JOURNAL OF BACTERIOLOGY, July 1978, p. 114-1230021-9193/78/0135-0114$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 135, No. 1

Printed in U.S.A.

Control of Ammonium Assimilation in Rhizobium 32H1ROBERT A. LUDWIG

Department ofBiology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received for publication 8 February 1978

The symbiotic, nitrogen-fixing bacterium Rhizobium sp. 32H1 is a specializedammonium producer during symbiosis. However, during free-living growth, Rhi-zobium 32H1 assimilates ammonium very poorly. Two pathways of ammoniumassimilation exist in enteric bacteria. One is mediated by glutamate dehydrogen-ase, and the other is mediated by glutamine synthetase-glutamate synthase. Theformer pathway is altogether inoperative in Rhizobium 32H1; the latter pathwayoperates at a slow rate and is under strict negative control by ammonium itself.Rhizobium 32H1 glutamine synthetase activity is modulated by both repression-derepression and reversible adenylylation. For a biochemical process lacking analternative pathway, such a regulatory pattern exacerbates the very process. Thissuggests that Rhizobium 32H1 restricts its own ammonium assimilation tomaximize the contribution of fixed nitrogen to the host plant during symbiosis.

Enteric bacteria such as Escherichia coli pre-fer ammonium ions as a nitrogen source. Am-monia is converted directly to glutamine by theenzyme glutamine synthetase (GS; L-gluta-mate:ammonia ligase, ATP requiring, EC6.3.1.2) or, alternatively, directly to glutamateby the enzyme glutamate dehydrogenase (GDH;EC 1.4.1.4). In the present work I describe stud-ies on the ammonium-assimilation system of thesymbiotic, nitrogen-fixing bacterium Rhizobium"cowpea" 32H1. These results indicate that, al-though Rhizobium 32H1 is a good ammoniumproducer, the free-living bacterium is a poorammonium utilizer.This seeming contradiction is all the more

interesting in light of the elaborate mechanismby which rhizobia synthesize ammonium viasymbiotic nitrogen fixation. These bacteria in-fect the roots of leguminous plants and inducethe development of a root nodule in which thebacteria proliferate and finally differentiate intoactive, nitrogen-fixing "bacteroid" forms. Notonly does symbiotic nitrogen fixation entail afairly complex developmental process (7), butthe energetics of reduction of dinitrogen to am-monia are unfavorable, requiring 12 to 15 ATPsper mol of ammonium reduced (15). Duringsymbiosis, ammonium assimilation has been re-duced, at seeming expense to the organism, sothat it might maximize the contribution of am-monium (as fixed nitrogen) to the host plant.Interestingly, the present work shows that poorammonium-assimilating ability extends to free-living rhizobia as well. This suggests that rhizo-bia are committed to the evolutionary benefitsof symbiosis.

In enteric bacteria, ammonium assimilationoccurs in either of two ways depending on therelative supplies of ammonium and energy avail-able. With excess ammonium, GDH reductivelyaminates 2-ketoglutarate to yield glutamate (seereaction 1 below), whereas, with limited ammo-nium, GS and glutamate synthase (GOGAT; L-glutamine:2-oxoglutarate aminotransferase, EC2.6.1.53) operate in concert to yield the same netresult (see reactions 2 and 3 below) (17). How-ever, in the latter case, 1 mol of ATP is hydro-lyzed per mol of net glutamate formed.

2-ketoglutarate + NH4+ -DH L-glutamate (1)NADPH

L-glutamate + NH4'GS

+ ATP -4 L-glutamine + ADP, Pi

L-glutamineGOGAT

+ 2-ketoglutarate -4 2 L-glUtamateNADPH

(2)

(3)

(NADPH is reduced nicotinamide adenine di-nucleotide phosphate.) The regulation patterncorresponds to the relatively high and low Kmvalues for ammonium exhibited by GDH andGS, respectively (18). When ammonium is pres-ent in excess relative to the energy source(s),GDH is derepressed and GS is repressed; theconverse is true when ammonium is limited (4,17).GS itself plays an important regulatory role in

this process. Glutamine auxotrophs ofKlebsiellaaerogenes, deficient in GS activity, constitu-tively derepress GDH. Such mutants also fail to

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AMMONIUM ASSIMILATION IN RHIZOBIUM 32H1

derepress the synthesis of other ammonium-yielding, amino acid-degradative enzymes suchas those of histidine or proline utilization duringammonium-limited growth (4).Not only is GS in enteric bacteria subject to

a complex pattem of feedback inhibition, but, inaddition, its catalytic and regulatory propertiesare further modulated by reversible adenylyla-tion (11). By a series of enzymatic reactions anAMP moiety may be reversibly attached to eachGS subunit (22). Native GS is a 600,000-molec-ular weight dodecamer composed of 12 identicalpolypeptides of 50,000 molecular weight (9). GSmagnesium-dependent catalytic activity is in-versely proportional to the number of AMPmoieties present (from 0 to 12). As might beexpected, under ammonium excess, GS is highlyadenylylated and, under ammonium limitation,it is highly deadenylylate (9).The present work shows that ammonium as-

