Alginate synthesis in Pseudomonas aeruginosa: environmental regulation of the algC promoter

Abstract

The exopolysaccharide alginate is a maJor virulence factor ofPseudomonas aeruginosa strains that infect the lungs of cystic fibrosis patients. The synthesis of alginate is almost uniquely associated with the pathogenicity ofP. aeruginosa within the environment of the cystic fibrosis lung. The gene a4gC is one of the essential alginate biosynthetic genes and codes for the enzyme phosphomannomutase. In this report, we present data on the transcriptional regulation of algC expression. The activity of the algC promoter is modulated by the response regulator, AlgR1, a member of the two-component signal transduction protein family, which also regulates other alginate-specific promoters. In both mucoid (alginate-positive) and nonmucoid (alginate-negative) P. aeruginosa strains, transcriptional activation ofalgC increased with the osmolarity of the culture medium. This osmolarity-induced activation was found to be dependent on AlgRl. AlgRl was found to interact directly with the algC promoter. Deletion mapping, in conjunction with mobility shift assays, showed that AlgRl specifically bound with two regions ofalgC upstream DNA. A fragment spanning nucleotide positions -378 to -73 showed strong specific binding, while a fragment located between positions -73 and +187 interacted relatively weakly with AlgR1. Phosphorylation of the AlgRl protein resulted in the stimulation of its in vitro ability to bind to the algC promoter region (a fragment spanning nucleotides -378 to -73). Transcription from the aIgC promoter, which has significant homology with the RNA polymerase o-54 (RpoN) recognition sequence, decreased in an rpoN mutant of P. aeruginosa.

AlgR functions in algC expression and virulence in Pseudomonas syringae pv. syringae

“…1, boxed sequences). The two binding sites, located at 413 and 75 bp upstream of the predicted ATG in P. syringae FF5, resemble algC-ABS1 and algC-ABS2 in P. aeruginosa (Fujiwara et al, 1993;Zielinski et al, 1992). Within the P. syringae algC coding region, one putative ABS was located 45 bp downstream from the predicted translational start site, a location that is similar to algC-ABS3 in P. aeruginosa (Fujiwara et al, 1993).…”

“…Sequence analysis of the algC promoter region CCGTTCGTCN 5 was previously reported as a consensus sequence recognized and bound by AlgR, and AlgR-binding sites (ABS) were identified in the promoter regions of algD and algC from P. aeruginosa (Kato & Chakrabarty, 1991;Zielinski et al, 1992). The nucleotide sequence of the region upstream of the translational start site of P. syringae algC contained two putative ABS (Fig.…”

“…In P. aeruginosa, AlgR is required for expression of the algC promoter (PalgC) and binds to PalgC at multiple sites (Zielinski et al, 1991(Zielinski et al, , 1992Fujiwara et al, 1993). The presence of multiple, putative ABS in the algC promoter region of several P. syringae strains (Fig.…”

“…The alginate genes are normally silent in P. aeruginosa but are specifically activated during growth of this organism in the lungs of CF patients. Activation involves two critical promoters, PalgC (Zielinski et al, 1991(Zielinski et al, , 1992 and PalgD (Deretic et al, 1987). The algC and algD promoters, while mapping at two separate locations on the P. aeruginosa chromosome, are nevertheless both responsive to environmental signals such as high osmolarity, starvation for nitrogen and/or carbon, heat shock and oxidative stress (Shankar et al, 1995).…”

“…AlgC also participates in rhamnolipid production, presumably by catalysing the conversion of glucose 6-phosphate to glucose 1-phosphate, the first step in the deoxy-thymidine-diphospho-L-rhamnose (dTDP-L-rhamnose) pathway (Olvera et al, 1999). In P. aeruginosa, both algD and algC are under positive control of AlgR, which functions as a response-regulator member of the two-component signal transduction system and binds to multiple sites upstream of algC and algD (Deretic et al, 1989;Kato & Chakrabarty, 1991;Mohr et al, 1992;Zielinski et al, 1992). P. aeruginosa AlgR was previously overproduced, purified, and shown to be required for transcriptional activation of the algD and algC promoter regions (Kato & Chakrabarty, 1991;Mohr et al, 1992;Zielinski et al, 1992).…”