Journal of Applied Bacteriology 1982, 52, 201-207

Glutamate as a differential nitrogen source for thecharacterization of acetylene-reducing Rhizobium strains

500 6

904/06/81

T. KANESHIRO & M.A. KURTZMAN Northern Regional Research Center,Agricultural Research, Science and Education Administration, US Department oj Agriculture,1815 North University Street, Peoria, Illinois 61604, USA

Received 8 January 1981 and accepted 1 June 1981

KA ESHIRO, T. & KURTZMAN, M.A. 1982. Glutamate as a differential nitrogen sourcefor the characterization of acetylene-reducing Rhizobium strains. Journal of AppliedBacteriology 52,201-207.

A majority (36 of 44) of Rhizobium japonicum strains tested reduced acetyleneasymbiotically when grown on an agar medium containing 0'1% (w/v) L-glutamateas a sole nitrogen (N) source. Glutamate as N source led to pinpoint colonies anduniform glutamine synthetase activity of three selected, slow-growing acetylene­reducing strains (R. japonicum L-259 and 110 and a cowpea-type Rhizobium 32H 1).The three test strains were characterized further by antibiotic resistance, colony type,cellular morphology, and differential growth on different sources. The evidencesuggests that, in an agar medium, glutamate creates a growth condition leading toacetylene reduction activity, pinpoint colonies and pleomorphism.

Dinitrogen (N 2) fixation by slow-growingrhizobia in liquid (Evans & Keister 1976) andagar-surface (Kaneshiro et ai. 1980) cultures ismeasured conveniently by means of the acetylenereduction assay. Many slow-growing rhizobia,however, do not reduce acetylene (Kaneshiroet at. 1978); therefore, existing taxonomic criteriaare not adequate to distinguish acetylene-redu­cing (AR ) and non-reducing (AR -) strains.Rhizobium strains are generally identified by theirintrinsic resistance to specific antibiotics (Elkan1971; Josey et at. 1979; Pain 1979).

In contrast to fast-growing rhizobia (Werner& Berghauser 1976), glutamate is taken uprapidly and efficiently by slow-growingRhizobium japonicum strains (USDA-BeltsvilleStrains 83 and 85). Consequently, glutamate hasbeen evaluated as a N source capable of elicitingAR + activity in 46 slow-growing rhizobia. Inliquid cultures, AR + strains appear to growunder micro-aerophilic conditions and to reduceacetylene in the presence of L-glutamate (Keister& Ranga Rao 1977). A preliminary study of AR +

0021-8847/82/04OO-D20 1 $02.00© 1982 The Society for Applied Bacteriology

strains (NRRL L-22, L259, L-302, L-311, andNitragin 32H1) further indicated that aerobicgrowth as measured by turbidity could be morereadily limited by L-glutamate as a N sourcerather than by either gluconate as a carbon (C)source or yeast extract as a vitamin source.

Pankhurst & Craig (1978) have shown thatcells of the cowpea-type strain, 32H 1, grown ina soft layer of agar containing glutamine, reduceacetylene and display pleomorphism similar tothat of symbiotic bacteroid-cells. The authorshave found that surface-grown cultures expressAR + activity more consistently than do liquidcultures. In addition, surface growth permitsobservation of colony morphology and simul­taneous performance of physiological and taxo­nomic tests. Accordingly, this report describes analternative procedure to characterize AR + agarcultures and a comparison of 32H 1 with R.japonicum strains. In place of glutamine,glutamate is shown to be a stable, effective Nsource for the agar-surface growth of AR +

rhizobia. The AR and AR - test strains havebeen further characterized by their antibioticresistance and asymbiotic growth on three differ­ent N sources to ascertain any correlation amongAR + strains.

Purchased by U. . Dept.Agriculture for Official U

202

Materials and Methods

T. Kaneshiro and M. A. Kurtzman

CHARACTERIZATION OF STRAINS

STRAINS

NRRL strains designated by the prefix L(Kaneshiro et al. 1978) were obtained from L. K.Nakamura (Agricultural Research ServiceCulture Collection, Peoria, Illinois 61604). Acowpea-type strain, Nitragin 32H 1, was kindlyprovided by J. C. Burton (Nitragin Co.,Milwaukee, Wisconsin 53209). The USDA­Beltsville strain 110 (Keyser & Weber 1979) wasprovided by D.F. Weber (Beltsville AgriculturalResearch Center, USDA-Beltsville CultureCollection, Beltsville, Maryland 20705).