imilation in Rhizobium 32H1 differs signifi-cantly from that of enteric bacteria in at leastfour respects (see Table 1). (i) Both 32H1 GSand GOGAT exhibit low catalytic activities un-der all growth conditions. However, GS totalactivity is present at high levels in 32H1, sug-gesting that rhizobial GS has a low turnovernumber relative to that of enteric bacteria. (ii)GDH does not assimilate ammonium in 32H1; itis a strictly catabolic enzyme. (iii) Rhizobial GSbecomes repressed at lower ammonium concen-trations than does enteric bacterial GS. (iv)32H1 GS becomes highly adenylylated at loweranmonium concentrations than does entericbacterial GS.

MATERIALS AND METHODSMedia and selection of auxotrophs. Rhizobium

"cowpea" 32H1 was cultured at 300C in defined mini-mal medium containing arabinose as the carbon sourceand the indicated nitrogen sources (0.1%) as describedpreviously (16). (Nitrogen sources included glutamate,glutamine, and ammonium [NH4+].) Glutamate auxo-trophs were isolated by penicillin-lysozyme enrich-ment of mutagenized cultures growing in minimal me-dium plus NH4' as described previously (16).

Preparation of ceils and cell extracts. GS as-says were conducted with Rhizobium 32H1 whole cellsor crude cell extracts. Whole cells growing in theexponential phase at 30°C were harvested by additionof cetyltrimethylammonium bromide to 0.1 mg/ml,and aeration at 300C was continued for 10 min. Cellswere chilled, centrifuged at 40C, suspended in theinitial volume of 0.01 M imidazole-chloride (pH 7.0) at40C, centrifuged, and suspended to a 10-fold concen-tration in imidazole-chloride. Whole-cell enzyme as-says were conducted immediately. Cell extracts wereprepared from whole cells to which KCI (0.1 M) anddithiothreitol (0.1 mM) had been added by ultrasonicdisruption for a total of 4 min in 15-s bursts at 40C.Cell debris was pelleted by centrifugation for 60 min

at 40C at 10,000 x g, the supernatant fluid constitutedthe crude cell extract. Enzyme assays on crude cellextracts were conducted immediately. GS was purifiedfrom crude cell extracts by the method of Streicherand Tyler (26).Enzyme assays. The GS assay procedures mea-

sured GS catalytic activity, total GS activity, or degreeof adenylylation in both enteric bacteria and rhizobia.These assays will hereafter be referred to as the bio-synthetic assay (forward reaction), the y-glutamyltransferase assay, and the ratio of transferase activitiesin the presence and absence of 60 mM Mg2e (24),respectively. Transferase activity measured in thepresence of 60 mM Mg2e is inversely proportional tothe adenylylation state and is completely inhibitedwhen the enzyme is fully adenylylated (25). Therefore,at the appropriate pH, the ratio of transferase activi-ties measured in the presence and absence of Mg2eyields an average adenylylation value for the enzyme(24). GS biosynthetic activity (forward reaction) wasmeasured as described for K. aerogenes (1). For Rhi-zobium 32H1, the forward reaction was conducted atpH 7.7 and extrapolated to pH 8.5, where activity wasmaximal for 100-fold-concentrated whole cells. TheRhizobium 32H1 GS forward reaction specifically re-quired Mg2e and was independent of added Mn2".GS transferase activity was measured as described

previously (1, 16,25). Rhizobium 32H1 GS transferaseactivity was measured at pH 7.0 (see Fig. 3 for a graphof GS transferase activity versus pH) and specificallyrequired Mn2", AsO43, and ADP. Transferase activityof fully adenylylated Rhizobium 32H1 GS, as definedoperationally by that of ammonium-shocked cultures(see text), was completely inhibited by the addition of60 mM Mg2e. Average adenylylation values (En) werecalculated by using both the ratio of GS transferaseactivities in the presence and absence of 60 mM Mg2e,and the relative absorbances at 260 and 290 nm (forpurified GS preparations) (25). This calculation wasmade by analogy to enteric bacterial GS for compari-son purposes only and assumed a dodecameric struc-ture composed of identical subunits, which is stillhypothetical, for Rhizobium 32H1 GS.

Catabolic (EC 1.4.1.2) and assimilatory (EC 1.4.1.4)GDH activities were measured in crude cell extractsby glutamate-dependent reduction of oxidized nicotin-amide adenine dinucleotide (NAD+), followed spectro-photometrically at 340 nm, and 2-ketoglutarate- andammonium-dependent oxidation of NADPH, respec-tively (17). Reactions were conducted at 370C and pH7.6. No 2-ketoglutarate- or ammonium-dependent as-similatory GDH activity was detected in 100-fold-con-centrated cell extracts, using eitherNADPH or NADHas the cofactor under any growth conditions tested.Biosynthetic GDH activity in Rhizobium 32H1 ex-tracts specifically required NAD+ as a cofactor.GOGAT activity was measured as described previ-

ously (17), using 100-fold-concentrated crude cell ex-tracts. Activity was measured at pH 7.6 and requiredNADH specifically as a cofactor. Extracts exhibitedno glutamine-dependent oxidation of NADPH.Gel electrophoresis. Sodium dodecyl sulfate elec-

trophoresis of proteins in polyacrylamide slab gels wasconducted as described by Laemmli (14). Electropho-resis in 7.5% acrylamide-0.2% methylene-bis-acryl-

