Nucleotide sequence and expression of a phosphate-regulated gene encoding a secreted hemolysin of Pseudomonas aeruginosa

Abstract

A 3.3-kilobase-pair fragment of Pseudomonas aeruginosa DNA containing the phospholipase C (heat-labile hemolysin) gene was sequenced, and the location of the gene was determined. The gene product contains at its NH2 terminus a 38-amino acid sequence which structurally resembles the signal peptides of other secreted proteins but is unusually long and positively charged (6+). The location of the translation start codon was determined by constructing a series of plasmids in which the promoter of a transcription vector was ligated to Pseudomonas DNA containing deletions at the 5' end of the gene. The plasmids were used to transform Escherichia coli, and the resulting clones were assayed for hemolysin activity. In addition, sizes of truncated proteins produced by mutants with translation terminators introduced at specific sites were analyzed in E. coli maxicells. The gene is transcribed, starting just upstream of the hemolysin gene, as an mRNA of approximately 2,800 bases. Analysis of the nucleotide sequence, analysis of mutants in maxicells, and transcriptional studies indicate that the hemolysin is part of an operon composed of two genes. Phosphate regulation of the operon is at the transcriptional level. The location of the 5' end of the transcript was determined by S1 mapping.