GL UT AMATE-MAN NITOL-GL UCON ATE(GMG) MEDIUM

The GMG growth medium, adjusted to pH6,65, consisted of (g/l): L-glutamic acid (neutral­ized with NaOH after weighing) 1; potassium D­gluconate 10; D-mannitol 3; KH 2P04 6;MgS04 "7H 2 0 0·2; CaCI2 "2H 20 0·08; agar 15;and 20 ml of (Fe-Mo) citrate solution. The(Fe-Mo) citrate solution contained 33·5 mgFeC6 H s0 7 " 5H 20and 7 mg Na 2 Mo04 " 2H 20/20 ml and was adjusted to a slighty acidic pHwith citric acid for adequate refrigerated storage.For assay of acetylene reduction, the GMGmedium contained 11 g agar/I.

OTHER MEDIA

Agar slant cultures of rhizobia were maintainedon a modified (Evans & Keister 1976) yeastextract-mannitol-soil extract (YMS) medium atpH 6·7 containing (g/l): yeast extract 1; mannitol5; potassium gluconate 5; agar 16; and a 20%(v/v) soil extract solution (YMGS). Generally,mannitol and gluconate are considered effectiveC substrates for the multiple, degradative meta­bolism and growth displayed by many types ofrhizobia (Arias et at. 1979; Keele et at. 1970;Kuykendall & Elkan 1976).

Maintenance cultures were transferred to aliquid medium (Kaneshiro et at. 1978) containing(g/l): L-glutamic acid 1; mannitol 5; potassiumgluconate 5; KH 2 P04 3; and yeast extract 0·3.Minor mineral salts were added in amountssimilar to the GMG medium. The liquid cultureswere incubated aerobically for 2-5 d at 25°C ona rotary shaker (approximately 200 rev/min) andwere subsequently left unshaken for up to 3 days.

Static liquid cultures of three AR + test strainsand eight AR - strains were diluted serially in0'85% (w/v) NaCI to yield about 50-700 colony­forming units (cfu counted by a Biotran IIautomated colony counter, New BrunswickScientific Co.) per YMGS plate and spread overagar media containing various Nand C combina­tions. The final concentrations (g/l) of the variousNand C sources for the detection of agar-surfacegrowth were: glutamic acid 1; mannitol 3;potassium gluconate 10; casamino acids 1·5; andyeast extract 0·3. With the exception of YMGS,the mineral salts for all media were the same asin the GMG medium. After 5-10 d incubationat 28°C, colony characteristics, cellular mor­phology, and cfu were tabulated to assessphenotypes.

Antibiotic sensitivity of cultures (estimated tocontain 1-3 x 105 cfu on each plate) wasmeasured on YMGS medium by using 7-mmdiameter antibiotic discs (Difco, Detroit,Michigan). Concentrations per disc were:kanamycin 10 j1g; neomycin 10 j1g; penicillin G10 units; polymyxin B 100 units; streptomycin10 j1g; and tetracycline 10 j1g. After 4-8 d incuba­tion, the lethal or inhibited growth diameters(minus the 7 mm discs) were measured (mm), butare tabulated here as either sensitive (s) orresistant (r) to the specific antibiotic.

GLUTAMINE SY THETASE (EC 6.3.1.2)ASSAY

Agar-surface cultures, grown aerobically in flatbottles on either VMS or GMG medium for 4 dat 28°C, were suspended in 1% (w/v) KCI solutionand centrifuged at 4°C. The packed cells weresuspended in a pH 7·0 buffered mixture (15%cellular matter by wet weight) at 4°C containing10 mmoljl imidazolechloride, 1 mmoljl MnCI 2 ,

and 1 mmol/l mercaptoethanol (Shapiro & Stadt­man 1970). Cell extracts (1-2'5 ml) were preparedby ultrasonic disruption (Branson, model S-llO)for a total of 4 min in 30-s bursts. Cellular debriswas then removed by centrifugation for 8 min at12000g, and protein in cell extracts was deter­mined colorimetrically by the method of Lowryet at. (1951). The forward, biosynthetic activityof glutamine synthetase was measured byhydroxamate produced from glutamate andhydroxylamine in the presence of adenosine