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amide "running gels" was conducted at 2.5 V/cm for18 h at room temperature. Purified E. coli RNA po-

lymerase holoenzyme (Boebringer Mannheim), puri-fied E. coli GS (E1o) (gift of S. Streicher), and purifiedRhizobium 32H1 GS preparations were run in parallelon adjacent tracks in slab gels. Polypeptides weresubsequently stained with Coomassie brilliant blueR250, and the relative electrophoretic mobilities(REMs) were calculated as fractions of the mobility ofbromophenol blue tracking dye. Molecular weights ofprotein subunits were calculated from a plot of logmobility versus molecular weight, using accepted mo-lecular weights for E. coli RNA polymerase subunitsas standards.

Native protein-polyacrylamide gel electrophoresiswas conducted identically to sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the followingmodifications: sodium dodecyl sulfate was absent fromall buffers; polyacrylamide gels contained 5% acryl-amide, and 0.133% methylene-bis-acrylamide mono-mers; and electrophoresis was conducted at 40C withcontinuous pumping of electrophoresis buffer betweenthe upper and lower buffer reservoirs. Gels were sub-sequently assayed in situ for y-glutamyl transferaseactivity qualitatively in the presence and absence ofadded ADP and AsO43-. The positions of the bandseliciting transferase activity were noted, and electro-phoretic mobilities were measured relative to thebromophenol blue tracking dye.

RESULTS

GS-GOGAT is the sole mode of ammo-mium assimilation in Rhizobium 32H1.Rhizobium 32H1 possessed no assimilatoryNADPH-dependent GDH activity under anygrowth conditions. However, it did possess astrictly catabolic, NAD+-dependent GDH activ-ity (Table 1). NADH failed to catalyze the re-verse reaction, ammonium assimilation, in crude

cell extracts even at extremely high substrateconcentrations (100 mM) of ammonium and 2-ketoglutarate. Catabolic GDH activity alteredlittle in response to changes in ammonium levelsin growth media (Table 1), differing markedlyfrom the induction of assimilatory GDH in en-teric bacteria under conditions of excess ammo-nium. NAD+-linked enzymes are associated withbiosynthetic pathways (10). Strain 32H1 thuspossesses a GDH which enables it to use gluta-mate as an energy source as well as a nitrogensource but does not allow it to assimilate am-

monium.GOGAT is also NADH linked in strain 32H1,

whereas in enteric bacteria the enzyme isNADPH linked. In this regard, 32H1 GOGAT isanalogous to that of eucaryotes such as Saccha-romyces cerevisiae which are also NADH de-pendent (20). However, the specific activities ofGOGAT present in 32H1 extracts were quite lowwhen compared with those of S. cerevisiae orbacteria possessing NADPH-dependent GO-GAT activities (20 to 50 U/mg in extracts).Furthermore, the levels ofGOGAT in 32H1 werenot altered appreciably under conditions ofeither nitrogen starvation or surfeit. Low levelsof GOGAT might allow 32H1 to utilize gluta-mine as an energy source, which was observedin this organism, with a minimum of regulation.This would presumably occur by glutaminase-mediated deamidation of glutamine to gluta-mate, followed by catabolic GDH oxidativedeamination of 2-ketoglutarate, whence it entersthe citric acid cycle.

Further evidence that GS-GOGAT was thesole mode of ammonium assimilation in strain32H1 came from an analysis ofmutants defective

TABLE 1. Levels of ammonium-assimilation enzymes in Rhizobium and K. aerogenesGS activityb

GDH activitybStraiGrowhmeTransferase GOGAT activ-

Strain Growth mediuma Biosyn- E tybthetic _Mg2+ +60 mM ND AP

Mg2+ NAD+ NADPH

Rhizobium 32H1+ A, Git 84 2,700 1,500 5 1.0 NDc 1.22A, Glt, NH4+ 16 650 340 6 1.7 ND 1.17

Rhizobium 32H1 glt-6 A, Glt 49 1,600 830 5 1.0 ND <0.1

K. aerogenes MK53d G, Glt 925 2,000 1,850 1-2 ND 24 49G, Gln 765 1,600 1,530 1-2 e - -

G, NH4+ 160 450 320 3-4 ND 250 109G, NH4+, Gin 33 160 67 6-8 - -

a A, Arabinose; Glt, glutamate; G, glucose; Gln, glutamine. See text."Units per minute per milligram of protein.c ND, Not detectable.d From Brenchley et al. (4) for comparison purposes.e, Not measured.

