CLONING AND SEQUENCE-ANALYSIS OF A HEMOLYSIN-ENCODING GENE FROM PSEUDOMONAS-PAUCIMOBILIS

We report the cloning, expression and nucleotide (nt) sequence of a be ta-hemolysin-encoding gene, termed hlyA, from Pseudomonas paucimobilis . A genomic DNA library of the pseudomonad was constructed in Escheric hia coli using the plasmid vector, pUC19. The hlyA gene was cloned by screening for a beta-hemolytic phenotype in E. coli transformants and was mapped to a 1100-bp PstI-SmaI fragment. The nt sequence analysis o f the 1100-bp insert revealed a 789-bp open reading frame which is pre ceded by a 10-nt purine-rich sequence with a possible ribosome-binding site of GGA. The ORF terminates with a single UGA stop codon and is i mmediately followed by a large inverted repeat with 27-bp arms which m ay serve as a Rho-factor-independent transcriptional terminator. The h lyA gene codes for a protein of 263 amino acids (aa) residues with a d educed relative molecular mass (M(r)) of 29 695 and a predicted pI val ue of 11.5. Expression of hlyA from recombinant DNA in E. coli occurre d regardless of insert orientation in the vector and produced a 29-kDa protein. Confirmation of P. paucimobilis as the source of the cloned hlyA gene was determined by DNA hybridization. A search of various nt and aa sequence databases revealed no homologues to hlyA or its encode d protein.