The poor growth of <i>Rhodospirillum rubrum</i> mutants lacking P<sub>II</sub> proteins is due to an excess of glutamine synthetase activity

Zhang, Yaoping; Pohlmann, Edward L.; Conrad, Mary; Roberts, Gary P.

doi:10.1111/j.1365-2958.2006.05251.x

Cited by 21 publications

(18 citation statements)

References 94 publications

(144 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…We randomly mutagenized a cbbM mutant (UR5251) with the Tn5 transposon and selected for fast-growing mutants in MG liquid medium under anaerobic conditions. The sites of the Tn5 insertion in 10 suppressors were identified by DNA sequencing as described previously (33,71), and 8 out of 10 Tn5 insertions were found to be located in different sites in cbbR, cbbF, or cbbP ( Table 2). This result strongly suggests that the disruption of other cbb genes in the CBB cycle can restore normal growth to a cbbM mutant under these anaerobic, photoheterotrophic growth conditions.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate

et al. 2011

Self Cite

View full text Add to dashboard Cite

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first step of CO 2 fixation in the Calvin-Benson-Bassham (CBB) cycle. Besides its function in fixing CO 2 to support photoautotrophic growth, the CBB cycle is also important under photoheterotrophic growth conditions in purple nonsulfur photosynthetic bacteria. It has been assumed that the poor photoheterotrophic growth of RubisCO-deficient strains was due to the accumulation of excess intracellular reductant, which implied that the CBB cycle is important for maintaining the redox balance under these conditions. However, we present analyses of cbbM mutants in Rhodospirillum rubrum that indicate that toxicity is the result of an elevated intracellular pool of ribulose-1,5-bisphosphate (RuBP). There is a redox effect on growth, but it is apparently an indirect effect on the accumulation of RuBP, perhaps by the regulation of the activities of enzymes involved in RuBP regeneration. Our studies also show that the CBB cycle is not essential for R. rubrum to grow under photoheterotrophic conditions and that its role in controlling the redox balance needs to be further elucidated. Finally, we also show that CbbR is a positive transcriptional regulator of the cbb operon (cbbEFPT) in R. rubrum, as seen with related organisms, and define the transcriptional organization of the cbb genes.The purple nonsulfur photosynthetic bacterium Rhodospirillum rubrum is metabolically diverse and can grow under photoautotrophic, photoheterotrophic, and chemoheterotrophic conditions. It can use different kinds of nitrogen and carbon sources, including N 2 and CO 2 , through effective metabolic systems such as the nitrogenase and the Calvin-BensonBassham (CBB) cycles (2,17,21,30). Both the nitrogenase system and the CBB cycle are very energy-demanding processes and are therefore usually tightly regulated and very sensitive to environmental signals (12,20,38,40,47).The main role of the CBB cycle is to fix CO 2 into organic carbon under photoautotrophic conditions where CO 2 serves as the sole carbon source. Thus, most cbb genes have their highest expression levels under these conditions (2,15,28,36,47). However, under photoheterotrophic conditions in the presence of organic carbon such as malate or acetate, the CBB cycle also functions as a major electron sink in many photosynthetic bacteria, and it is assumed that this property maintains the redox balance in the cell (18,24,34,57,61,62,67). Recently, it was shown that the CBB cycle acts as an electronaccepting process to recycle the excess reduced cofactors under non-N 2 -fixing conditions in Rhodopseudomonas palustris (39). Thus, an R. rubrum cbbM mutant in which the CBB cycle is blocked by the elimination of its key enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) (encoded by cbbM), not only fails to grow under photoautotrophic conditions but also grows poorly under photoheterotrophic conditions (66). Similar phenotypes have been seen for RubisCOdeficient strains of Rhodobacter sphaeroides and Rhodob...

show abstract

Section: Resultsmentioning

confidence: 99%

“…Nx r Gm r Km r colonies were purified on SMN selection plates and then grown in MG medium again to verify their photoheterotrophic growth. The sites of the Tn5 insertions in 10 transconjugants were identified by DNA sequencing, as described previously (33,71).…”

Section: Methodsmentioning

confidence: 99%

The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate

et al. 2011

Self Cite

View full text Add to dashboard Cite

show abstract

“…This regulatory cascade is not even conserved in other enterobacteria (36). In Rhodospirillum rubrum, the other model system for which the metabolic defect is known, the poor-growth phenotype is due to an excess of GS activity (47). In S. elongatus, the gene that encodes GS (glnA) is activated by NtcA and GS activity is associated with P II deficiency in S. elongatus.…”