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AMMONIUM ASSIMILATION IN RHIZOBIUM 32H1

in the utilization ofammonium as the sole nitro-gen source (Table 1). Compared to that of thewild type, growth of the mutants ranged fromslightly slower on ammonium as the sole nitro-gen source to none at all on ammonium. Amutant of the latter phenotype, 32H1 glt-6,lacked detectable GOGAT activity (Table 1),and revertants which regained the ability togrow on ammonium exhibited restored GOGATactivity (data not presented). Catabolic GDHlevels inglt-6 remained virtually unchanged, andthe glt-6 mutant failed to grow on ammoniumeven at concentrations as high as 20 mM, con-

firming that GDH cannot mediate ammoniumassimilation in 32H1.Repression ofGS by ammonium and glu-

tamine. GS protein (transferase activity), un-like GOGAT and GDH, was present at levelssimilar to those found in enteric bacteria (Table1). GS was fully derepressed when strain 32H1was grown in minimal medium containing glu-tamate as the nitrogen source. Glutamate alsohappens to be a preferred nitrogen source for32H1 (unpublished data). Although the total GScontent, as measured by transferase activity, of32H1 grown in glutamate as the sole nitrogensource was high, biosynthetic activity was low(Table 1).The GS content in strain 32H1 was very sen-

sitive to the external ammonium concentration.When 32H1 was grown in minimal medium plusglutamate to which increasing concentrations ofammonium were added, GS activity was re-pressed at ammonium concentrations above 0.1mM (Table 2). Biosynthetic GS activity paral-leled the extent of GS derepression, although itwas quite low when compared with the biosyn-

TABLE 2. Rhizobium 32H1 GS levels when grown inminimal-glutamate plus the indicated amounts of

ammonium or glutamine

GS Bio- GS Bio-tran syn- [Gluta- trans- syn-

(M)2 ferase GS mine] ferase thetic(MM) activity tivGac (mM) activity GS ac-(%tiv%) tivity

-a 100 100 - 100 100

0.01 100 0.01 1000.02 970.05 930.10 91 90 0.1 51 580.20 330.50 281.0 24 21 1.0 24 212.0 245.0 24

10.0 24 21 10.0 14 15a None.

thetic/transferase activities ratio for derepressedGS of enteric bacteria (Table 1).Glutamine also repressed GS in strain 32H1

(Table 2, Fig. lb). Such glutamine-mediatedrepression required slightly higher concentra-tions of glutamine than of ammonium whenadded to cultures in which GS was fully dere-pressed (Fig. la and b). This may be the resultof differential transport rates of glutamine andammonium. GS repression was more completeat high concentrations of glutamine than of am-monium. This was a substantially different re-sult from that obtained with enteric bacteria, inwhich GS was derepressed during growth onglutamine as the nitrogen source (Table 1).Although GS was derepressed in 32H1 cul-

tures grown in minimal medium plus glutamateand 0.1 mM ammonium, this concentration ofammonium repressed GS synthesis when di-rectly added to a fully derepressed culture (Fig.la). As can be inferred from Fig. 1, the concen-tration of added ammonium eventually de-creased through assimilation to a level sufficientto derepress GS. Therefore, the ammonium con-centration sufficient to repress GS is at most 0.1mM. Other experiments (unpublished data)showed that fully GS-derepressed cultures ex-hibited transient repression of GS upon additionof 10 AM ammonium. These results show that32H1 GS is extremely sensitive to repression byammonium.Adenylylation of GS induced by ammo-

nium. Both repression and inactivation of GSwere observed when ammonium was added to32H1 cultures which were fully derepressed forGS (ammonium shocked). GS transferase activ-ity exhibited a slow decay versus timne afterammonium shock (Fig. 1 and 2). This slow-decaycurve is assumed to represent repression andturnover of GS. GS transferase activity mea-sured in the presence of Mg2" exhibited rapidinactivation after ammonium shock. Fifty per-cent inactivation was reached in about 10 min.The same inactivation curve was obtained inthe GS biosynthetic assay. These results areanalogous to those obtained with enteric bacte-ria and suggest that the inactivation resultedfrom adenylylation. The half-time required foradenylylation of GS in 32H1 was on the order ofminutes, whereas it was on the order of secondsfor that of enteric bacteria. This difference mayreflect the inherently slower response of theadenylylation enzymes of 32H1. Alternatively, itmay accurately reflect a slower net intracellularammonium accumulation in 32H1 after ammo-nium shock. Similar transient adenylylationcurves, however, were obtained for ammonium-shocked cultures for which ammonium was sup-plied at concentrations as low as 10,M.

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17 hrtime after addition

FIG. 1. Rhizobium 32H1I GS levels after (a) am-monium shock and (b) glutamine shock. Cultures of32HI+ growing exponentially in minimal mediumplus 0.1% glutamate at 30°C were shocked at timezero with: (a) 0.1 mM (0), 1.0 mM (U, and 10 mM(A) ammonium. Aliquots were removed at the timesindicated, and relative GS transferase activity(-Mg2") was measured. Upon addition of 0.1 mMammonium, GS becomes repressed. However, at latertimes the ammonium concentration decreasedthrough assimilation and GS again became dere-pressed. A more highly transient GS repression curveis obtained when cultures are shocked with 0.01 mMammonium. In (b), similar results were obtained uponshock with 0.1 mM (0), 1.0 mM (, and 10 mM (A)glutamine. GS transferase (-Mg2") was measured atthe times indicated. In both (a) and (b), 100% activityequaled 2550 U/mg ofprotein.

