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The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P. oleovorans. Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices. The topology of alkane hydroxylase was studied in E. coli using protein fusions linking different amino-terminal fragments of the alkane hydroxylase (AlkB) to alkaline phosphatase (PhoA) and to beta-galactosidase (LacZ). Four AlkB-PhoA fusions were constructed using transposon TnphoA. Site-directed mutagenesis was used to create PstI sites at 12 positions in AlkB. These sites were used to create AlkB-PhoA and AlkB-LacZ fusions. With respect to alkaline phosphatase and beta-galactosidase activity each set of AlkB-PhoA and AlkB-LacZ fusions revealed the expected complementary activities. At three positions, PhoA fusions were highly active, whereas the corresponding LacZ fusions were the least active. At all other positions the PhoA fusions were almost completely inactive, but the corresponding LacZ fusions were highly active. These data predict a model for alkane hydroxylase containing six transmembrane segments. In this model the amino terminus, two hydrophilic loops, and a large carboxyl-terminal domain are located in the cytoplasm. Only three very short loops near amino acid positions 52, 112, and 251 are exposed to the periplasm.

The pncB gene from Escherichia coli, which encodes nicotinic acid phosphoribosyltransferase (EC 2.4.2.11), was cloned on a 1.5-kilobase TaqI-EcoRI fragment. Its position on the E. coli chromosome was determined at 20.8 min between the asnS and pepN loci. The nucleotide sequence of the gene and the transcription and translation initiation sites were determined. Expression of pncB on a multicopy plasmid leads to a 25-fold increase in nicotinic acid phosphoribosyltransferase activity. Growth of E. coli in the presence of nicotinic acid leads to strong repression of nicotinic acid phosphoribosyltransferase activity, indicating that the cloned pncB sequence contains its own control sequences. It is shown that increased nicotinic acid phosphoribosyltransferase activity effects a 5-fold increase in the intracellular concentration of NAD. The cloned pncB gene can therefore be used as a tool to raise intracellular cofactor levels.