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

Abstract

The AmtB protein transports uncharged NH 3 into the cell, but it also interacts with the nitrogen regulatory protein P II , which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P II homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to NH + 4 addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB 1 and amtB 2 , have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB 1 are defective in their response to both NH + 4 addition and darkness, while mutants lacking AmtB 2 show little effect on the regulation of nitrogenase activity. These responses to darkness and NH + 4 appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB 1 is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB 1 -P II regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P II is proposed. Other results demonstrate a degree of specificity in interaction of AmtB 1 with the different P II homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P II proteins regulate processes involved in nitrogen acquisition and utilization.

Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum: dependence on manganese, 2-oxoglutarate and the ADP/ATP ratio

“…It has been shown that an R. rubrum mutant lacking amtB1 has an impaired nitrogenase 'switch-off' response and fails to sequester GlnJ to the membrane (Wang et al, 2005;Zhang et al, 2006b). Also, a similar phenotype has been observed in an Azospirillum brasilense mutant lacking amtB (Huergo et al, 2006).…”

“…Based on this observation we assumed that the proportion of AmtB1 in the membrane was much larger than that of AmtB2, and therefore that the latter would not interfere with our analysis. In addition, AmtB2 has never been shown to have any role in GlnJ sequestration to the membrane (Zhang et al, 2006b). These considerations, together with the recent observation that PII mutants in R. rubrum can develop suppressor mutations (Zhang et al, 2006a), for instance in amtB1, led us to perform this study in wild-type cells rather than in a PII mutant strain.…”

“…In the case of R. rubrum, a number of studies have suggested that the regulation of nitrogen fixation and assimilation processes is intimately linked to cellular energy status (Zhang et al, 2001(Zhang et al, , 2006b. Different studies by several research groups have addressed this question by measuring the fluctuations of cellular adenine nucleotide pools (mainly ADP and ATP) upon addition of ammonium to nitrogenfixing cultures or after subjecting cells to darkness (Li et al, 1987;Nordlund & Höglund, 1986;Paul & Ludden, 1984).…”

“…In R. rubrum, three PII paralogues have been identified (Zhang et al, 2001), termed GlnB, GlnK and GlnJ, which are subjected to UMP modification at Tyr51 . Although the amino acid sequence of the three proteins is very similar, they show both distinct and overlapping functions in the cell, including post-translational regulation of nitrogenase and GS as well as activation of gene transcription (Wang et al, 2005;Zhang et al, 2001Zhang et al, , 2005Zhang et al, , 2006b. At the molecular level, the effect of PII proteins on the post-translational regulation of GS has been analysed in greater detail in R. rubrum .…”

“…The glnB gene is co-transcribed with glnA (the gene encoding GS) (Johansson & Nordlund, 1996), while both glnJ and glnK are linked to genes encoding putative ammonium transporters of the Amt family, amtB1 and amtB2, respectively (Zhang et al, 2006b). It has been shown that an R. rubrum mutant lacking amtB1 has an impaired nitrogenase 'switch-off' response and fails to sequester GlnJ to the membrane (Wang et al, 2005;Zhang et al, 2006b).…”