Plurification and further characterizationof 32H1 GS. 32H1 GS purified by the methodof Streicher and Tyler (in preparation) migratedas a single polypeptide of molecular weight65,000 on sodium dodecyl sulfate-polyacryl-amide gel electropherograms (Tables 3 and 4)compared with 50,000 for the E. coli GS subunits

(22). Electrophoresis of purified 32H1 GS andthe corresponding crude cell extracts from (i)derepressed, (ii) repressed, and (iii) derepressed,ammonium-shocked cultures was also con-ducted on polyacrylamide gels which retain pro-teins in the native state. In both crude cellextracts and purified GS preparations, transfer-ase activity bands with similar REMs were ob-served. In crude cell extracts, a second transfer-ase activity band with a higher REM was ob-served; this band was not evident in purified GSpreparations. However, this band exhibited y-glutamyl transferase activity independent ofadded ADP and arsenate. Crude cell extracts so

0 20 40 60 80 100 120 mintime after NH4

FIG. 2. Ammonium shock of Rhizobium 32H1 GS-derepressed cultures. As for Fig. 1, a Rhizobium 32HIculture growing exponentially at 30"C in minimalmedium plus 0.1% glutamate as a nitrogen sourcewas shocked at time zero by addition of 1.0 mMammonium. Aliquots were removed, and GS transfer-ase was measured without added Mg2" (0) and inthe presence of 60 mM Mg2" () as a function ofinitial GS transferase activity. Simnilar repression-inactivation curves were obtained upon addition of0.01, 0.1, or 10.0 mM ammonium. However, uponaddition of 0.01 mM ammonium, both repression andinactivation ofGS occurred only transiently. GS bio-synthetic activity (data not presented) after 1.0 mMammonium shock exhibited an inactivation curvesimilar to that ofGS transferase activity plus 60mMMg2+, except that biosynthetic activities were muchlower (Table 1). 100%6 activity equaled 2,700 U/mg ofprotein.

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AMMONIUM ASSIMILATION IN RHIZOBIUM 32H1

TABLE 3. REM: SDS-acrylamide gelelectrophoresis

Polypeptide Mol wt REM

E. coli RNA po- 165,000; 160,000 0.181; 0.184lymerase,B' and/3

E. coli RNA po- 96,000 0.275lymerase 8

E. coli RNA po- 38,000 0.4,38lymerase a

E. coli GS (Ei-o) 50,000 0.387Rhizobium 32H1 65,000 0.344GS, purified

TABLE 4. REM: native protein-gel electrophoresis(pH 8.80)

y-Glutamyl transferase activityProtein +ADP, -ADP,

+AS043- -As043- REM

E. coli GS (Ei-o) + - 0.42Rhizobium 32H1+,

crude cell extractBand I + - 0.33Band II + + 0.57

Rhizobium 32H1+ GS, + - 0.33purified

assayed also indicated the presence of both /B-aspartyl- and y-glutamyl transferase activities,independent of ADP and arsenate, which con-stituted 7% of the total GS (ADP-dependent)activities in both repressed and derepressed cellextracts. The ADP-independent aspartyl andglutamyl transferase activity-pH curves coincideand are nonadditive in the presence ofsaturatinglevels of both glutamine and asparagine sub-strates. This suggests that the higherREM bandobserved in such electropherograms is an aspar-aginase (glutaminase).No differences in the REM of purified 32H1

GS from both repressed and derepressed cul-tures could be detected upon electrophoresis inthe native protein-polyacrylamide gels at eitherpH 8.8 or 7.6. Purified E. coli GS (molecularweight, 600,000) migrated more rapidly thanthat of 32H1 (Tables 3 and 4). The low REM of32H1 GS, as compared to that E. coli GS, at pH8.8 is consistent with a dodecameric structurecomposed of 65,000-molecular weight subunits,although this remains to be demonstrated.32H1 GS exhibited the same activity-versus-

pH curve when crude cell extracts and purifiedGS preparations were examined (Fig. 3). Theactivity-versus-pH profile for derepressed GSdid not demonstrate the activity stimulation atthe progressively high pH (above pH 7.0) asso-ciated with fully deadenylylated GS from K.

aerogenes (1). Interestingly, the pH profile ofadenylylated GS from K. aerogenes (1) is vir-tually superimposable on that of both repressedand derepressed GS isolated from 32H1. Therepressed GS from 32H1 merely showed lessactivity than the derepressed GS at any givenpH value. This suggests that 32H1 GS is alwayspartially adenylylated even when the organismis grown in minimal medium containing gluta-mate as the nitrogen source. In contrast, growthof K. aerogenes in the analogous medium con-stituted a condition of extreme GS derepression-deadenylylation (ammonium limitation) (Table1).

