Download Metabolic regulation of nitrogen fixation in Rhodospirillum rubrum

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Biochemical Society Transactions (2006) Volume 34, part 1
Metabolic regulation of nitrogen fixation in
Rhodospirillum rubrum
H. Wang and A. Norén1
Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden
Abstract
Nitrogenase activity in Rhodospirillum rubrum is post-translationally regulated by DRAG (dinitrogenase
reductase glycohydrolase) and DRAT (dinitrogenase reductase ADP-ribosylation transferase). When a sudden
increase in fixed nitrogen concentration or energy depletion is sensed by the cells, DRAG is inactivated and
DRAT activated. We propose that the regulation of DRAG is dependent on its location in the cell and the
presence of an ammonium-sensing protein.
Introduction
Rhodospirillum rubrum is a photosynthetic, nitrogen-fixing
bacterium within the group of α-proteobacteria, which has
the capacity to survive under a wide range of environmental
conditions. Nitrogenase, which catalyses the nitrogen fixation reaction, is expressed when cells are grown anaerobically
in the light with a carbon source (i.e. malate) but also
under other conditions, e.g. fermentative growth in the dark.
Reduction of N2 to ammonia is a highly energy-demanding
reaction which needs to be tightly regulated in the cell. In
R. rubrum, nitrogenase is regulated both at the transcriptional
as well as at the metabolic level. The metabolic regulation is
a modification of one of the two subunits of dinitrogenase
reductase, which has been shown to be an ADP-ribosylation
of Arg101 , and, subsequently, nitrogenase becomes reversibly
inactivated [1,2]. The short-term inactivation of nitrogenase
is carried out by DRAT (dinitrogenase reductase ADPribosylation transferase) and the reversible activation/
demodification by DRAG (dinitrogenase reductase glycohydrolase) in a ‘switch-off/on’ mode. A similar system
for short-term regulation of nitrogenase has also been
found in other photosynthetic bacteria as well as species of
Azospirillum and archaea (Methanococcus).
The ‘switch-off’ effect
A nitrogen-fixing R. rubrum culture is not modified/
‘switched-off’ until a sudden change in fixed nitrogen (increased concentrations) or energy status appears. Neilson and
Nordlund [3] showed that upon addition of low concentrations of ammonium, asparagine or glutamine, nitrogenase
was reversibly inactivated within minutes, and this phenomenon was also observed in later studies when R. rubrum cells
Key words: dinitrogenase reductase activating glycohydrolase (DRAG), membrane association,
dinitrogenase reductase ADP-ribosylation transferase (DRAT), nitrogen fixation, nitrogenase,
Rhodospirillum rubrum.
Abbreviations used: DRAG, dinitrogenase reductase glycohydrolase; DRAT, dinitrogenase
reductase ADP-ribosylation transferase.
1
To whom correspondence should be addressed (email [email protected]).
C 2006
Biochemical Society
were subjected to darkness or NAD+ [4]. The regulatory
cycle is shown in Figure 1.
Regulation of the DRAG/DRAT system
These two enzymes are encoded by genes that are within the
same operon (draT/draG/draB) and the expressed proteins
have to work in a reciprocal manner; therefore it is postulated
that both DRAG and DRAT themselves have to be posttranslationally regulated in vivo, but the signal transduction
pathways involved in the regulation of DRAG and DRAT
have not yet been identified in detail. Upon cell breakage,
the regulation is lost and both DRAG and DRAT are found
to be always active. No in vitro experiments have been able to
show inactivation of active DRAG. We have shown previously
that the internal pool of NAD+ is of importance for the regulation of DRAT since NAD+ can act as a switch-off effector
and the NAD(P)+ concentration increases when effectors like
glutamine and ammonium are added to the cells, or they are
grown in the dark [4]. The regulation of DRAG/DRAT is
surprisingly observed in a mutant of Klebsiella pneumoniae
harbouring the R. rubrum draG/draT genes on a plasmid;
thus it is likely that the DRAT/DRAG regulatory molecules/
proteins are present in K. pneumoniae and are of a more
prevalent nature among nitrogen-fixing organisms [6]. We are
presently focusing on the regulation of DRAG in R. rubrum
and postulate that the interaction with the chromatophore
membrane is part of the mechanism for DRAG regulation.
PII proteins in R. rubrum
An important nitrogen and carbon signal transducer protein
in R. rubrum is, as in many other organisms, the PII protein encoded by the glnB gene. Zhang et al. [7] have identified
a role for the three PII -like proteins in R. rubrum, GlnB, GlnJ
and GlnK. GlnB is required for activation of NifA activity,
whereas GlnJ and GlnK do not appear to be involved in this
process. GlnJ expression is regulated by the NtrBC two-component regulatory system, whereas the expression of GlnB
and GlnK is NtrBC-independent. glnB is co-transcribed
with glnA, glnJ with amtB1, and glnK with amtB2. Mutations
The 11th Nitrogen Cycle Meeting 2005
Figure 1 Regulatory cycle of dinitrogenase reductase (data taken from [5])
of glnB, glnJ and glnK in all combinations were constructed
by Zhang et al. [7] and revealed that GlnB, GlnJ or both
were involved in the regulation of the DRAT/DRAG system
and surprisingly this included both the response to fixed
nitrogen (ammonium) and energy (dark) switch off. All three
PII proteins show high amino acid sequence similarity to each
other (64–69%).
Membrane association of DRAG
DRAG is a well-characterized protein that has been purified
and sequenced, but still the regulation of DRAG is not yet
clarified. Two models of regulation have been put forward
and both of those include protein–protein interactions as
a mode of regulation. We are advocating an interaction
with a membrane protein, based on the fact that DRAG is
always found associated with the membrane when the cells
are subjected to switch-off and subsequently harvested and
broken. DRAG is considered as a peripheral membrane protein since it is easily removed by 0.5 M NaCl and the sequence
does not reveal any strong hydrophobic stretches, indicating
that there is no transmembrane helix structure present. The
addition of guanine nucleotides can also dissociate the protein from the membrane [8], and Halbleib and Ludden [9]
also showed the re-association of DRAG with DRAG-free
chromatophore membranes. Furthermore, if DRAG activity
is regulated by association with the chromatophores, as we
suggest, a specific membrane-protein interaction is likely to
occur. Results from liposome–DRAG interaction studies by
Halbleib and Ludden [9] support a protein–protein inter-
action, since no lipid interactions with DRAG could be observed. Two specific high-molecular-mass protein complexes
are formed when treating DRAG-containing chromatophores with chemical cross-linkers. These complexes have
been found to react with anti-DRAG antibodies in Westernblot experiments [10]. Localization studies of DRAG in
R. rubrum show that the protein is sequestered to the membrane during switch-off but this sequestration also is affected
by other proteins involved in nitrogen metabolism. Mutant
studies confirm the reversibility of the DRAG membrane
association in R. rubrum.
References
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Received 23 September 2005
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