Mucoid Pseudomonas aeruginosa in cystic fibrosis: mutations in the muc loci affect transcription of the algR and algD genes in response to environmental stimuli

Abstract

Increased levels of alginate biosynthesis cause mucoidy in Pseudomonas aeruginosa, a virulence factor of particular importance in cystic fibrosis. The algR gene product, which controls transcription of a key alginate biosynthetic gene, algD, is homologous to the activator members of the two-component, environmentally responsive systems (NtrC, OmpR, PhoB, ArcA, etc). In this report, we show that mutations in the muc loci, (muc-2, muc-22, and muc-23, in the standard genetic P. aeruginosa strain PAO, as well as a mapped muc allele in an isolate from a cystic fibrosis patient) affect transcription of algD and algR. This influence was strongly dependent on environmental factors. Regulation by nitrogen was observed in all strains examined, but the absolute transcriptional levels, determining the mucoid or nonmucoid status, were strain (muc allele)-dependent. Increased concentrations of NaCl in the medium, an osmolyte which is elevated in cystic fibrosis lung secretions, resulted in an increased algD transcription and mucoid phenotype in a muc-2 strain; the same conditions, however, produced a nonmucoid phenotype in the muc-23 background and abolished algD transcription. Mutations in the muc loci may cause mucoidy by deregulating the normal response of the alginate system to environmental stimuli.

AlgR-binding sites within the algD promoter make up a set of inverted repeats separated by a large intervening segment of DNA

“…2 NH4, minimal medium with 0.2% ammonium sulfate as the nitrogen source; NO3, minimal medium with 0.2% potassium nitrate as the nitrogen source. b CDO activity was determined in sonic extracts of PA0568 (muc-2) grown under conditions as previously described (10,34). All values expressed as mean milliunits per milligram of protein ± standard error.…”

“…For transcriptional fusion studies, appropriate DNA fragments were cloned into the transcriptional fusion vector pVDX18 and conjugated into P. aeruginosa PA0568 (muc-2), and exconjugants were selected on Pseudomonas isolation agar (Difco) supplemented with carbenicillin (300 ,g/ml). The media and growth conditions for induction with NaCl or alternative nitrogen sources (nitrate instead of ammonia) have been previously described (10,30). Promoter activity was assayed by determining the activity of catechol 2,3-dioxygenase (CDO), the gene product of xylE used as a reporter gene, as previously described (10,23,30).…”

“…The media and growth conditions for induction with NaCl or alternative nitrogen sources (nitrate instead of ammonia) have been previously described (10,30). Promoter activity was assayed by determining the activity of catechol 2,3-dioxygenase (CDO), the gene product of xylE used as a reporter gene, as previously described (10,23,30). One unit of CDO is defined as the amount of enzyme required to convert 1 ,umol of catechol into 2-hydroxymuconic semialdehyde in 1 min at 24°C.…”

“…A key step in the overproduction of alginate and the establishment of mucoidy is the strong transcriptional activation of the algD gene encoding GDPmannose dehydrogenase (8)(9)(10)30). This enzyme catalyzes the oxidation of GDPmannose into GDPmannuronic acid, a direct precursor of alginate (8,33).…”

“…This enzyme catalyzes the oxidation of GDPmannose into GDPmannuronic acid, a direct precursor of alginate (8,33). The algD promoter is subject to complex transcriptional regulation and has become a benchmark for monitoring molecular events which govern expression of the alginate system (10,15,17,39). One of the regulatory elements (12-14, 21, 39) positively controlling algD is AlgR (9), a response regulator from a superfamily of bacterial signal transduction systems (7).…”