Section: Discussionmentioning

confidence: 99%

Mutations atpipXSuppress Lethality of P_II-Deficient Mutants ofSynechococcus elongatusPCC 7942

et al. 2009

View full text Add to dashboard Cite

The P II proteins are found in all three domains of life as key integrators of signals reflecting the balance of nitrogen and carbon. Genetic inactivation of P II proteins is typically associated with severe growth defects or death. However, the molecular basis of these defects depends on the specific functions of the proteins with which P II proteins interact to regulate nitrogen metabolism in different organisms. In Synechococcus elongatus PCC 7942, where P II forms complexes with the NtcA coactivator PipX, attempts to engineer P II -deficient strains failed in a wild-type background but were successful in pipX null mutants. Consistent with the idea that P II is essential to counteract the activity of PipX, four different spontaneous mutations in the pipX gene were found in cultures in which glnB had been genetically inactivated.Cyanobacteria are phototrophic organisms that perform oxygenic photosynthesis. Autotrophic growth requires the constant assimilation of ammonium via the coordinated action of glutamine synthetase (GS) and glutamate synthase, also known as the GS-GOGAT cycle, resulting in consumption of 2-oxoglutarate (34). Due to the lack of 2-oxoglutarate dehydrogenase in cyanobacteria, synthesis of 2-oxoglutarate represents the final step in the oxidative branch of the trichloroacetic acid cycle and directly links 2-oxoglutarate levels to nitrogen assimilation (35). Thus, 2-oxoglutarate accumulates during nitrogen starvation, making this metabolite an excellent indicator of the intracellular carbon-nitrogen balance (12,25).In cyanobacteria, multiple metabolic and developmental processes are induced by nitrogen starvation. NtcA, the global regulator for nitrogen control, activates genes involved in nitrogen assimilation, heterocyst differentiation, and acclimation to nitrogen starvation (20,30,41). 2-Oxoglutarate, the signal of nitrogen deficiency, stimulates binding of NtcA to target sites (45), transcription activation in vitro (44), and complex formation between the global nitrogen regulator NtcA and its coactivator factor PipX, a regulatory protein conserved in cyanobacteria (5, 9). The interaction between PipX and NtcA is known to be relevant for maximal activation of NtcA-dependent genes under nitrogen limitation (9, 10). PipX-deficient cultures of Synechococcus elongatus PCC 7942 showed reduced activity of nitrogen assimilation enzymes, retarded nitrogen induction, a slower rate of nitrate consumption, and when subjected to nitrogen starvation, retarded phycobilisome degradation and faster reduction of the chlorophyll content (10).The homotrimeric P II protein is one of the most conserved and widespread signal transduction proteins in nature and plays key roles in nitrogen assimilatory processes (28). P II proteins contain three binding sites (one per subunit) for 2-oxoglutarate and ATP, and their primary function is to regulate, by direct protein-protein interactions, the activity of proteins implicated in nitrogen metabolism (reviewed in reference 13). In cyanobacteria, several proteins are k...

show abstract

“…The luminescence was measured with a TD-20/20 luminometer (Turner Biosystems, Sunnyvale, CA) with a 2-s delay period and a 10-s integration period. The ATP concentration was expressed as nmol per mg of dry weight, based on previous data that the dry weight of R. rubrum was 0.28 Ϯ 0.01 mg per ml of culture at an optical density at 600 nm (OD 600 ) of 1 (76).…”

Section: Methodsmentioning

confidence: 99%

Effect of Perturbation of ATP Level on the Activity and Regulation of Nitrogenase in Rhodospirillum rubrum

2009

Self Cite

View full text Add to dashboard Cite

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.The P II family of regulatory proteins is one of the most broadly distributed in nature, being found in all three domains of life (1,20,39,51). P II was originally found to be involved in the regulation of glutamine synthetase (GS) activity by interaction with adenylyltransferase (ATase or GlnE, encoded by glnE) (63, 65). However, P II has subsequently been shown to regulate many other processes involving nitrogen assimilation and metabolism, by directly interacting with different proteins, termed P II receptors (51). In many organisms, more than one P II homolog has been identified. The "housekeeping" homolog is usually referred to as GlnB, encoded by glnB, with other secondary homologs named GlnK, GlnK 2 , GlnJ, GlnY, GlnZ, or Pz (1,12,47,69,78). In a few species of Archaea, a distinct group of P II proteins was named NifI, which is encoded by the nifH-linked gene (39).Because of its physiological diversity, the photosynthetic bacterium Rhodospirillum rubrum is a good model organism in which to study the role of P II . It has three P II homologsGlnB, GlnK, and GlnJ-that play distinct and overlapping functions by interacting with at least six receptors in the cell (71,78,81). Some of these are common to other bacteria, such as GlnE (ATase) that is involved in the regulation for GS activity, the two-component regulatory protein NtrB, and the ammonium transporter (gas channel) A...

show abstract

The poor growth of Rhodospirillum rubrum mutants lacking P_II proteins is due to an excess of glutamine synthetase activity

Cited by 21 publications

References 94 publications

The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate

The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate

Mutations atpipXSuppress Lethality of P_II-Deficient Mutants ofSynechococcus elongatusPCC 7942

Effect of Perturbation of ATP Level on the Activity and Regulation of Nitrogenase in Rhodospirillum rubrum

Contact Info

Product

Resources

About

The poor growth of Rhodospirillum rubrum mutants lacking PII proteins is due to an excess of glutamine synthetase activity

Cited by 21 publications

References 94 publications

The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate

The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate

Mutations atpipXSuppress Lethality of PII-Deficient Mutants ofSynechococcus elongatusPCC 7942

Effect of Perturbation of ATP Level on the Activity and Regulation of Nitrogenase in Rhodospirillum rubrum

Contact Info

Product

Resources

About

The poor growth of Rhodospirillum rubrum mutants lacking P_II proteins is due to an excess of glutamine synthetase activity

Mutations atpipXSuppress Lethality of P_II-Deficient Mutants ofSynechococcus elongatusPCC 7942