB gene, was able to grow on n-triacontane as the sole C source. A SoCg alkB disruption mutant that is completely unable to grow on n-triacontane was obtained, demonstrating the role of an AlkB-type AH system in degradation of solid n-alkanes.
| S-EPMC3067205 | biostudies-literature
Project description:Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30 degrees C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.
| S-EPMC1907095 | biostudies-literature
Project description:Gram-positive actinomycete Rhodococcus jostii RHA1 is able to grow on C10 to C19 n-alkanes as a sole source of carbon and energy. To clarify, the n-alkane utilization pathway-a cluster of 5 genes (alkBrubA1A2BalkU) which appeared to be involved in n-alkane degradation-was identified and the transcriptional regulation of these genes was characterized. Reverse transcription-PCR analyses revealed that these genes constituted an operon and were transcribed in the presence of n-alkane. Inactivation of alkB led to the absence of the ability to utilize n-undecane. The alkB mutation resulted in reduction of growth rates on C10 and C12 n-alkanes; however, growths on C13 to C19 n-alkanes were not affected by this mutation. These results suggested that alkB was essential for the utilization of C10 to C12 n-alkanes. Inactivation of alkU showed the constitutive expression of alkB. Purified AlkU is able to bind to the putative promoter region of alkB, suggesting that AlkU played a role in repression of the transcription of alk operon. The results of this study indicated that alkB was involved in the medium-chain n-alkanes degradation of strain RHA1 and the transcription of alk operon was negatively regulated by alkU-encoded regulator. This report is important to understand the n-alkane degradation pathway of R. jostii, including the transcriptional regulation of alk gene cluster.
| S-EPMC6921075 | biostudies-literature
Project description:A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C(4) to C(14)), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Delta) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Delta strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.
| S-EPMC92174 | biostudies-literature
Project description:The genus Acinetobacter encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges. Acinetobacter sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned mutS from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of mutS resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of mutS also abolished the marker-specific variations in transforming efficiency seen in mutS(+) recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual Acinetobacter strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the Acinetobacter amino acid sequences.
| S-EPMC95523 | biostudies-literature
Project description:Alkane monooxygenases in Nocardioides sp. strain CF8 were examined at the physiological and genetic levels. Strain CF8 can utilize alkanes ranging in chain length from C(2) to C(16). Butane degradation by butane-grown cells was strongly inhibited by allylthiourea, a copper-selective chelator, while hexane-, octane-, and decane-grown cells showed detectable butane degradation activity in the presence of allylthiourea. Growth on butane and hexane was strongly inhibited by 1-hexyne, while 1-hexyne did not affect growth on octane or decane. A specific 30-kDa acetylene-binding polypeptide was observed for butane-, hexane-, octane-, and decane-grown cells but was absent from cells grown with octane or decane in the presence of 1-hexyne. These results suggest the presence of two monooxygenases in strain CF8. Degenerate primers designed for PCR amplification of genes related to the binuclear-iron-containing alkane hydroxylase from Pseudomonas oleovorans were used to clone a related gene from strain CF8. Reverse transcription-PCR and Northern blot analysis showed that this gene encoding a binuclear-iron-containing alkane hydroxylase was expressed in cells grown on alkanes above C(6). These results indicate the presence of two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8.
| S-EPMC93262 | biostudies-literature
Project description:Alkanes are oxidized in Acinetobacter sp. strain ADP1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase. rubA and rubB encode rubredoxin and a NAD(P)H-dependent rubredoxin reductase. We demonstrate here that single base pair substitutions in rubA or rubB lead to defects in alkane degradation, showing that both genes are essential for alkane utilization. Differences in the degradation capacity for hexadecane and dodecane in these mutants are discussed. Two genes, estB and oxyR, are located downstream of rubB, but are not necessary for alkane degradation. estB encodes a functional esterase. oxyR encodes a LysR-type transcriptional regulator, conferring resistance to hydrogen peroxide. rubA, rubB, estB, and oxyR constitute an operon, which is constitutively transcribed from a sigma70 promoter, and an estB-oxyR containing message is also transcribed from an internal promoter.
| S-EPMC93931 | biostudies-literature
Project description:Alcanivorax sp. strain NBRC 101098 was isolated from seawater in Japan. Strain NBRC 101098 is able to degrade various types of n-alkanes. Here, we report the complete genome of strain NBRC 101098.
| S-EPMC4132616 | biostudies-literature