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 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.
Leech
Andrew J
AJ
Department of Microbiology and Immunology, P.O. Box 980678, Medical College of Virginia Campus of Virginia Commonwealth University, 1101 E. Marshall St., 5-047 Sanger Hall, Richmond, VA 23298-0678, USA. deohman@vcu.edu
Sprinkle
April
A
Wood
Lynn
L
Wozniak
Daniel J
DJ
Ohman
Dennis E
DE
D000464:Alginates; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D020723:Glucuronic Acid / Q000235:genetics; D006603:Hexuronic Acids; D020125:Mutation, Missense; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000464:Alginates; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D006603:Hexuronic Acids; D014157:Transcription Factors; C072862:AlgB protein, Pseudomonas; D020723:Glucuronic Acid