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-XylS-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 rubredoxinand rubredoxin reductase-encoding genes on the Acinetobacter sp. strain ADP1 chromosome.

In Acinetobacter sp. strain ADP1, alkane degradation depends on at least five essential genes. rubAB andxcpR are constitutively transcribed. Here we describe inducible transcription of alkM, which strictly depends on the presence of the transcriptional activator AlkR. alkRitself is expressed at a low level, while a chromosomally locatedalkM::lacZ fusion is inducible by middle-chain-length alkanes from heptane to undecane, which do not support growth of ADP1, and by long-chain-length alkanes from dodecane to octadecane, which are used as sources of carbon and energy. The putative AlkM substrate 1-dodecene is also an effective inducer. Products of alkane hydroxylase activity like 1-dodecanol prevent induction of alkM expression. alkM is expressed only in stationary phase, suggesting its dependence on at least one other regulatory mechanism.

Polyphosphate kinase (Ppk) catalyzes the formation of polyphosphate from ATP. We cloned the ppk gene (2,073 bp) fromAcinetobacter sp. strain ADP1; this gene encodes a putative polypeptide of 78.6 kDa with extensive homology to polyphosphate kinase from Escherichia coli and other bacteria. Chromosomal disruption of ppk by inserting a transcriptionally fusedlacZ does not affect growth under conditions of phosphate limitation or excess. β-Galactosidase activity expressed from the single-copy ppk::lacZ fusion is induced 5- to 15-fold by phosphate starvation. An increased amount ofppk transcript (2.2 kb) was detected when cells were grown at a limiting phosphate concentration. Primer extension analysis revealed a regulated promoter located upstream of a second, constitutive promoter. Potential similarities of this regulation with the effects of PhoB and PhoR of E. coli are discussed.

Alkanes are oxidized in Acinetobacter sp. strain ADP1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase. rubA andrubB encode rubredoxin and a NAD(P)H-dependent rubredoxin reductase. We demonstrate here that single base pair substitutions inrubA 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, estBand 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, andoxyR constitute an operon, which is constitutively transcribed from a ς70 promoter, and anestB-oxyR containing message is also transcribed from an internal promoter.

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