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 Si mapping.Phospholipase C (PLC) (phosphatidylcholine cholinephosphohydrolase) from Pseudomonas aeruginosa has been studied from different viewpoints: (i) as a hemolytic toxin that may contribute to the virulence of an opportunistic pathogen (22,23,44), (ii) as a secreted protein (10,12,25,48), and (iii) as a part of the phosphate regulon (13,14,19). Extracellular PLC hemolysins are also produced by organisms such as Clostridium perfringens (alpha toxin) and Staphylococcus aureus (beta toxin). In contrast, there is a different class of bacterial hemolysins produced by S. aureus (alpha toxin), Escherichia coli, and other organisms which are thought to have a nonenzymatic mechanism of action (18).The mechanism of pathogenicity for P. aeruginosa is complex, and the role of the hemolysin is not quite clear. PLC has been implicated as a virulence determinant in the pathogenesis of lung infection (22), and in another study, PLC production by urinary tract isolates was greater than that by lung, blood, or other isolates (4). Liu (23) observed that the PLC hemolysin and alkaline phosphatase are produced together during growth in low Pi and repressed during growth in high P, and proposed that these enzymes function cooperatively as a phosphate-scavenging mechanism. P. aeruginosa plcA mutants have been isolated which are deficient in the production of PLC and several other exported phosphate-repressible proteins (13, 17). Another class of mutants (plcB) hyperproduce most phosphaterepressible proteins and are also altered in Pi transport (14,17).PLC is also interesting for its properties as a secreted protein. The transport of proteins through the inner membrane of bacteria has been extensively studied in E. coli (for recent reviews, see references 33, 36, 41) bu...

Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

“…CDase produces sphingosine and the latter lipid downregulates macrophages that are stimulated by bacterial lipopolysaccharides from Gram-negative bacteria [21] and, hence, the bacterium may escape the host defence system [22]. It is noteworthy in that respect that the P. aeruginosa PA01 SMase gene [31] is located 347 bp downstream of the CDase gene on the same DNA strand. SMase and CDase can ideally work in tandem when both enzymes are simultaneously expressed by Pseudomonas.…”

Nucleotide sequence and expression in Escherichia coli of the gene coding for sphingomyelinase of Bacillus cereus

“…The mature phosphatidylcholine-hydrolyzing phospholipase C consists of 245 amino acid residues with a molecular mass of 28 520 Da which is considerably smaller than that of the mature sphingomyelinase, and it showed no significant sequence homology with the sphingomyelinase. Furthermore, these phospholipases C of B. cereus shows no sequence homology with two other microbial phospholipases that have been characterized; that is, the E. coli detergent-resistent phospholipase A [6] and the P. aeruginosa phospholipase C [42]. It is interesting, in connection with the infection by B. cereus of eukaryotic cells, that the genes coding for the phosphatidylcholine-hydrolyzing and sphingomyelin-hydrolyzing enzymes form a gene cluster, although regulation of the gene expression remains to be characterized.…”

Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa The GenBank accession numbers for the sequences reported in this paper are AF055999, AF127222, and AF127223.

“…P. aeruginosa secretes several factors that have the potential to facilitate the acquisition of haem from exogenous sources. A heat-labile haemolysin, phospholipase C, was purified from P. aeruginosa culture supernatants (Berka & Vasil, 1982) and the corresponding gene, plcH, was subsequently cloned (Pritchard & Vasil, 1986). In addition, P. aeruginosa secretes a heat-stable glycolipid haemolysin composed of rhamnose and β-hydroxydecanoate ; the genes required for the production of these rhamnolipids are regulated by quorum sensing and iron starvation (Ochsner et al, 1994 ;Ochsner & Reiser, 1995).…”