DNA sequence determination and functional characterization of the OCT‐plasmid‐encoded <i>alkJKL</i> genes of <i>Pseudomonas oleovorans</i>

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<jats:title>Summary</jats:title><jats:p>The <jats:italic>alkBFGHJKL</jats:italic> and <jats:italic>alkST</jats:italic> operons encode enzymes that allow <jats:italic>Pseudomonas putida (oleovorans)</jats:italic> to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the <jats:italic>alkBFGHJKL</jats:italic> operon encoding the AlkJ, AlkK and AlkL polypeptides.</jats:p><jats:p>The <jats:italic>alkJ</jats:italic> gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane‐bound and converts aliphatic medium‐chain‐length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in <jats:italic>P. putida</jats:italic> alcohol dehydrogenase (AlcA) mutants.</jats:p><jats:p>AlkK is homologous to a range of proteins which act by an ATP‐dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long‐chain fatty acid CoA ligases. The <jats:italic>alkK</jats:italic> gene complements a <jats:italic>fadD</jats:italic> mutation in <jats:italic>Escherichia coli</jats:italic>, which shows that it indeed encodes an acyl‐CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm.</jats:p><jats:p>AlkL is homologous to OmpW, a <jats:italic>Vibrio cholerae</jats:italic> outer membrane protein of unknown function, and a hypothetical polypeptide encoded by <jats:italic>ytt4</jats:italic> in <jats:italic>E. coli.</jats:italic> AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The <jats:italic>alkL</jats:italic> gene product was found in the outer membrane of <jats:italic>E. coli</jats:italic> W3110 containing the <jats:italic>alk‐genes.</jats:italic> The <jats:italic>alkL</jats:italic> gene can be deleted without a clear effect on growth rate. Its function remains unknown.</jats:p><jats:p>The G+C content of the <jats:italic>alkJKL</jats:italic> genes is 45%, identical to that of the <jats:italic>alkBFGH</jats:italic> genes, and significantly lower than the G+C content of the OCT‐plasmid and the <jats:italic>P. putida</jats:italic> chromosome.</jats:p>

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