Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins

Abstract

We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase (Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp-J near 0', with the gene order cys-59-xcp-1-proB, and loci xcp-2, xcp-3, and xcp-31 at 35', with the gene order trpC,D-xcp-31xcp-31-xcp-2-argC. Loci xcp4 and xcp41 through xcp44 were cotransducible with proA at 40'; loci xcp-5, xcp-51, xcp-52, and xcp53 were located at 55', with the gene order leu-10-trpF-met-9010xcp-53-xcp-51xcp-511xcp-52, and xcp-6 was located at 65' to 70', between catA and mtu-9002. Nine mutations (xcp-2, xcp-3, xcp-31, xcp4, and xcp4J through xcp45) caused decreased production of extracellular enzymes. Six strains with mutations xcp-1, xcp-5, xcp-51, xcp-52, xcp-53, and xcp-6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins, such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme. The formation and release of extracellular enzymes has been studied mostly in gram-positive bacteria such as Bacillus spp. (6), but there is comparatively little information about gram-negative organisms in this respect. Pseudomonas aeruginosa produces several extracellular enzymes and toxins, and some of these proteins are of importance in the pathogenicity of Pseudomonas infections (25, 34). Most strains produce at least two proteases, designated elastase and alkaline protease (29-31). Lipase, staphylolytic enzyme, alkaline phosphatase, and phospholipase C are also found extracellularly in broth media (7, 47, 48). Exotoxin A is a ribosylating enzyme which inactivates the ribosomal protein EF-2 in eucaryotic cells (17). Previous studies with protease-deficient mutants of P. aeruginosa PAKS-1 showed (48) that the mutations caused pleiotropic effects with decreased activities of several extracellular enzymes; i.e., they were exoprotein deficient. Differences in phenotypic properties between mutants indicated that a number of genetic loci were involved. This study on exoprotein-deficient Pseudomonas mutants was undertaken to determine the location of chromosomal * Corresponding author.

Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria.

“…Description of a chromosomal fragment required for protein secretion Previously a Pseudomonas DNA fragment was cloned that complemented the protein secretion mutations xcp-5, and 51-55 on the 55 min region of the chromosome (Filloux et al, 1989). Transposon insertions in the cloned DNA fragment, suppressing the complementation of different secretion mutations (Filloux et al, 1987;Wretlind and Pavlovskis, 1984) were isolated and physically mapped (Filloux et al, 1989). Three insertion spots were identified, which are represented by the insertions 77 and 26 on a 4 kb EcoRI fragment and 76 on a 5.8 kb EcoRI fragment ( Figure 1).…”

“…Pseudomonas aeruginosa is a particular case among Gramnegative bacteria because of its ability to secrete a great variety of proteins including lipase, phospholipase, alkaline phosphatase, exotoxin A, alkaline protease and elastase. Mutants have been isolated which are pleiotropically defective in the secretion of most of these proteins (Filloux et al, 1987;Wretlind et al, 1977;Wretlind and Pavlovskis, 1984). The mutations (xcp) were mapped in at least three different loci, at min 0, 55 and 65 of the chromosomal map.…”

Protein secretion in Pseudomonas aeruginosa

“…One mutation, xcp-6, was located at min 47. The latter mutation affected the secretion of alkaline phosphatase and phospholipase, but not of elastase, lipase and exotoxin A [6]. This suggests that in addition to the common components of the secretion apparatus, specific components may be required for limited subsets of exoproteins.…”

“…To understand the mechanism of protein secretion across the outer membrane, xcp mutants have been isolated that are pleiotropically defective in the production of several extracellular proteins [6,17,18]. Some of the mutants obtained (mutations xcp-2, xcp-3, xcp-31, xcp-4 and xcp-41 through xcp-45) did not accumulate the exoproteins intracellularly [6]. These mutants, which are only affected in exoprotein synthesis on plates, but not in broth, probably contain a mutation in a regulatory gene, and will therefore not further be discussed here.…”

“…Genetic studies have indicated the existence of at least two distinct secretion pathways in this organism, i.e. a pathway specific for alkaline protease and a general secretion pathway used by (most of) the other exoproteins [6]. These pathways are homologous to the conserved one-step and two-step mechanisms, respectively, which are widely distributed among Gram-negative bacteria.…”