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The NtrC family regulator AlgB, which controls alginate biosynthesis in mucoid Pseudomonas aeruginosa, binds directly to the algD promoter.

ABSTRACT: Alginate production in mucoid (MucA-defective) Pseudomonas aeruginosa is dependent upon several transcriptional regulators, including AlgB, a two-component response regulator belonging to the NtrC family. This role of AlgB was apparently independent of its sensor kinase, KinB, and even the N-terminal phosphorylation domain of AlgB was dispensable for alginate biosynthetic gene (i.e., algD operon) activation. However, it remained unclear whether AlgB stimulated algD transcription directly or indirectly. In this study, microarray analyses were used to examine a set of potential AlgB-dependent, KinB-independent genes in a PAO1 mucA background that overlapped with genes induced by d-cycloserine, which is known to activate algD expression. This set contained only the algD operon plus one other gene that was shown to be uninvolved in alginate production. This suggested that AlgB promotes alginate production by directly binding to the algD promoter (PalgD). Chromosome immunoprecipitation revealed that AlgB bound in vivo to PalgD but did not bind when AlgB had an R442E substitution that disrupted the DNA binding domain. AlgB also showed binding to PalgD fragments in an electrophoretic mobility shift assay at pH 4.5 but not at pH 8.0. A direct systematic evolution of ligands by exponential enrichment approach showed AlgB binding to a 50-bp fragment located at bp -224 to -274 relative to the start of PalgD transcription. Thus, AlgB belongs to a subclass of NtrC family proteins that can activate promoters which utilize a sigma factor other than sigma(54), in this case to stimulate transcription from the sigma(22)-dependent PalgD promoter.

SUBMITTER: Leech AJ

PROVIDER: S-EPMC2223705 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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The NtrC family regulator AlgB, which controls alginate biosynthesis in mucoid Pseudomonas aeruginosa, binds directly to the algD promoter.

Leech Andrew J AJ Sprinkle April A Wood Lynn L Wozniak Daniel J DJ Ohman Dennis E DE

Journal of bacteriology 20071102 2

Alginate production in mucoid (MucA-defective) Pseudomonas aeruginosa is dependent upon several transcriptional regulators, including AlgB, a two-component response regulator belonging to the NtrC family. This role of AlgB was apparently independent of its sensor kinase, KinB, and even the N-terminal phosphorylation domain of AlgB was dispensable for alginate biosynthetic gene (i.e., algD operon) activation. However, it remained unclear whether AlgB stimulated algD transcription directly or indi ...[more]

PMID: 17981963

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Project description:UNLABELLED:Pseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (?5 µM), not under iron-replete conditions (?10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections. IMPORTANCE:Pseudomonas aeruginosa is a leading model for the investigation of biofilms. While data have been generated about the role of iron in alginate-independent (Psl/Pel) biofilm development, there is a paucity of data regarding the role of iron in alginate production and its associated mucoid phenotype. We demonstrate that biologically relevant levels of iron that exist in the airway mucus of cystic fibrosis (CF) patients have a substantial influence on production of alginate and the overt mucoid phenotype, pathognomonic of P. aeruginosa infections in CF. Mucoid mutants of non-CF P. aeruginosa isolates are mucoid only under iron limitation and do not express increased levels of alginate under iron-replete growth conditions. However, a significant number of long-term CF isolates lost their iron-regulated expression of increased alginate production and mucoidy and became iron constitutive for these properties. In contrast to the formation of Psl-type biofilms, increasing iron limitation ultimately leads to an iron-constitutive expression of alginate and mucoidy.

Project description:Chronic pulmonary infection with Pseudomonas aeruginosa is a common and serious problem in patients with cystic fibrosis (CF). The P. aeruginosa isolates from these patients typically have a mucoid colony morphology due to overproduction of the exopolysaccharide alginate, which contributes to the persistence of the organisms in the CF lung. Most of the alginate biosynthetic genes are clustered in the algD operon, located at 34 min on the chromosome. Alginate biosynthesis begins with the formation of an activated monomer, GDP-mannuronate, which is known to occur via the products of the algA, algC, and algD genes. Polymannuronate forms in the periplasm, but the gene products involved in mannuronate translocation across the inner membrane and its polymerization are not known. One locus of the operon which remained uncharacterized was a new gene called algK between alg44 and algE. We sequenced algK from the mucoid CF isolate FRD1 and expressed it in Escherichia coli, which revealed a polypeptide of the predicted size (52 kDa). The sequence of AlgK showed an apparent signal peptide characteristic of a lipoprotein. AlgK-PhoA fusion proteins were constructed and shown to be active, indicating that AlgK has a periplasmic subcellular localization. To test the phenotype of an AlgK-mutant, the algK coding sequence was replaced with a nonpolar gentamicin resistance cassette to avoid polar effects on genes downstream of algK that are essential for polymer formation. The algKdelta mutant was nonmucoid, demonstrating that AlgK was required for alginate production. Also, AlgK- mutants demonstrated a small-colony phenotype on L agar, suggesting that the loss of AlgK also caused a growth defect. The mutant phenotypes were complemented by a plasmid expressing algK in trans. When the algKdelta mutation was placed in an algJ::Tn501 background, where algA was not expressed due to polar transposon effects, the growth defect was not observed. AlgK- mutants appeared to accumulate a toxic extracellular product, and we hypothesized that this could be an unpolymerized alginate precursor. High levels of low-molecular-weight uronic acid were produced by the AlgK- mutant. When AlgK- culture supernatants were subjected to dialysis, high levels of uronic acids diffused out of the dialysis sac, and no uronic acids were detectable after extensive dialysis. In contrast, the mucoid wild-type strain produced only polymerized uronic acids (i.e., alginate), whereas the algKdelta algJ::Tn501 mutant produced no uronic acids. Thus, the alginate pathway in an AlgK- mutant was blocked after transport but at a step before polymerization, suggesting that AlgK plays an important role in the polymerization of mannuronate to alginate.

Dataset Information

The NtrC family regulator AlgB, which controls alginate biosynthesis in mucoid Pseudomonas aeruginosa, binds directly to the algD promoter.

Publications

The NtrC family regulator AlgB, which controls alginate biosynthesis in mucoid Pseudomonas aeruginosa, binds directly to the algD promoter.

Similar Datasets

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