Characterization of a TetR/AcrR family transcriptional repressor gene NpR3597 in Nostoc punctiforme

Nostoc punctiforme is a filamentous cyanobacterium capable of differentiating its vegetative cells into spore-like akinetes that can withstand desiccation and cold. The NpR3597 gene was previously identified in a time-course DNA microarray experiment to be up-regulated during akinete formation, and up-regulated in dividing cells of the filament as indicated by GFP transcriptional reporter strains. Sequence similarity to characterized proteins indicates that this is a putative tetracycline repressor family protein similar to TetR/AcrR found in a vast array of bacteria species that represses divergently transcribed genes encoding for ABC transport proteins. The mechanism of AcrR repression typically involves binding to an inverted repeat in the promoter of the divergently transcribed gene, inhibiting attachment of RNA polymerase. Although no phenotype could be determined for a NpR3597 deletion mutant strain, an over-expression strain bearing multiple copies of this gene on a plasmid under control of its own promoter caused pigmentation changes. Desiccation-revival tests showed that over-expression of NpR3597 results in nonfunctional akinetes. Spectrophotometric examination of the over-expression strain indicated that the levels of phycocyanin, a light harvesting protein subunit of the photosynthetic phycobilisome apparatus, was significantly higher than normal. Through a second round of microarray analysis, it was found that over-expression of NpR3597 correlated to increased transcription of genes involved in phycocyanin synthesis as well as other photosynthetic genes, and down-regulation of upstream divergently transcribed multi-drug resistance efflux pump gene NpF3598. Strong down-regulation of a number of additional genes with inverted repeat sequences in their upstream intergenic region, particularly another multi-drug resistance efflux pump gene NpF1932, was also observed. To determine the binding site for NpR3597, a conserved inverted repeat sequence found upstream of both NpF3598 and NpF1932 was used in Electric Mobility Gel Shift Assays (EMSA). A plasmid encoding for a 6xHistidine tagged NpR3597 was constructed, and the His-tagged protein purified. DNA fragments containing or lacking the putative inverted repeat binding sites were generated by PCR, end-labeled with biotin and used for EMSA with the purified protein. Only fragments containing the following motif ANNNNACNN - N2 - CNGTNTAGT in their inverted repeat sequence exhibited a gel mobility shift, indicating NpR3597 likely acts as a dimer and represses transcription by binding to these inverted repeat (IR) sequences.

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