(Received for publication, July 22, 1994; and in revised form, November 11, 1994)
From the
The control of glutamine synthetase (GS), the first enzyme in
the main pathway used by Rhizobium meliloti to assimilate
ammonia, is central to cellular nitrogen metabolism. R. meliloti is unusual in having three distinct types of GS, including a
unique GS, GSIII, that differs considerably from both GSI, which
resembles other bacterial GS proteins and GSII, which resembles the GS
found in eukaryotes. We show here that GSIII can be
post-translationally modified in vivo by ADP-ribosylation at
an arginine residue. PO
attached to GSIII
during bacterial growth as part of the modifying group could be removed
by treatment with snake venom phosphodiesterase or by turkey
erythrocyte ADP-ribosylarginine hydrolase. Treatment of modified GSIII
with hydroxylamine at neutral pH releases a chromophore that has the
retention time of ADP-ribose when analyzed by reversed-phase high
performance liquid chromatography. ADP-ribosylation inhibits GSIII
activity.
Symbiotic nitrogen fixation takes place in root nodules that
form when roots of some legumes are infected by soil bacteria from the
genera Rhizobium, Azorhizobium, or Bradyrhizobium. In symbiosis, the bacteria receive carbon
compounds from the host plant, and instead of assimilating ammonia that
they produce by nitrogen fixation, they release the ammonia to the host
plant. This behavior differs from that of free-living, nitrogen-fixing
bacteria, which tightly couple nitrogen fixation and ammonia
assimilation. Ammonia assimilation in Rhizobium uses the
glutamate synthase cycle, in which glutamine synthetase (GS) ()acts in conjunction with glutamate synthase to produce
glutamate, at the expense of ATP and reducing power (Miflin and Lea,
1980). Glutamine and glutamate produced by these enzymes can be used in
the synthesis of other nitrogen-containing compounds. Regulation of the
GS-glutamate synthase pathway occurs primarily by changing the amount
or specific activity of GS, and, in enteric bacteria, the control of GS
synthesis and specific activity are central to the regulation of cell
nitrogen metabolism (Reitzer and Magasanik, 1987).
Many rhizobia differ from enteric bacteria by having more than one GS (Darrow, 1980). Rhizobium meliloti has three distinct GS proteins (DeBruijn et al., 1989; Shatters et al., 1993), and although these GS isozymes are regulated in response to available nitrogen sources, to oxygen concentration, and to the presence of the other enzymes, the contribution of each isozyme to total GS activity is still poorly understood.
GSI resembles the single GS found in enteric
bacteria in its structure, sequence, and regulation by
post-translational modification (Darrow, 1980; Somerville and Kahn,
1983). Escherichia coli glutamine synthetase is a classic
example of an enzyme whose activity is regulated post-translationally
(Rhee et al., 1985; Stadtman et al., 1990). Each
subunit of the dodecameric GS complex can be adenylylated at a single
tyrosine near the carboxyl terminus, and the bound AMP inhibits the
activity of that subunit. Adenylylation and deadenylylation of E.
coli GS is carried out by the P protein, which in
turn is regulated by proteins that ultimately are sensitive to the
ratio of glutamine to
-ketoglutarate (Reitzer and Magasanik,
1987). This regulatory cascade also controls GS synthesis at the level
of transcription by influencing the phosphorylation of NR
(NtrC), a transcriptional activator (Stock et al.,
1989). There is evidence that Rhizobium GSI is also modified,
probably by adenylylation (Darrow, 1980). GSI-type proteins in other
bacteria are also modified by adenylylation (Woehle et al.,
1990).
A second kind of modification, ADP-ribosylation, has recently been shown for Rhodospirillum rubrum GS, although the physiological importance of the modification is not yet clear (Woehle et al., 1990). ADP-ribosylation of proteins is common (Moss and Vaughn, 1990) and is involved in regulating enzyme activity, protein synthesis, cell proliferation, and both DNA and RNA metabolism. Arginine, cysteine, and glutamate residues can serve as ADP-ribose acceptors. Many ADP-ribosyl transferases are produced by parasites and are involved in disrupting host metabolism. For example, cholera toxin ADP-ribosylates an intestinal guanine nucleotide-binding regulatory protein (Fishman, 1990), activating an adenylate cyclase activity, disrupting ion transport, and ultimately leading to diarrhea. In bacteria, one of the best described modifications of a protein used within the cell is ADP-ribosylation of nitrogenase. Reversible ADP-ribosylation of a specific arginyl residue of the dinitrogenase reductase subunit inhibits nitrogenase activity when ammonia becomes available (Pope et al., 1985). Although E. coli GS is not ADP-ribosylated in vivo, Moss et al. (1990) have shown that the enzyme can be inactivated in vitro by ADP-ribosylation at arginine 172.
