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

Alginate production in mucoid (MucA-defective) Pseudomonas aeruginosa is dependent upon several transcriptional regulators, including AIgB, a two-component response regulator belonging to the NtrC family. This role of AlgB was apparently independent of its sensor kinase, King, 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 AIgB 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 AIgB promotes alginate production by directly binding to the algD promoter (PalgD). Chromosome immunoprecipitation revealed that Alga bound in vivo to PalgD but did not bind when AlgB had an R442E substitution that disrupted the DNA binding domain. AIgB 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 AIgB binding to a 50-by fragment located at by -224 to -274 relative to the start of PalgD transcription. Thus, AIgB 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 PaIgD promoter.