Glutamate for rhizobial growth 203

Table 1. Growth media and antibiotic resistance of 11 Rhizobium strains in comparison to their acetylene­reducing activity

RRL strain number {USDA- Growth on medium* Antibiotic resistance (r)tBeltsville synonym; Rhizobiumsp. or original host-plant) YMGSt CA GMG Km m Pen G Pm B Str Tet

AR + strains§(32H 1; Crotalaria paulina )** + pp*L-259 (26; R. japonicum) ac pp pp(11 0; R. japonicum) + pp

AR - strainsL-21 (3037; Crotalaria striata) ac pp pp sL-238 (3; R. japonicum) + rL-242 (7; R. japonicum) + sL-245 (10; R.japonicum) + sL-246 (11; R. japonicum) pp pp pp sL-281 (51; R.japonicum) + rL-335 (3275; Vigna sinensis) + rL-341 (3298; Vigna

sesquipedalis) ac pp pp

* Growth characteristics expressed as positive (+); sparse (_); negative (- ); positive with slight acid formationto bromothymol blue indicator (ac); or positive but pinpoint colonies (pp).

t Abbreviations for media: yeast extract-mannitol-gluconate-soil extract (YMGS); casamino acids minimal(CA); glutamate-mannitol-giuconate medium (GMG).

t Sensitive (s) strains gave 5-35-mm inhibition zone. Abbreviations for antibiotics: kanamycin (Km); neomycinm); penicillin G (Pen G); polymyxin B (Pm B); streptomycin (Str); tetracycline (Tet).§ Acetylene-reducing (AR +) and non-reducing (AR -) strains after aerobic growth on both CA and GMG media... itragin Co. strain.

triphosphate at pH 7·15 and 3rC (Bender et ai.1977; Bergersen & Turner 1978; Ludwig 1978).Reverse y-glutamyl transferase activity of cellextracts was measured at pH 7·0 (Ludwig 1978)and 37°C as described by Shapiro & Stadtman(1970); relative adenylylation was estimated fromthe ratio of y-glutamyl transferase activities inthe absence and presence of 60 mmoljl Mg2 +.

ACETYLE E REDUCTIO ASSA Y A D

TOTAL CELLULAR PROTEI

Surface cultures were grown aerobically (5'5 mlof agar media inoculated with 0·3 ml of liquidcultures) inside cotton plugged serum vials of20·5 ml total capacity. After 3 d incubation at28°C, the vials were sealed with silicone rubbercaps, flushed with argon, and then filled with 0·1atm acetylene-0'9 atm argon. After further micro­aerobic incubation for 4 d, ethylene formed inthe 15 ml gas phase was measured by gaschromatography over a Poropak R column(Burris 1974). This procedure (Kaneshiro et at.1980) allowed analysis of AR + cultures within7 d and without disturbing surface growth. Themicro-aerobic cultures produced ethylene at alinear rate for approximately 3-4 d, after an

initial lag period of about 1 d. The rhizobialcells were then washed off the agar surface with0'85% NaCI and collected by centrifugation todetermine the amount of total cellular proteinper vial. The cellular protein fraction wasmeasured colorimetrically after digestion with1 mol/l NaOH (Kaneshiro ef al. J978).

Results

GLUTAMATE AS SOURCE FOR

DIFFERE TIAL GROWTH

Glutamate or aspartate, which can serve as solesources for three AR Rhizobium strains

(32H 1, L-259 and 110), led to similar numbersof cfu as observed on YMGS plates after 5-10 dof incubation at 28°C (Table 1). Casamino acids,which might also serve as a N source, did notconsistently produce cfu. Supplementation ofcasamino acids with yeast extract (300 mg/l)failed to enhance the growth of strains 32H 1 and110. Even though only three (L-21, L-246 andL-341) of the eight AR - strains grew on theGMG medium, both AR - and AR + strains wereslow growing as pinpoint and discretely roundcolonies. Therefore, all eight AR - strains had to