Relative UV absorbance at 260 and 290 nmalso yields a measurement of E. coli GS ade-nylylation (24). Purified 32H1 GS has an absorb-ance ratio (260 nm/290 nm) of 1.25 when purifiedfrom GS-derepressed cultures and 1.27 whenpurified from GS-repressed cultures. By analogy

6-0 6-5 7.0 7.5 8-0pH

FIG. 3. pH-versus-activity curve for Rhizobium32H1 GS. GS waspurified from GS-repressed (0) andGS-derepressed (U) Rhizobium 32HI cultures as de-scribed in the text. GS transferase activity (-Mg2")was measured at variouspH values. Similar activity-versus-pH curves were obtained for the correspondingcrude cell extracts, but with obviously lower specificactivities. Compare this figure with the activity-ver-sus-pH curves for the GS of K. aerogenes (Fig. 2 ofreference 4). 32HI GS from both GS-repressed andGS-derepressed cultures resembles adenylylated (andnot deadenylylated) GS from K. aerogenes in its ac-tivity-versus-pH profile.

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to purified E. coli GS, this yields En = 5.6 for32H1 repressed GS. This supports the hypothe-sis that 32H1 is always partially adenylylated.Deadenylylation of 32H1 GS by

SVD. Snake venom phosphodiesterase (SVD)can catalyze the removal of 5'-AMP residuesfrom adenylylated proteins (26). Crude cell ex-tracts from 32H1 cultures grown in (i) GS-dere-pressed, (ii) GS-repressed, and (iii) GS-dere-pressed, ammonium-shocked media, along withpurified, highly adenylylated E. coli GS (Ego),were treated with SVD (Fig. 4). 32H1 GS-re-pressed extracts regained both biosynthetic ac-tivity and transferase-plus-Mg2e activity linearlywith time of SVD incubation. GS from ammo-nium-shocked cultures regained both activitiesat a faster rate, which supports the hypothesisthat ammonium-shocked GS is more highly ad-

SVD treatment

FIG. 4. Deadenylylation of 32HI GS with SVD.SVD (Boehringer Mannheim) was employed to dead-enylylate Rhizobium 32HI GS in crude extracts andpurified GS preparations. 32HI GS (0.3 U) from a

GS-derepressed (0), GS-repressed (U), or GS-dere-pressed, ammonium-shocked culture (A) was in-cubated with 0.01 U of SVD in 0.1 M tris-(hydroxymethyl)aminomethane-hydrochloride (pH8.8)-1.0 mMMgCl2 for the indicated times at 37°C, atwhich points aliquots were removed and GS transfer-ase (+Mg2+) was measured. E. coli GS (E10) was alsotreated with SVD (0) as a control.

J. BACTERIOL.

enylylated than that from GS-repressed cul-tures. However, GS-derepressed extracts, hy-pothesized to be partially adenylylated, failed tofurther deadenylylate even after extensive SVDtreatment. The decay of activity with incubationtime may represent degradation produced byother activities present in SVD preparations.Furthermore, the activity-versus-pH profile forderepressed GS was not altered after SVD treat-ment. If adenylyl residues were protected fromdeadenylylation in vivo, they might also be ex-pected to be protected from SVD attack in vitro.Therefore, the SVD treatment experiment nei-ther supports nor refutes the hypothesis.Growth of 32H1 on ammonium as the

nitrogen source. Enteric bacteria prefer am-monium as a nitrogen source; in contrast Rhi-zobium 32H1 assimilates ammonium poorly. Tomeasure growth on ammonium, cells were grownat 300C to 5 x 108 ml-' in minimal medium plusglutamate. Cultures were pelleted by centrifu-gation, suspended in phosphate buffer, pelleted,and resuspended. Cells were then diluted intofresh miniimal medium (1:100) plus the indicatedamounts of ammonium sulfate as the sole nitro-gen source and grown at 300C (Table 5). Tocalculate generation times, portions were re-moved at various times and plated for viablecells per milliliter on minimal medium plus glu-tamate. GS transferase activities were measuredat 96 h after dilution, at which time all cultureswere growing exponentially. Portions were fil-tered through nitrocellulose filters (0.45 jLm),and the cells on the filters were assayed for GStransferase activity.When strain 32H1 was grown in minimal me-

dium plus ammonium, the shortest observedgeneration time was 24 h, as opposed to 8.3 hwith glutamate as the nitrogen source. Further-more, the growth rate in minimal medium plusammonium was maximal at an ammonium con-centration of only 0.5 mM. Higher concentra-tions of ammonium inhibited growth of 32H1(Table 5). This inhibition did not result fromnonspecific ionic effects, since control culturescontaining the optimal ammonium concentra-tion plus increasing amounts of K2S04 failed toinhibit growth. The highest GS specific activitieswere present in cultures initially containing 0.1mM ammonium (Table 5), which was also theconcentration when GS was fully derepressed(Table 2).These results support the hypothesis that am-

monium assimilation via GS is the rate-limitingstep in the slow growth of strain 32H1, usingammonium as the nitrogen source. Since growthwas optimal at an initial ammonium concentra-tion of 0.5 mM, whereas GS specific activity washighest at 0.1 mM ammonium, net ammonium

