THE ALKANE OXIDATION SYSTEM OF PSEUDOMONAS-OLEOVORANS - INDUCTION OF THE ALK GENES IN ESCHERICHIA-COLI W3110(PGEC47) AFFECTS MEMBRANE BIOGENESIS AND RESULTS IN OVEREXPRESSION OF ALKANE HYDROXYLASE IN A DISTINCT CYTOPLASMIC MEMBRANE SUBFRACTION

The alkane hydroxylase system of Pseudomonas oleovorans, which catalys es the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxylase AlkB, is an in tegral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5-2% AlkB relative to the total cell protein, both in P. oleovora ns and in recombinant Escherichia coli DH1. We present a study on the induction and localization of the alkane hydroxylase in E. coliW3110, which appears to be an interesting host strain because it permits expr ession levels of AlkB of up to 10-15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild-ty pe W3110. Freeze-fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytop lasm; these occurred much more frequently in cells expressing alkB tha n in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressi ng alkB by density gradient centrifugation showed the customary cytopl asmic and outer membranes, as well as a low-density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both b y SDS-PAGE and enzyme activity measurements. A typical cytoplasmic mem brane protein, NADH oxidase, was absent from the low-density membrane fraction. alkB expression in W3110 changed the composition of the phos pholipid headgroup in the membrane, as well as the fatty acid composit ion of the membrane. The major changes occurred in the unsaturated fat ty acids: C16:1 and C18:1 increased at the expense Of C17:0cyc and C19 :0cyc.