Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism

Abstract

A 6.5-kb DNA fragment carrying the functions required for specific secretion of the extracellular alkaline protease produced by Pseudomonas aeruginosa was cloned. The whole 6.5-kb DNA fragment was transcribed in one direction and probably carried three genes involved in secretion. The expression in trans of these genes, together with the apr gene, in Escherichia coli allowed synthesis and secretion of the alkaline protease, which was extensively investigated by performing pulse-chase experiments under various conditions. We demonstrated the absence of a precursor form, as well as the independence of alkaline protease translocation from SecA. The absence of secretion genes impaired alkaline protease secretion; the protein then remained intracellular and was partially degraded.

Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion

“…No appreciable amount of proteolytic or elastolytic activity was detected in toluene cellular extracts, indicating the lack of intracellular accumulation of active enzymes (Table 1). This observation was in agreement with earlier work (11,15,16,18,29,30). Examination of both types of MVs for protease activity demonstrated the association of active enzyme.…”

“…In contrast, elastolytic activity was detected exclusively in culture supernatants and was not affected by removal of vesicles from cell-free culture supernatants. Previous studies have demonstrated that the enzyme is secreted as a proenzyme that becomes active only as it is released into the supernatant (15,28). For this Figure 6A and B show gentamicintreated cells labelled for PLC.…”

Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus

“…In each case the protease is secreted via an ABC transporter, often linked to the protease structural gene in the same operon. However, the genetic organization of the protease (prt) operon varies, as does the number of proteases secreted (Ahn et al, 1999;Akatsuka et al, 1995Akatsuka et al, , 1997Duong et al, 1992;Ghigo & Wandersman, 1992a, b;Guzzo et al, 1991b;Kawai et al, 1999).…”

Reliable Enzyme‐Linked Immunosorbent Assay Systems for Pathogenic Factors of Pseudomonas aeruginosa Alkaline Proteinase, Elastase, and Exotoxin A: A Comparison of Methods for Labeling Detection Antibodies with Horseradish Peroxidase

“…We and other groups have cloned the elastase structural gene, lasB (3,7,36) and the alkaline proteinase gene, aprA (1,10,28). There are several reports describing the methods used to determine the concentration of the pathogenic factors produced by P. aeruginosa (2, 4, 5, 13, 16-18, 20, 30, 32).…”