The role of the GAF and central domains of the transcriptional activator VnfA in Azotobacter vinelandii

Abstract

VnfA is a transcriptional activator that is required for the expression of the structural genes encoding nitrogenase-2 in Azotobacter vinelandii. VnfA consists of three domains: an N-terminal regulatory domain termed GAF, including a Cys-rich motif; a central domain from the AAA+ family; and a C-terminal domain for DNA binding. Previously, we reported that transcriptionally active VnfA harboring an Fe-S cluster (presumably of the 3Fe-4S type) as a prosthetic group and the Cys-rich motif were possibly associated with coordination of the Fe-S cluster. In the present study, we have investigated the roles of the GAF and central domains in the regulatory function of VnfA using truncated variants: DN15 VnfA and DGAF VnfA that lack the N-terminal 15 residues and whole GAF domain, respectively, and GAF VnfA consisting of only the GAF domain. DN15 VnfA and DGAF VnfA lost the ability to bind the Fe-S cluster, whereas GAF VnfA was still able to bind to the cluster, consistent with the hypothesis that the Cysrich motif is essential for Fe-S cluster binding. The GAF domain showed an inhibitory effect on the transcriptional activity of VnfA, which was reversed in the presence of the Fe-S cluster, and reactivated upon disassembly of the cluster. The inhibitory activity of the GAF domain acts on the NTPase activity of the central domain, whereas the binding ability of VnfA to DNA was not significantly affected, when VnfA retains its tetrameric conformation. The results imply that a major pathway, by which VnfA function is regulated, operates via the control of NTPase activity by the GAF domain. Structured digital abstractl VnfA binds to VnfA by molecular sieving (View Interaction 1, 2) Database VnfA has been submitted to the Swiss-Prot database under accession number: C1DI41.

Complete genome sequence of Anabaena variabilis ATCC 29413

“…Tischer, in 1964 in Mississippi, who called it Anabaena flos-aquae MSU A-3 7 [1]. It was submitted to the Indiana University Culture Collection ( Anabaena flos-aquae IUCC 1444) and was then submitted by C.P Wolk as Anabaena variabilis to ATCC in 1976 ( Anabaena variabilis ATCC 29413).…”

“…A. variabilis ATCC 29413 was first isolated as a freshwater strain in 1964 in Mississippi by R.G. Tischer, who called the strain Anabaena flos-aquae A-37 [1]. He was primarily interested in the extracellular polysaccharide produced by this strain [2-4], which was subsequently called Anabaena variabilis by Healey in 1973 [5].…”

Regulation of V‐nitrogenase genes in Anabaena variabilis by RNA processing and by dual repressors

“…In A. vinelandii, the vnf genes are regulated by a transcriptional activator VnfA (Walmsley et al, 1994;Nakajima et al, 2010;Yoshimitsu et al, 2011). Little is known about the regulation of nitrogenase gene expression in cyanobacteria except that the genes are expressed late in heterocyst differentiation and that there are no genes similar to the NifA/VnfA activators that are present in the Proteobacteria.…”

Fe–S proteins that regulate gene expression

“…While both apo- and holo-forms bind target DNA, holo-VnfA exhibits increased ATPase activity that, in turn, is required for VnfA to activate transcription. In contrast, in the absence of the [3Fe-4S] cluster, the GAF domain inhibits ATPase activity and thus VnfA-mediated regulation [184]. Therefore, the presence of the cluster is proposed to block inhibition by the GAF domain.…”