GSII, which is similar to eukaryotic GS enzymes in its structure and sequence (Carlson and Chelm, 1986; Shatters and Kahn, 1989), is regulated transcriptionally and possibly post-translationally (Manco et al., 1992). GSII enzymes are uncommon in bacteria and have been reported only in the plant associated bacteria, (Brady)rhizobium and Agrobacterium (Darrow and Knotts, 1977; Fuchs and Keister, 1980), and in actinomycetes, including Frankia, a genus that can carry out symbiotic nitrogen fixation with various plants (Edmands et al., 1987).
GSIII is a newly described type of GS (Espin et al., 1990, Shatters et al., 1993) that is abundant
in R. meliloti mutants that lack GSI and GSII. Purified GSIII
from R. meliloti has unusual kinetic properties, including a
very low affinity for ammonium and glutamate and no transferase
activity (Shatters et al., 1993). Analysis of the amino acid
sequence of GSIII (Chiurazzi et al., 1992) ()shows
that GSIII can be distinguished from both the GSI and GSII families of
GS proteins but that amino acids that are highly conserved in
comparisons between GSI and GSII proteins are also found in GSIII.
Transcription of the glnT gene that encodes GSIII is
regulated, and the amount of GSIII activity differs in different growth
media. We report here that R. meliloti GSIII can be modified
by ADP-ribosylation and that the modification appears to inactivate
GSIII biosynthetic activity. Post-translational control could therefore
play a role in regulating the contribution of GSIII to cellular
glutamine synthesis.
To prepare supernatants from
the trichloroacetic acid precipitation for HPLC analysis, 250 mM ammonium acetate, 10 mM EDTA, pH 9.0, was added to the
supernatant to give a final volume of 5 ml. Each sample was loaded onto
a 0.5 ml of dehydroboronyl-Bio-Rex 70 column (obtained from Dr. Michael
Jacobson, University of Kentucky) and washed with 10 ml of 250 mM ammonium acetate, 10 mM EDTA, 1 M guanidine, pH
9.0, and 10 ml of 250 mM ammonium acetate, 10 mM EDTA. Samples were eluted with 1 ml of HO, and the
volume was reduced to approximately 200 µl in a vacuum centrifuge
(Savant). Each sample was then analyzed by reversed-phase HPLC on a 3.9
150-mm Nova-Pak C18 column with 100 mM KH
PO
, pH 6.0, 2% methanol eluent at 0.5
ml/min flow rate. Reaction products were detected by UV A
. Standards were run under the same conditions.
Figure 1:
PO
is associated with GSIII in vivo and can be removed by
snake venom phosphodiesterase in vitro. GSIII protein labeled in vivo was immunoprecipitated with GSIII-specific antibody
and protein A-Sepharose. Protein released from the protein A at low pH
was treated with various enzymes and analyzed by SDS-PAGE and
autoradiography.
P-Labeled GSIII was treated with water (lane A), alkaline phosphatase (lane B), snake venom
phosphodiesterase (lane C), calf spleen phosphodiesterase (lane D), and both alkaline phosphatase and snake venom
phosphodiesterase (lane E).
Figure 2:
The modification of GSIII may be AMP or
ADP-ribose. P-Labeled GSIII was treated with snake venom
phosphodiesterase, and the trichloroacetic acid-soluble products were
analyzed by thin layer chromatography as described. The positions of
unlabeled ATP, ADP, AMP, and
PO
standards in adjacent lanes are
indicated.
Immunoprecipitated P-labeled GSIII protein was subjected to chemical
treatments known to distinguish ADP-ribose linked to various amino
acids, and the released products were analyzed by SDS-PAGE and
reversed-phase HPLC. HCl treatment can release
P-label in
ADP-ribose that is linked to glutamate or aspartate, Hg
treatment can release
P-label in ADP-ribose from
cysteine and hydroxylamine (NH
OH), pH 7.0, treatment can
release
P-label in ADP-ribose from arginine
(Cervantes-Laurean et al., 1993). NH
OH treatment
completely removed
P-label from immunprecipitated GSIII
(data not shown), but the other treatments had no effect. This suggests
that the modification was at an arginine residue. HPLC analysis of the
trichloroacetic acid-soluble products produced by HCl,
Hg
, or NH
OH treatment of labeled GSIII
revealed that only the NH
OH treatment produced a new
compound (Fig. 3). One product of NH
OH treatment had
a retention time (5.68) similar to that of an ADP-ribose standard
(5.72) suggesting that GSIII was modified by ADP-ribose. The additional
peak seen in the HPLC profile of the NH
OH treated sample
was also observed when an ADP-ribose standard was treated with
NH
OH. We suggest that this second peak is an ADP-ribose
derivative (probably inosine diphosphate ribose) created during the
NH
OH treatment.
Figure 3:
Trichloroacetic acid-soluble products of
chemical treatments. Soluble products released from GSIII by treatment
with (A) water, (B) 0.1 M HCl, (C)
1 mM Hg(OAc), or (D) 1 M NH
OH (pH 7.0). Each sample was analyzed by
reversed-phase HPLC on a 3.9
150-mm Nova-Pak
C18
column with 100 mM KH
PO
, pH 6.0, 2%
methanol eluent at 0.5 ml/min flow rate. Reaction products were
detected by UV A
. The mobilities of AMP and
ADP-ribose standards are shown on the left.