204 T. Kaneshiro and M. A. Kurtzman

Table 2. Acetylene-reducing activity of 44 Rhizobium japonicum strains onGMG medium

RRL strain

L-243, L-255, L-256, L-259,L-273, L-279, L-287, L-298,L-299, L-302, L-307, L-308

L-241, L-248, L-250, L-251,L-252, L-262, L-264, L-265,L-271, L-272, L-276, L-277,L-278, L-284, L-285, L-286,L-288, L-290, L-291, L-292,L-294, L-297, L-301, L-306

L-238, L-246, L-253, L-281,L-289, L-295, L-296, L-303

USDA-Beltsvillesynonym·

8,20,21,26,41,47, 58, 74, 83,94,115,116

6,13,15,16,17,29, 31, 33, 39,40, 44, 45, 46,54, 55, 56, 59,61,62,63,67,71, 85a, 114

3, 11, 18, 51,60,68,70,111

Acetylene reductionper vial

AR+ (>0'15 Jimol)

AR- (no reduction)

• USDA-Beltsville strains are listed in the same respective order as theNRRL strains.

be identified by their specific failure to reduceacetylene.

A previous survey of 44 R. japonicum strainsgrown on a casamino acids minimal medium(Kaneshiro et al. 1978) indicated that 32 wereAR . On the GMG agar medium, 36 of the 44strains reduced acetylene (Table 2). Strains L-252,L-273, L-285, L-287 and L-291 gave acetylene­reducing activities when glutamate, instead ofcasamino acids, was the N source for growth.Strain L-303, which reduced acetylene whengrown on casamino acids, was exceptional inthat it did not reduce acetylene after growth onGMG medium. Thus, the GMG medium appearsto provide better growth conditions prior to assayfor acetylene reduction.

Using strain 32H 1, which grew particularlywell with glutamate as a source (Ludwig 1978),a level of 1-2 gil of glutamate was found to beoptimal (Fig. 1)for the production of both cellularprotein and ethylene (1'6Iimoljmg protein in 4 d).Glutamate concentrations > 2 gil appeared toinhibit ethylene production without inhibitingsignificant growth.

CHARACTERIZATION OF THREE AR +

RHIZOBIA

Morphology

Besides pinpoint colony formation, the threeAR + strains grown on GMG agar showed atendency toward pleomorphism, yielding abun­dant V-, Y-, and other odd-shaped cells. Pleo-

morphic cells, which grew under microaerophilicconditions in a soft-agar layer (Pankhurst &Craig 1978), displayed acetylene-reducingactivity. It was observed that aerobically growncells of pinpoint colonies were also mor­phologically variable (Fig. 2b) when examinedby phase-contrast microscopy, in contrast to theuniform rods observed in YMGS cultures (Fig.2a). Because AR - and AR + strains were bothpleomorphic in either GMG or other nutrient­deficient media, pleomorphism derived fromaerobic cultures, by itself, could not differentiatethe two types.

Utilization of C and N sources

Because glutamate promoted aerobic growth(Table 1) and subsequent acetylene reduction,its effect when mixed with gluconate-mannitolC sources was re-evaluated (Table 3). Withoutany source in the medium, a gluconate­mannitol combination led to minimal, visiblegrowth and no AR + activity. Glutamate plusone other C source stimulated cellular proteinsynthesis (1'7-2'5 mg per vial) but did not produceconsistently high AR + activity. A combinationof glutamate-mannitol-gluconate gave consis­tent AR + activity (0'2-1'9 limol ethylenelmgprotein) for all AR strains tested. In anothertrial using 0'1% (wIv) aspartate instead ofglutamate, similar amounts of acetylene werereduced. Casamino acids and yeast extract as Nsources, however, gave cultures with different

Glutamate for rhizobial growth 205

Table 3. Acetylene reduction and total cellular proteinof strains 32H 1, L-259 and 110 after aerobic growth

....J 3·0 3'0 0> on various nitrogen (N) and carbon (C) source0 E combinationsE~

c(I)

Strain(I) 0c ;X-.... 5.- Nand C sourcesCl> ">. 2'0 X ........ 2·a 0 combined in 32HI L-259 110.J::.

