Cloning and sequence analysis of a hemolysin-encoding gene from Pseudomonas paucimobilis

Michael F. Minnick, David C. Scherer

Research output: Contribution to journalArticlepeer-review

Abstract

We report the cloning, expression and nucleotide (nt) sequence of a β-hemolysin-encoding gene, termed hlyA, from Pseudomonas paucimobilis. A genomic DNA library of the pseudomonad was constructed in Escherichia coli using the plasmid vector, pUC19. The My A gene was cloned by screening for a β-hemolytic phenotype in E. coli transformants and was mapped to a 1100-bp Pstl-Smal fragment. The nt sequence analysis of the 1100-bp insert revealed a 789-bp open reading frame which is preceded 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 immediately followed by a large inverted repeat with 27-bp arms which may serve as a Rho-factor-independent transcriptional terminator. The hlyA gene codes for a protein of 263 amino acids (aa) residues with a deduced relative molecular mass (Mr) of 29695 and a predicted pI value of 11.5. Expression of hlyA from recombinant DNA in E. colioccurred 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 encoded protein.

Original languageEnglish
Pages (from-to)57-63
Number of pages7
JournalGene
Volume130
Issue number1
DOIs
StatePublished - Aug 16 1993

Keywords

  • Cytolysin
  • nucleotide sequence
  • opportunistic pathogen
  • pseudomonad
  • recombinant DNA

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