Cloning and sequence analysis of a hemolysin-encoding gene from Pseudomonas paucimobilis.
We report the cloning, expression and nucleotide (nt) sequence of a beta-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 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 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 (M(r)) of 29,695 and a predicted pI value of 11.5. Expression of hlyA from recombinant DNA in E. coli occurred 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.
Minnick
M F
MF
Division of Biological Sciences, University of Montana, Missoula 59812.
Scherer
D C
DC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011549:Pseudomonas / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli