Analysis of the promoters and upstream sequences of nifA1 aud nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN

Abstract

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. R. capsulatus nifA1 and nifA2 encode identical NIFA proteins that activate transcription of nifHDK and other nif genes. In this study, we report that nifA1-lacZ and nifA2-lacZ fusions are repressed in the presence of NH3 and activated to similar levels under nitrogen-deficient conditions. This nitrogen-controlled activation was dependent on R. capsulatus ntrC (which encodes a transcriptional activator) but not rpoN (which encodes an RNA polymerase sigma factor). We have used primer extension analyses of nifA1, nifA2 and nifH and deletion analyses of nifA1 and nifA2 upstream regions to define likely promoters and cis upstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed that ntrC but not rpoN is required for nifA1 and nifA2 activation, and that nifA1 and nifA2 do not possess typical RPON-activated promoters.

PIISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control

“…In K. pneumoniae, nifLA transcription is nitrogen regulated by NtrC and is therefore dependent on GlnB (52,95), while in Azotobacter vinelandii, nifLA transcription is independent of nitrogen sources (24,186). In both Herbaspirillum seropedicae and Rhodobacter capsulatus, nifA expression is regulated with respect to nitrogen by NtrC, and hence GlnB may be involved (57,72,215). By contrast, nifA expression in Azospirillum brasilense is independent of NtrC and only partially repressed by either ammonium or oxygen, maximal repression being achieved by the synergistic effect of both effectors (57).…”

New Roles for Conserved Regions within a ς54-dependent Enhancer-binding Protein

“…Third, there exists a distinct class of proteins that contain domains homologous to the central domain of 54 activators but activate forms of holoenzyme that lack 54 ; these have sequences that differ primarily in the C3 region (e.g. Rhodobacter capsulatus NtrC) (11,12). Mutations within C3 generally cause a lack of energy coupling; however, the proposed roles of individual conserved amino acids have varied when different activators were studied (1,2).…”

The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters

“…In the absence of both fixed nitrogen and oxygen, transcription of the genes encoding the nitrogenase polypeptides (nifHDK) and other nifgenes required for the synthesis of nitrogenase is activated by NifAl or NifA2 (25) at promoters recognized by the alternative sigma factor RpoN (CF54, ON, or NtrA). Induction of nifAl and nifA2 occurs when fixed nitrogen is limited and requires ntrC (nifRl) but not rpoN (nifR4) (14,29). R. capsulatus glnB is proposed to be a negative regulator of nif gene transcription, since glnB mutants express nitrogenase constitutively with respect to fixed nitrogen although oxygen repression is still present (22).…”

“…We decided to initially analyze the basis of the ntrC control at glnB by determining the transcription start site(s). RNA was isolated from R. capsulatus strains essentially as described previously (14). Strains were first cultured in the light in minimal medium plus 7.5 mM NH4' and then induced anaerobically overnight in minimal medium containing either NH4' (7.5 mM), glutamate (10 mM), or no fixed nitrogen.…”