CLONING AND HETEROLOGOUS EXPRESSION OF A GENE ENCODING AN ALKANE-INDUCED EXTRACELLULAR PROTEIN INVOLVED IN ALKANE ASSIMILATION FROM PSEUDOMONAS-AERUGINOSA

Pseudomonas aeruginosa PG201 produces a 16-kDa extracellular protein i n media containing n-hexadecane as a carbon source but not in media co ntaining glycerol or glucose. This protein was purified, and the N-ter minal amino acid sequence was determined. The amino acid composition o f the protein was found to be very similar to that of the so-called pr otein-like activator for n-alkane oxidation (PA) from P. aeruginosa S7 B1. This extracellular protein was previously characterized (K. Hisats uka, T. Nakahara, Y. Minoda, and K. Yamada, Agric. Biol. Chem. 41:445- 450, 1977) and found to stimulate the growth of P. aeruginosa on n-hex adecane and to possess emulsifying activity. To study the role(s) of t he PA protein and to make it accessible for possible future applicatio ns, we have cloned the PA-encoding (pra) gene and determined its nucle otide sequence. This analysis revealed a protein-coding region of 162 amino acids, with the first 25 residues being reminiscent of those of a typical bacterial signal sequence. The pva gene was inactivated by i nsertional mutagenesis, and the resulting strain was found to lack ext racellular PA protein and to be retarded in its growth in n-hexadecane -containing media. These results are consistent with the growth stimul atory role of the PA protein. The pra gene was expressed in Escherichi a coli, and substantial amounts of the recombinant protein were found in the extracellular growth medium. The recombinant protein was purifi ed by metal chelate affinity chromatography. The ability to produce se creted PA protein by E. coli provides a simple and safe means to analy ze its function(s) in alkane assimilation in the future.