Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum

Abstract

Homologs of ntrB and ntrC genes from Rhodospirillum rubrum were cloned and sequenced. A mutant lacking ntrBC was constructed, and this mutant has normal nitrogenase activity under nif-derepressing conditions, indicating that ntrBC are not necessary for the expression of the nif genes in R. rubrum. However, the posttranslational regulation of nitrogenase activity by ADP-ribosylation in response to NH 4 ؉ was partially abolished in this mutant. More surprisingly, the regulation of nitrogenase activity in response to darkness was also affected, suggesting a physiological link between the ntr system and energy signal transduction in R. rubrum. The expression of glutamine synthetase, as well as its posttranslational regulation, was also altered in this ntrBC mutant.In enteric bacteria the general nitrogen regulation (ntr) system controls a variety of nitrogen assimilation pathways (29). The ntr system involves the products of at least five genes: ntrA, ntrB, ntrC, glnB, and glnD. The products of ntrB and ntrC belong to the family of two-component regulators: NTRB is a histidine kinase that phosphorylates NTRC under nitrogenlimiting conditions, and the phosphorylated form of NTRC then acts as transcriptional activator of glnA (encoding glutamine synthetase [GS]) and other operons involved in nitrogen assimilation.Homologs of the ntrBC genes have been found in many nitrogen-fixing bacteria, and their roles in nitrogen fixation have been best characterized in Klebsiella pneumoniae. In this organism, NTRB and NTRC are required for transcription of nifLA regulatory genes, with NIFA activating the transcription of other nif operons (25). In some diazotrophs, such as Azotobacter vinelandii, Bradyrhizobium japonicum, and Azospirillum brasilense, NTRB and NTRC are not essential for nif gene expression, but the mutations in ntrBC have detectable effects on some other aspects of nitrogen assimilation, such as nitrate utilization and GS activity (20,22,35).Rhodospirillum rubrum is a purple nonsulfur, photosynthetic, nitrogen-fixing bacterium, and the posttranslational regulatory system that regulates nitrogenase activity is best characterized in this organism. This regulatory system involves reversible mono-ADP-ribosylation of dinitrogenase reductase, and regulation is performed by two enzymes: dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductaseactivating glycohydrolase (DRAG). DRAT modifies dinitrogenase reductase and thereby inactivates the enzyme. DRAG removes the ADP-ribose from dinitrogenase reductase and restores its activity (21).The DRAT-DRAG system has been characterized in other nitrogen-fixing bacteria, such as A. brasilense (9, 42) and Rhodobacter capsulatus (24), and it negatively regulates nitrogenase activity in response to exogenous NH 4 ϩ or to energy limitation in the form of darkness (in the cases of R. rubrum and R. capsulatus) or anaerobiosis (in A. brasilense). Both DRAT and DRAG activities are themselves subject to posttranslational regulation under these conditions (10,14,19,...

GlnD Is Essential for NifA Activation, NtrB/NtrC-Regulated Gene Expression, and Posttranslational Regulation of Nitrogenase Activity in the Photosynthetic, Nitrogen-Fixing Bacterium Rhodospirillum rubrum

“…Previous studies showed that NtrBC are not essential for nif expression in R. rubrum, indicating that R. rubrum has a different regulatory mechanism for nif expression from that seen in K. pneumoniae (59). In R. rubrum, transcription of nifA is not regulated but NifA activity is tightly controlled in response to NH 4 ϩ (62).…”

“…The NtrB-NtrC regulatory system is tightly controlled by the status of P II modification in E. coli (40). However, the NtrB-NtrC system is less well characterized in R. rubrum, although an ntrBC mutant has been examined (59). It was expected that some NtrB-NtrCregulated gene expression should be altered in R. rubrum glnD mutants.…”

Control of Nitrogenase Reactivation by the GlnZ Protein in Azospirillum brasilense

“…It is possible, therefore, that the PII protein is also involved in the signal pathway leading to the reversible ADP-ribosylation of the iron protein in A. brasilense. ntrBC mutants of A. brasilense (17,24) and R. rubrum (20) failed to inactivate the iron protein by ADP-ribosylation upon addition of NH 4 ϩ but did switch off under anaerobiosis and in darkness, respectively. These results suggested that the ammonium-and energy-triggered pathways for nitrogenase activity control are different.…”

“…Addition of L-methione-DL-sulfoximine (MSX), a specific glutamine synthetase inhibitor, to derepressed cells prevented nitrogenase switch-off by ammonium, suggesting that glutamine synthetase or glutamine may be part of the signal pathway to DRAT and DRAG (4,10,13,26). Moreover, ntrC mutants of A. brasilense and R. rubrum are incapable of ADPribosylation (17,20,25) (15) at 30°C at 120 rpm. Plasmid pJI1 was constructed by cloning a 0.37-kbp SacI-HindIII DNA fragment containing the A. brasilense glnZ gene into the vector pLAFR3.18.…”

Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides

“…With respect to NSP bacteria, previous studies had indicated that single nucleotide mutations occur at a number of key loci in genes that encode elements of the nitrogen regulatory cascade (Drepper et al, 2003;Hübner et al, 1993;Paschen et al, 2001;Wall & Gest, 1979;Zhang et al, 1995;Zinchenko et al, 1997), as well as at other places on the chromosome (Edgren & Nordlund, 2004;Kern et al, 1998;Masepohl et al, 2002). These changes alter basic control mechanisms resulting in potential derepression of the nitrogenase complex during normal repressive growth conditions.…”