Gene expression is regulated mainly at the transcriptional level. Transcription regulators are regulatory proteins that activate or repress RNA synthesis by modulating RNA polymerase activity in response to the prevailing physiological conditions. A unique class of transcription regulators comprises the enhancer binding proteins (EBPs) that activate transcription of sigma 54-dependent promoters using a mechanism similar to that used by regulatory proteins of eukaryotes. The EBPs bind to specific upstream activation sequences (UAS) and through ATP hydrolysis catalyse the isomerization of the closed  to open complex and transcription initiation. Two sigma 54 transcription regulators of Herbaspirillum seropedicae, the NifA and NtrC proteins, have already been characterized. A search in the Herbaspirillum seropedicae Genome Sequencing Program (Genopar) database identified 11 probable sigma 54 transcription regulators. The DNA binding domain, including the helix-turn-helix (HTH) motif, of the EBP named ORF216.0019 of H. seropedicae was cloned into the expression vector pET28a yielding plasmid pALIP 216. The DNA binding domain was overexpressed in Escherichia coli BL21lDE3 and purified by affinity chromatography on a Hitrap Chelating column. DNA aptamers for ORF 216.0019 were isolated by mixing the purified DNA-binding domain and H. seropedicae genomic DNA partially digested with Sau3AI. DNA-protein complexes were purified in a mini-spin agarose NTA-Ni²⁺ column and the DNA fragments were subsequently cloned into pUC18 digested with BamHI and sequenced. The sequences obtained suggested that protein ORF216.0019 regulates osmY which encodes an osmotic sensor, and prhA, which encodes a putative type III secretion system regulator.

Supported by CNPq, MCT, Fundação Araucária and Paraná Tecnologia