Effect of perturbation of ATP level on the activity and regulation of nitrogenase in Rhodospirillum rubrum

Abstract

Nitrogenase activity in Rhodospirillum rubrum and in some other photosynthetic bacteria is regulated in part by the availability of light. This regulation is through a posttranslational modification system that is itself regulated by P(II) homologs in the cell. P(II) is one of the most broadly distributed regulatory proteins in nature and directly or indirectly senses nitrogen and carbon signals in the cell. However, its possible role in responding to light availability remains unclear. Because P(II) binds ATP, we tested the hypothesis that removal of light would affect P(II) by changing intracellular ATP levels, and this in turn would affect the regulation of nitrogenase activity. This in vivo test involved a variety of different methods for the measurement of ATP, as well as the deliberate perturbation of intracellular ATP levels by chemical and genetic means. To our surprise, we found fairly normal levels of nitrogenase activity and posttranslational regulation of nitrogenase even under conditions of drastically reduced ATP levels. This indicates that low ATP levels have no more than a modest impact on the P(II)-mediated regulation of NifA activity and on the posttranslational regulation of nitrogenase activity. The relatively high nitrogenase activity also shows that the ATP-dependent electron flux from dinitrogenase reductase to dinitrogenase is also surprisingly insensitive to a depleted ATP level. These in vivo results disprove the simple model of ATP as the key energy signal to P(II) under these conditions. We currently suppose that the ratio of ADP/ATP might be the relevant signal, as suggested by a number of recent in vitro analyses.

FIG. 1.

In vivo analysis of ATP levels in R. rubrum strain UR2379 (wild type with plasmid pUX2524 carrying the luciferase reporter system). (A) Effect of the concentration of polymyxin B on the RLU at 4 mM luciferin. The values are normalized to the value obtained when polymyxin B concentration was at 0 mM. (B) Effect of the concentration of luciferin on the RLU in the absence of polymyxin B. The values are normalized to the maximum value obtained, when the luciferin concentration was 4 mM. Aliquots (10 μl) of cells were mixed with 100 μl of luciferase assay buffer solution, and the luminescence signal was measured with a luminometer for 2 min. Each point represents an average of at least three replicate assays, with a standard deviation of <10%.

FIG. 2.

Effect of FCCP and CCCP on ATP pools (A) and nitrogenase activity (B) in R. rubrum strain UR2379 (wild type carrying the luciferase reporter system). At time zero, FCCP (○) or CCCP (□) was added to MG-grown cultures at final concentrations of 25 and 30 μM, respectively. At the times indicated, 1-ml aliquots of cells were withdrawn anaerobically and assayed under illumination for nitrogenase activity for 2 min. Initial nitrogenase activity (100%) in UR2379 was ∼700 nmol of ethylene produced per h per ml of cells at an OD₆₀₀ of 1.0. Similarly, 10-μl aliquots of cells were mixed with 100 μl of luciferase assay buffer, and the luminescence signal was measured with a luminometer. The initial ATP concentration is ∼85,000 RLU/ml of cells at an OD₆₀₀ of 1.0. Each point represents an average of at least three replicate assays, with standard deviation of <10%.

FIG. 3.

Changes in nitrogenase activity and in vivo ATP concentration of R. rubrum UR2379 (wild type carrying the luciferase reporter system) in response to dark-light shifts (panel A) or the addition of NH₄⁺ (B). (A) UR2379 was grown in MG medium for about 2 days, and cells were shifted to dark at time zero and returned to light at 60 min. The nitrogenase activity (•) and ATP concentration (○) were measured as described in Fig. 2. Similar initial nitrogenase activity and ATP concentration were seen as in Fig. 2. (B) UR2379 was grown in MN⁻ medium for about 2 days, and NH₄Cl was added at final concentration of 10 mM at time zero. The nitrogenase activity (•) and ATP concentration (○) was measured as described in Fig. 2. The initial nitrogenase activity (100%) in UR2379 was ∼200 nmol of ethylene produce per h per ml of cells at an OD₆₀₀ of 1.0. The initial ATP concentration was ∼76,000 RLU per ml of cells at an OD₆₀₀ of 1.0.

FIG. 4.

Growth curve of UR2 (wild type) (•), UR2187 (purE mutant) (□), and UR2188 (purE mutant) (○) in MG with different concentrations of adenine: 0 mM (A), 0.05 mM (B), 0.1 mM (C), and 0.5 mM (D).

FIG. 5.

Western immunoblots of GlnB in UR2 (wild type) and UR2187 (purE mutant). Cells were grown in MG medium with 0.1 mM adenine for about 2 days, with OD₆₀₀ at 2.0 to 2.5. Then, 1-ml portions of the same culture were rapidly extracted by TCA precipitation of derepressed cultures of UR2 and UR2187 (lane 1), 60 min after a shift to dark (lane 2), and 10 min after a return to light (lane 3). Protein samples were loaded on low cross-linker SDS-Tricine gels and immunoblotted with antibody to R. rubrum GlnB. Arrow M indicates the position of the modified subunit, and arrow U indicates the position of the unmodified subunit.