Alkane Hydroxylase from Acinetobacter sp. Strain ADP1 Is Encoded by alkM and Belongs to a New Family of Bacterial Integral-Membrane Hydrocarbon Hydroxylases

Abstract

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.

Molecular screening for alkane hydroxylase genes in Gram‐negative and Gram‐positive strains

“…Three integral membrane non-haem iron alkane hydroxylase sequences have been deposited in the sequence databases: AlkB (P. oleovorans GPo1) (Eggink et al, 1987a), AlkM (Acinetobacter sp. ADP1) (Ratajczak et al, 1998a) and AlkBpm [Stenotrophomonas (Pseudomonas) maltophilia N246] (Lee et al, 1996). In addition, an alkane hydroxylase gene homologue is present on the chromosome of Mycobacterium tuberculosis H37Rv.…”

“…other Acinetobacter strains, which can be explained by the presence of related genes with levels of DNA sequence identity to the alkM sequence ranging from 68% to 80%, which is enough to detect a signal in Southern blotting. (Ratajczak et al, 1998a). The ORFs were labelled alkR and alkM respectively.…”

“…To establish the function of alkM, we transformed an alkM-negative mutant of Acinetobacter sp. ADP1 (WH405) (Ratajczak et al, 1998a) with pWHEB7, which contains only alkM (A. calcoaceticus EB104). As a positive control, plasmid pWH968, containing the Acinetobacter sp.…”

“…In addition, the alkane hydroxylase AlkB is the prototype of a distinct class of non-haem iron integral membrane monooxygenases that includes the Pseudomonas putida mt-2 xylene monooxygenase (Suzuki et al, 1991) and the alkane hydroxylase of Acinetobacter sp. ADP1 (Ratajczak et al, 1998a).…”

“…A comparison between the alkane hydroxylases of P. oleovorans GPo1 (Eggink et al, 1987a) and Acinetobacter sp. ADP1 (Ratajczak et al, 1998a) allowed us to identify such regions, basically centred around two histidine clusters that were previously shown to be essential for the function of the structurally related desaturases (Shanklin et al, 1994). Using this method, we could show that a large proportion of bacteria able to grow on n-alkanes used in this study possess genes that are related to the P. oleovorans gene alkB, which encodes the catalytic component of the alkane hydroxylase system, whereas such genes cannot be ampli®ed from strains that are unable to grow on n-alkanes.…”