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TABLE 5. Growth ofRhizobium 32HI+ onammonium as the nitrogen source

GS transfer-[Glutamate] [(NH4)2SOJ b eneatiaon

(MM) (MM) time (h) at (relative sp30"C act)10.0 30.6 0.24

-a 1i0 29.4 0.33- 0.5 24.1 0.40- 0.2 26.4 0.67- 0.1 37 0.92- 0.05 42 0.90- 0.02 56 0.62- 0.01 120 0.53

5.9 mM 8.3 hr 1.00a-, No glutamate present.

uptake might be rate limiting during exponentialgrowth at initial anmonium concentrations be-low 0.5 mM.

DISCUSSIONTo reiterate the results presented here: Rhi-

zobium 32H1 exhibits a strikingly different pat-tern of response to ammonium ions than doenteric bacteria. 32H1 ssimilates ammoniumpoorly. It exhibits low levels of both biosyntheticGS as well as GOGAT, and it does not evenpossess a GDH for assimilation. Furthernore,GS is highly sensitive to ammonium repressionand adenylylation in 32H1. These propertieseffectively diminish ammonium assimilationrates under all growth conditions. When entericbacteria modulate ammonium assimilation byGS repression-adenylylation in ammonium ex-cess, they do so to switch to the energeticallyfavorable GDH-mediated asimilation. Rhizo-bium 32H1, on the other hand, simply switchesoff ammonium assimilation under the analogousconditions; it possesses no alternative pathwayto GS-GOGAT.Brown and Dilworth (5) have suggested that

GDH is capable of ammonium assimilation inexcess ammonium in several Rhizobium strainstested. It is possible that some strains ofrhizobiamay utilize GDH assimilatorily, although 32H1does not. Kondorosi et al. (13) found that an R.meliloti 41 mutant unable to grow on ammo-nium was GOGAT- and that the wild-type strainpossessed no assimilatory GDH.

Interestingly, although GS biosynthetic activ-ity was low in strain 32H1, GS total activity waspresent at levels similar to those of enteric bac-teria (Table 1). This suggests that GS is presentat high levels for regulatory purposes. Previ-ously, Ludwig and Signer (16) demonstratedthat GS is necessary for the synthesis of nitro-genase in free-living cultures of 32H1. A gluta-mine auxotroph, 32H1 gln-5, defective in GS

also fails to induce nitrogenase both in cultureand in nodules. One class of Gln+ revertants ofgln-5 regains both catalytic and regulatory func-tions. However, if GS is present at high levelsin 32H1 for regulatory purposes, such regula-tion must differ from that of wild-type entericstrains. 32H1 GS appears always to be partiallyadenylylated; yet, in K. aerogenes cultures inwhich GS is similarly adenylylated (En = 6)histidase synthesis is repressed. Furthermore,when a K. aerogenes culture in which GS isdepressed-deadenylylated (En = 1 to 2) is am-monia shocked (En =6 to 8), histidase synthesisimmediately switches from derepressed to re-pressed rates (8). However, in certain K. aero-genes mutants in which GS is synthesized atderepressed rates constitutively (G1nC pheno-types), GS is always highly adenylylated. Yetsuch derepressed, adenylylated GS derepresseshistidase constitutively as well (2). Therefore,Rhizobium 32H1+ may be analogous to the GlnCmutants of K. aerogenes in that high levels ofthe adenylylated 32H1+ GS are required forinduction of nitrogenase synthesis (16). This ar-gument might further predict that GS-repressed32H1+ cultures would allow induction of nitro-genase, since the GS adenylylation state is sim-ilar to GS-derepressed cultures. Only under con-ditions of ammonium shock, in which GS be-comes highly adenylylated, might nitrogenasenot be induced. Therefore, initiation of ammo-nium production by nitrogenase may be tanta-mount to ammonium shock. Any hypotheticalchange in adenylylation state of 32H1' GS afternitrogenase induction (and ammonium produc-tion) might be a mechanism for repression offurther rounds of nitrogenase synthesis. Thesehypotheses are being tested in this laboratory.

