Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa

Abstract

We have cloned a gene from Pseudomonas aeruginosa that stimulates the expression of exotoxin A. A recombinant library of genomic DNA from strain PA103 constructed with a broad-host-range plasmid vector containing chromosomal insert fragments generated by Sau3A was used to transform the hypotoxigenic mutant strain PA103-29. A recombinant plasmid, pFHK6, was isolated from a PA103-29 transformant which displayed increased toxin production. From pFHK6, which contained a 20-kilobase-pair chromosomal insert, a 3-kilobase-pair XhoI fragment was isolated and subcloned into the plasmid cloning vector pVK101 to give pFHK10. In toxigenic P. aeruginosa strains containing pFHK10, toxin expression was increased 10-fold and high levels of iron in the culture medium only partially inhibited the overproduction. Expression studies suggested that pFHK10 did not contain the toxin structural gene. In addition, Southern analysis with the 3-kilobase-pair XhoI fragment suggested that the putative toxin regulatory gene is common among different strains of P. aeruginosa including previously reported nontoxigenic strains.

Regulation of exotoxin A synthesis in Pseudomonas aeruginosa: characterization of toxA‐lacZ fusions in wild‐type and mutant strains

“…3B). However, these results appear to contrast with those reported by Hedstrom et al (1986) and with the results shown in Fig. 8B.…”

“…It is of interest that a strain which lacks the ETA structural gene (WR5) apparently still has all the factors necessary to regulate the synthesis of ETA at the transcriptional level. In this regard, Hedstrom et al (1986) have shown that this strain carries sequences homologous to the regulatory gene toxR. However, whether this gene or its product and the ETA promoter are sufficient to regulate synthesis of ETA remains to be determined (see below).…”

“…8B indicate that when the toxR gene is controlled by the IacZ promoter as with pREX-19, the pattern of regulation by iron is now altered such that there is little or no difference in p-galactosidase synthesis or in ETA synthesis (data not shown) between high-and low-iron media. With regard to the alteration in the pattern of iron regulation observed by Hedstrom et al (1986), it is possible that in those studies synthesis of toxR was directed by the kanamycin resistance promoter because kan was insertionally inactivated by cloning of toxR into the X^ol site of kan in their vector piasmid.…”

Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production

“…The regulation of exotoxin A production in P. aeruginosa is a complicated process that involves several genes including regA, regB, fur, vfr, and lasR (Hedstrom et al, 1986;Hindahl et al, 1987;Wozniak et al, 1987;Wick et al, 1990a;Prince et al, 1991;West et al, 1994a;Gambello et al, 1993). The most extensively analysed of these genes is regA, which enhances toxA transcription.…”

Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa

“…These include lasR, a global regulator of protease production (Gambello et al, 1993), agmR, which is involved in regulation of glycerol metabolism genes (Po and Schweizer, 1993), anr, an oxygen responsive regulator (Sawers, 1991), fur, which regulates gene expression in response to iron levels (Prince et al, 1991;Prince, 1992), vfr, a cyclic AMP receptor homologue, (West et al, 1994), and most recently pvdS, an alternative sigma factor that regulates production of both iron uptake genes and ETA production (Ochsner et al, 1996). The regAB locus has the greatest and most direct effect on production of ETA (Hedstrom et al, 1986;Hindahl et al, 1987;Frank and Iglewski, 1988;Frank et al, 1989;Storey et al, 1991). The regA gene was cloned by complementation of a chemical mutant of P. aeruginosa defective in production of ETA, PA103-29 (Hedstrom et al, 1986).…”