'X~xQ; ::> growth medium· [Jimol C 2 H 4 and (mg cellular'0 ~ protein) per vial]c '0::>

C (0'2) (0'1) (0'2)0 I·a Gin-man 0 0 0E ::><t 0 Glu-gln 1·2 (1'7) 0·2 (2-4) 0·5 (2'3)E

<t Glu-man 0·01 (1'9) 0·8 (2'5) 0·1 (2'5)Glu-man-gln 3·1 (1,6) 2·3 (2'0) 0·3 (1-8)

0 CA-man-gln 0·1 (1'5) 3·7 (4'8) 0·1 (l'0)a I-a 2·a 3·a 4'0 5·0 6·a Y-man-gln 0·3 (0'9) 2·1 (2'0) 0·1 (1'0)

Glutamic acid (giL)

Fig. 1. Glutamic acid concentration (wjv, neutralizedwith NaOH) required for strain 32Hl growth (X, mgtotal cellular protein per vial) and acetylene-reducingactivity (0, Jimol ethylene per vial) in a glutamate­mannitol-gluconate (GMG) medium.

reducing activities than those from glutamate­grown cultures. For example, growth on 0'1%(w/v) yeast extract medium led to decreasedacetylene reduction (0'1-0'3 jlmol/mg protein) bystrains 32H1 and 110, but not by L-259. At ahigher 0'3% yeast extract concentration, strains32H1 and 110 showed no detectable growth in3-4 d, whereas strain L-259 still grew vigorouslyand reduced 2 jlmol acetylene per vial. Appar­ently, strain L-259 can grow and reduces acety-

• Abbreviation and final concentration (wjv): CA(casamino acids 0'15%); gin (potassium gluconate1%); glu (glutamic acid 0'1%); man (mannitol 0'3%);Y (yeast extract 0'1%).

lene when cultivated on media containing eitherglutamate or the mixed amino acids present incasamino acids. However, there is no explanationfor the low acetylene-reducing activities (maxi­mum of0·5 jlmol per vial) displayed by such AR +

strains of R. japonicum as 110 and L-241 in theGMG medium (Table 2).

Antibiotic resistance

None of the six antibiotics assayed (Table 1)selectively restricted growth of the three AR +

10 pm

Fig. 2. Phase-contrast photomicrographs of aerobic strain 32Hl: (a) rod-shaped cells grown on yeast extract­mannitol-gluconate-soil extract agar for 4 d; and (b) pleomorphic cells grown on GMG agar after 18 days. A dropof liquid-suspended cells was solidified on a glass-slide with a melted drop of 2% (w/v) Difco purified agar.

206 T. Kaneshiro and M. A. Kurtzman

strains. The AR + strains and a majority of theeight AR - strains were sensitive to kanamycin,penicillin G and streptomycin; they were resistantto neomycin, polymyxin B and tetracycline.Although an intrinsic resistance to specific anti­biotics (e.g. L-246 to streptomycin) might becharacteristic of each strain (Elkan 1971; Joseyet al. 1979), there was no detectable relationshipof resistance to either AR + activity or glutamaterequirement.

Glutamine synthetase activity

Because glutamine synthetase is a crucial enzymefor AR + activity and ammonium ion assimilationby cowpea-type rhizobia (Bergersen & Turner1978), the enzyme levels were also measured incellular extracts of the three strains (Table 4). Incontrast to cells grown on a YMS medium, theGMG-grown strains, 32H1 and 110, containedmore extractable protein per unit wet weightand higher: specific activities (j1mol glutamyl­hydroxamate/min/mg protein). As expected,glutamine synthetase of strain L-259 was activein both GMG and YMS cultures. In short,glutamate appears to stimulate aerobic AR +

cultures evenly when measured by their glutaminesynthetase activity.

Discussion

The well-known ability of slow-growing rhizobiato form pinpoint colonies was used by Trinick(1980) to distinguish between slow- and fast­growing strains. In a glutamate medium (GMG),

agar-surface cultures were also slow-growi~g,

produced pinpoint colonies and were active inacetylene reduction (AR +). More AR + cultureswere obtained on GMG (36 of 44 R. japonicumstrains) than on a casamino acids medium;whereas some AR - strains (e.g. L-238, L-242,L-245, L-281 and L-335) failed to grow on GMG.Consequently, it appears that the GMG agarmedium is partly selective for AR + strains.

