Distinct Roles of P _II -Like Signal Transmitter Proteins and amtB in Regulation of nif Gene Expression, Nitrogenase Activity, and Posttranslational Modification of NifH in Azoarcus sp. Strain BH72

Abstract

P II -like signal transmitter proteins, found in Bacteria, Archaea, and plants, are known to mediate control of carbon and nitrogen assimilation. They indirectly regulate the activity of key metabolic enzymes and transcription factors by protein-protein interactions with signal transduction proteins. Many Proteobacteria harbor two paralogous P II -like proteins, GlnB and GlnK, whereas a novel third P II paralogue (GlnY) was recently identified in Azoarcus sp. strain BH72, a diazotrophic endophyte of grasses. In the present study, evidence was obtained that the P II -like proteins have distinct roles in mediating nitrogen and oxygen control of nif gene transcription and nitrogenase activity. Full repression of nif gene transcription in the presence of a combined nitrogen source or high oxygen concentrations was observed in wild-type and glnB and glnK knockout mutants, revealing that GlnB and GlnK can complement each other in mediating the repression. In contrast, in a glnBK double mutant strain in the presence of only GlnY, nif gene transcription was still detectable, albeit at a lower level, on nitrate or 20% oxygen. As another level of control, nitrogenase activity was regulated by at least three types of mechanisms in strain BH72: covalent modification of dinitrogenase reductase (NifH), probably by ADP-ribosylation, and two other, unknown means. Functional inactivation upon ammonium addition (switchoff) required the putative high-affinity ammonium transporter AmtB and GlnK, but not GlnB or GlnY. Functional inactivation in response to anaerobiosis did not depend on AmtB, GlnK, or GlnB. In contrast, covalent modification of NifH required both GlnB and GlnK and AmtB as response to ammonium addition, whereas it required either GlnB or GlnK and not AmtB when cells were shifted to anaerobiosis. In a glnBK double mutant expressing only GlnY, NifH modification was completely abolished, further revealing functional differences between the three P II paralogues.

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

“…This model is also consistent with the previous data on the system: (i) DRAG has been detected in association with the membrane fraction of R. rubrum cells in which DRAG activity is downregulated (Norén & Nordlund, 1997); (ii) in A. brasilense, the reactivation of DRAG activity was altered in a mutant lacking GlnZ, a GlnK-like protein (Klassen et al, 2001); (iii) in R. capsulatus, DRAT interacts with both GlnB and GlnK in the yeast two-hybrid system (Pawlowski et al, 2003) and we have detected similar interactions between DRAT and all three P II homologues of R. rubrum (Zhu et al, 2006). Thus, although we specifically propose this scheme for the R. rubrum system, it is consistent with results seen with Azoarcus, R. capsulatus and Azospirillum brasilense as well, in which the DRAT-DRAG regulation is altered in mutants lacking P II or AmtB Klassen et al, 2001;Martin & Reinhold-Hurek, 2002;Yakunin & Hallenbeck, 2002). We note that rather different results have been seen in the case of R. capsulatus, where mutants lacking two AmtB homologues (AmtB and AmtY) affected the DRAT-DRAG response to NH z 4 , but not to darkness; the basis for this difference is unclear (Yakunin & Hallenbeck, 2002).…”

“…It has been proposed that the co-production of GlnK and AmtB could prevent AmtB from sequestering GlnB under nitrogen-deficient conditions, allowing GlnB to play its necessary roles in the cell (Blauwkamp & Ninfa, 2003). Mutations in amtB of Rhodobacter capsulatus and Azoarcus also show dramatic effects on the regulation of nitrogenase activity by NH z 4 , and it has been proposed that AmtB may be an ammonium sensor (Javelle et al, 2004;Martin & Reinhold-Hurek, 2002;Yakunin & Hallenbeck, 2002), but the effect might be indirect, since mutants lacking P II also showed an altered NH z 4 response Martin & Reinhold-Hurek, 2002). Given the centrality of P II to nitrogen regulation, the notion that AmtB might exert its effects through P II interaction is certainly attractive.…”

Horizontal transfer of the nitrogen fixation gene cluster in the cyanobacterium Microcoleus chthonoplastes

“…In addition, the nifcluster contains two genes proposed to be involved in nitrogen regulation, P-II and P-II', (Martin and Reinhold-Hurek, 2002) and a gene encoding a ferredoxin protein (fd) mediating electron transfer (Rubio and Ludden, 2008). Two copies of nifB were found, of which the first seems to be a partial and possibly non-functional gene (nifB').…”

Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus

“…rubrum, Azoarcus sp. strain BH72 and Azospirillum brasilense (Klassen et al, 2001;Martin & Reinhold-Hurek, 2002;Zhang et al, 2000Zhang et al, , 2001a. In all cases, the P II signal transduction proteins play an important role in regulating not only synthesis of nitrogenase but also activity of the enzyme complex in response to the cellular nitrogen status.…”

“…rubrum as well as in Azoarcus sp. BH72, GlnB and one of the two GlnKlike proteins (GlnJ and GlnK, respectively) are involved in regulation of DraT/DraG activity, whereas the second GlnK-like protein apparently has no function in this regulatory mechanism (Martin & Reinhold-Hurek, 2002;Zhang et al, 2001b).…”