Figure 4: GSIII protein in various strains. WSU650, ET8051 (pFB6162), and MX727 (pFB6162) were grown in the media indicated. Lysates were prepared and GSIII enzymatic activities were measured. Proteins were separated by SDS-PAGE, and a nitrocellulose blot of the gel was analyzed using anti-GSIII serum. The molecular mass of protein standards are indicated.
Does
modification activate or inactivate GSIII? We reasoned that if
ADP-ribosylated GSIII was the active form, ET8051 (pFB6162) and MX727
(pFB6162) would have comparable amounts of radioactivity when labeled
with PO
, but that if ADP-ribosylated GSIII was
the inactive form, there should be much more radioactivity in GSIII
from ET8051 (pFB6162) since a smaller fraction of this protein appears
to be active. When both strains were grown in
PO
, the immunoprecipitated GSIII from ET8051
(pFB6162) contained much more
P-label than MX727 (pFB6162)
at a molecular weight that corresponds to GSIII. These results suggest
that GSIII in ET8051 (pFB6162) was mostly in modified form and the
modified GSIII is less active.
Figure 5: ADP-ribosylarginine hydrolase treatment of GSIII. Partially purified GSIII proteins were treated with ARH as described then analyzed for GS activity as a function of time of incubation with ARH. The ordinate is the ratio of GS activity in samples incubated with ARH divided by the activity in samples treated with ARH buffer.
GSIII from R. meliloti is an unusual GS that is
found in a glnA glnII double mutant of R. meliloti 104A14. The discovery that GSIII could be labeled in vivo with [P]phosphate led us to investigate the
form of this post-translational modification and its role in GSIII
regulation. Our results suggest that ADP-ribosylation of GSIII at an
arginine inhibits GS activity. Two lines of evidence lead us to
conclude that the modifying group is ADP-ribose, the release of a
compound with the mobility of ADP-ribose from hydroxylamine-treated
protein and the sensitivity of the protein to ARH. The sensitivity of
the modification to ARH and to hydroxylamine but not to other chemical
treatments suggests that arginine is the modified amino acid.
Arginine-specific ADP-ribosylation of proteins is widely distributed in
bacterial viruses and prokaryotic and eukaryotic cells (Moss and
Vaughan, 1990) and is often involved in post-translational modification
of enzyme activity.
In R. meliloti, we have found GSIII only in a glnA glnII mutant that lacks both GSI and GSII, but large amounts of GSIII protein can be detected in this strain (Shatters et al., 1993). GSIII activity in these cells is different in different growth media, and both transcriptional and post-translational regulation are implicated in controlling GSIII activity. Expression of GSIII protein requires a functional NtrA protein, and transcription of the glnT region of R. meliloti 1021 is strongly dependent on the NtrC protein (DeBruijn et al., 1989), suggesting that glnT is transcriptionally regulated by the nitrogen regulatory system. The results presented here showing that GSIII can be inhibited by ADP-ribosylation suggest that GSIII activity can also be controlled post-translationally.
Woehle et
al.(1990) reported that GS in R. rubrum can be
adenylylated or ADP-ribosylated in vivo, but the physiological
significance of ADP-ribosylation was not established. Although the DNA
sequence of R. meliloti GSIII predicts a tyrosine
near the site of the tyrosine modified by adenylylation of GSI-type
enzymes, we do not detect the phosphotyrosine that would be expected
after acid hydrolysis of tyrosine-AMP (Foster et al., 1989).
The growth rate of WSU650, the strain from which GSIII was taken for
this analysis, is still dependent on both glutamate and ammonium
concentrations (Shatters et al., 1993), so the mutant could be
nitrogen-limited. Under these circumstances, adenylylation may not be
favored, and we cannot eliminate the possibility that GSIII is also
adenylylated under some conditions.
Based on a comparison of the specific activities of GSIII in WSU650, ET8051 (pFB6162), and MX727 (pFB6162), it appeared that the GSIII in WSU650 was not as active as it could have been. It may be that ADP-ribosylation of GSIII is not responding directly to a measure of the cell's nitrogen status or that GSIII modification occurs as the cells are being harvested and growth is limited.
Ammonium assimilation and its regulation are very different in R. meliloti and E. coli. The most obvious difference is that there are three glutamine synthetases in R. meliloti while E. coli has only one. The three types of GS in Rhizobium are regulated differently and may serve different purposes in the cell. GSI, the glnA gene product, is post-translationally modified by adenylylation; GSII, the glnII gene product, is transcriptionally regulated by the nitrogen regulatory system and there may be some post-translational regulation of this enzyme (Manco et al., 1992). glnT is apparently also transcriptionally regulated by the nitrogen regulatory system (Shatters et al., 1993) and we have shown here that its product, GSIII, can be inhibited by ADP-ribosylation. The complexity of these regulatory mechanisms may enable the cell to respond to different growth conditions and illustrates the importance of glutamine as a crucial biosynthetic precursor and ammonium donor for cellular nitrogen metabolism.