Role of Escherichia coli Nitrogen Regulatory Genes in the Nitrogen Response of the Azotobacter vinelandii NifL-NifA Complex

Abstract

The redox-sensing flavoprotein NifL inhibits the activity of the nitrogen fixation (nif)-specific transcriptional activator NifA in Azotobacter vinelandii in response to molecular oxygen and fixed nitrogen. Although the mechanism whereby the A. vinelandii NifL-NifA system responds to fixed nitrogen in vivo is unknown, the glnK gene, which encodes a PII-like signal transduction protein, has been implicated in nitrogen control. However, the precise function of A. vinelandii glnK in this response is difficult to establish because of the essential nature of this gene. We have shown previously that A. vinelandii NifL is able to respond to fixed nitrogen to control NifA activity when expressed in Escherichia coli. In this study, we investigated the role of the E. coli PII-like signal transduction proteins in nitrogen control of the A. vinelandii NifL-NifA regulatory system in vivo. In contrast to recent findings with Klebsiella pneumoniae NifL, our results indicate that neither the E. coli PII nor GlnK protein is required to relieve inhibition by A. vinelandii NifL under nitrogen-limiting conditions. Moreover, disruption of both the E. coli glnB and ntrC genes resulted in a complete loss of nitrogen regulation of NifA activity by NifL. We observe that glnB ntrC and glnB glnK ntrC mutant strains accumulate high levels of

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

“…NifL, NifA, and their mutant forms were expressed on a second plasmid from a constitutive promoter (32). The ␤-galactosidase assays were carried out as described (29,35) Protein Expression and Purification. Plasmids pTJ45, pDB737, and pIM15 were used for overexpression of N his6 NifL, NifA, and NifA-E356K (28)(29)(30).…”

“…However, the activity of NifA-E356K is constitutive and insensitive to NifL (Table 1, compare first and second rows). NifA-E356K exhibits considerably higher activity than wild-type NifA under anaerobic nitrogen-limiting conditions because NifL retains some inhibitory activity under these conditions (31,35). The nifL-R306C mutation effectively suppressed the constitutive activity of nifA-E356K, although a low level of NifA activity was detectable under anaerobic, nitrogen-limiting conditions.…”

“…1) was chosen to seek suppressor mutations because position 356 is conserved in NifA proteins but not in other members of the 54 -dependent activator family. To carry out the screening, we used a previously described twoplasmid system in E. coli, consisting of a nifHp-lacZ reporter and a second plasmid expressing nifL and nifA from a constitutive promoter (29,31,32,35). After random PCR mutagenesis of the region encoding the central and C-terminal domains of NifL and introduction of the mutant library into the nifA-E356K background, we screened for suppressors on 20 g͞ml 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside indicator plates containing excess fixed nitrogen under aerobic conditions.…”

The Amino-terminal GAF Domain of Azotobacter vinelandii NifA Binds 2-Oxoglutarate to Resist Inhibition by NifL under Nitrogen-limiting Conditions

“…A mutant form of Av GlnK, GlnK E44C, a 3 above). Lanes 3,6,9,12,15,18, and 21 contained NifA and ATP incubated for t ϭ 0, 2, 6, 10, 30, 60, and 100 min, respectively. Lanes 4,7,10,13,16,19, and 22 contained NifA, ATP, and 2-oxoglutarate incubated for t ϭ 0, 2, 6, 10, 30, 60, and 100 min, respectively.…”

“…The response to 2-oxoglutarate in vitro is within the physiological range observed in Escherichia coli, which varies from ϳ100 M under conditions of nitrogen excess to ϳ1 mM under conditions of nitrogen limitation (17). When expressed in E. coli, the A. vinelandii NifL-NifA system is also apparently responsive to the 2-oxoglutarate concentration in vivo (18). 2-Oxoglutarate is an intermediate in the tricarboxylic acid cycle and provides the carbon skeleton for nitrogen assimilation; hence 2-oxoglutarate signals the carbon status (19).…”