Altematively, the high GS specific activitiesobserved when 32H1 is grown on 0.1 mM am-monium as a nitrogen source might representthe induction of a second forn of GS, as hasbeen suggested by recent results (6). That reportdescribes two forms of GS in R. japonicum61A76 which are separable by virtue of differentisoelectric points. The second form of GS seemsto be induced in glutamate-grown cultures of61A76. However, the transferase activity of thissecond forn of GS is completely inhibited byadded magnesium, an anomalous result not ex-pected of a glutamate-induced, hypotheticallybiosynthetically active GS. Also, no adenylyla-tion of this second form of GS has been observedin cells grown in excess ammonium. If theseresults for 61A76 apply analogously to 32H1, thesecond form of GS must be susceptible, in somemanner, to inactivation after ammonium shockof derepressed cultures, since total cell GS bio-synthetic activity is rapidly decreased in an ex-

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122 LUDWIG

ponential fashion (Fig. 2). This argument wouldfurther suggest that the increase in transferaseactivity in the presence of 60 mM Mg2e in GS-derepressed 32H1 cultures reflects partial dead-enylylation of the conventional form of GS,which itself is either constitutive or only slightlyrepressible. However, another explanation forthe second form of GS observed in strain 61A76is that it is an asparagine synthetase, as has beenshown to be present in rhizobia by other workers(21). Such an asparagine synthetase might alsobe expected to possess -y-glutamyl transferaseactivity. However, asparagine synthetase cannotcontribute to net ammonium assimilation sinceasparagine does not substitute for glutamine inGOGAT-mediated net glutamate synthesis (un-published data). Resolution of these ambiguitiesawaits further study of the putative two formsof GS in several Rhizobium species.Conventional purification of GS from strain

32H1 yields a single form of the enzyme onnative protein-acrylamide gel electrophero-grams. This GS exhibits a single polypeptidesubunit on sodium dodecyl sulfate-acrylamidegel electropherograms, and it is analogous inmany respects to the GS of enteric bacteria. Thepoor catalytic properties of the enzyme and itsextreme susceptibility to repression and ad-enylylation by ammonium distinguish 32H1 GSmarkedly from that of enteric bacteria.

I favor the following model for the regulationofammonium assimilation in 32H1. Amino acidssuch as aspartate and glutamate are preferrednitrogen sources for the organism. Ammoniumcan be generated from these substrates by usingcatabolic GDH and can be transaminated toother amino acids. When 32H1 is forced to growon ammonium or nitrate (which is reduced toammonium), the organism does so, albeit ex-tremely slowly, utilizing GS and GOGAT. Underammonia excess, GS is adenylylated-repressedand ammonium assimilation (and growth) is dra-matically retarded even further. The slowgrowth on ammonium results from low catalyticactivity of GS, which is always partially ad-enylylated or otherwise structurally modified.Adenylylation therefore varies from partial tocomplete; ammonium assimilation varies frompoor to worse. GOGAT likewise is present at lowlevels under all growth conditions, furtherimpeding ammonium assimilation.

It has not been demonstrated that rhizobiaare net ammonium exporters during free-livinggrowth. However, much evidence has accumu-lated which implies that rhizobia do export am-monium to surrounding plant tissue as symbioticnitrogen-fixing bacteroids. 15N2 fixed by bacte-roids is exported as 15NH4 and not as labeledamino acids such as glutamate or glutamine (3).

Ammonium assimilation enzymes are present atlow levels in bacteroids. Correspondingly GS,GOGAT, and GDH have been observed at highlevels in bacteroid-associated nodule tissue,which suggests that assimilation occurs via plantenzymes (5, 19). According to this evidence, bac-teroids can be viewed as ammonium-producingorganelles to which the host plant supplies pho-tosynthate and facilitates low-level oxygentransport via leghemoglobin to activate bacte-roid oxidative phosphorylation.

Constraints on bacteroid ammonium assimi-lation allow maximum transport of fixed nitro-gen to the host plant. It is therefore less surpris-ing that free-living rhizobia mimic the ammo-nium-exporting behavior of bacteroids, but it isstill not clear why this should necessarily be thecase. The lack of a discrete ammonium-assimi-lation system which would facilitate such a proc-ess during free-living growth strongly suggeststhat rhizobia are evolutionarily committed tomaximizing the benefits to be derived from sym-biosis, apparently even to the detriment of free-living growth.

ACKNOWLE)GMENTSI acknowledge Ethan Signer, Harry Meade, and Elisabeth

Raleigh for discussions and helpful criticism, and StanleyStreicher for communication of results prior to publicationand for the gift of E. coli GS.

This research was supported by Public Health ServiceNational Research Service Award GM05266 to Robert Ludwigfrom the National Institute of General Medical Sciences andgrants from the National Science Foundation (PCM 7719214)and the American Cancer Society (VC 28G) to E. Signer. R.Ludwig is presently a fellow of the Rockefeller Foundation atthe Massachusetts Institute of Technology.

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19. Robertson, J. G., J. K. F. Farnden, M. P. Warburton,and J. M. Banks. 1975. Induction of glutamine synthe-tase during nodule development in lupins. Aust. J.Plant. Physiol. 2:265-272.

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22. Shapiro, B. M., and A. Ginsburg. 1968. Effects of spe-cific divalent cations on some physical and chemicalproperties of glutamine synthetase from E. coli. Tautand relaxed enzyme forms. Biochemistry 7:2153-2167.

23. Shapiro, B. M., and E. R. Stadtman. 1968. 5'-Adenylyl-0-tyrosine: the novel phosphodiester residue of ad-enylylated glutamine synthetase from E. coli. J. Biol.Chem. 243:3769-3780.

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