The GMG agar medium elicited AR growthof R. japonicum (L-259 and 110) cells that weredistinctly pleomorphic, similar to pleomorphic32H 1 cells that were AR + in a soft-agar layer(Pankhurst & Craig 1978). In agreement withKeister & Ranga Rao (1977) and Scott et al.(1979), glutamate-N and gluconate-C in the agarmedium were conducive to AR + activity. Thus,a N-C mixture ofglutamate-mannitol-gluconatecompounds favoured both growth and AR +

activity of the slow growers.Although glutamate inhibits assimilation of

ammonium ions (O'Gara & Shanmugam 1976)by slow-growing rhizobia, there is evidence thatexogenous glutamate inhibits neither AR +

(Keister & Ranga Rao 1977) nor glutaminesynthetase (Brown & Dilworth 1975) activities.Instead, glutamate appears to be a preferred Nsource for growth of 32H1 (Ludwig 1978), andthe formation of biosynthetic glutamine syn­thetase activity appears to be correlated withnitrogenase activity (Bergersen & Turner 1978).Thepresent study also indicates high biosyntheticglutamine synthetase activity (0'1-0'15 j1moljmin/mg protein) of strains 32H 1, L-259 and 110grown aerobically on agar with exogenous

Table 4. Glutamine synthetase activity of three AR + strains compared after growthon VMS and GMG agar media

Protein Biosyntheticcontent of y-Glutamyl glutamine

Growth cellular extract· transferase synthetase Relativemedium Strain (mg/ml) (Jimoljmin/mg protein) adenylylationt

GMG 32H1 2·8 8·2 0·14 1·7VMS 32H1 0·7 2·1 0·01 2·9GMG L-259 1·8 5·3 0·15 1·8VMS L-259 0·7 4·1 0·13 1·7GMG 110 1·8 7·0 0·10 1·8VMS 110 0·2· 0·8 0·01 1·9

• Cellular extract after sonic disruption and centrifugation. Strain 110 grown onVMS formed a slimy emulsion upon centrifugation, but its cellular debris was separablefrom the extract after sonic disruption.

:.. Ratio of transferase activity in the absence/presence of 60 mmol/l Mg 2 + by aprocedure similar to Bergersen & Turner (1978).

Glutamate for rhizobial growth 207

glutamate. Brown & Dilworth (1975) havesuggested that either glutamine or glutamatesupplied by the plant is assimilated by symbioticrhizobia to allow growth and development ofN 2-fixing capacity in root nodules. Such a systemwould seem amenable to analysis with free-livingAR + cultures on a GMG-type agar medium.

The mention of firms' names or trade productsdoes not imply that they are endorsed orrecommended by the US Department of Agricul­ture over other firms or similar products notmentioned.

References

ARIAS,A., CERVE A SKY, C, GARDIOL, A. & MARTI EZ­DRETS, G. 1979 Phosphoglucose isomerase mutantof Rhizobium meliloti. Journal oj Bacteriology 137,409-414.

BE DER, R.A, JA SSE ,K.A, RESNICK, AD., BLUME ­BERG, M., FOOR, F. & MAGASA IK, B. 1977 Bio­chemical parameters of glutamine synthetase fromKlebsiella aerogenes. Journal oj Bacteriology 129,1001-1009.

BERGERSE , F.1. & TURNER, G.L. 1978 Activity ofnitrogenase and glutamine synthetase in relation toavailability of oxygen in continuous cultures of astrain of cowpea Rhizobium sp. supplied with excessammonium. Biochimica et Biophysica Acta 538,406-416.

BROWN, CM. & DILWORTH, M.1. 1975 Ammoniaassimilation by Rhizobium cultures and bacteroids.Journal oj General Microbiology 86, 39-48.

BURRIS, R.H. 1974 Methodology. In The Biology ojNitrogen Fixation ed. Quispel, A. pp. 9-33.Amsterdam: orth-Holland Publishing Co.

ELKA ,G.H. 1971 Biochemical and genetical aspectsofthe taxonomy ofRhizobiumjaponicum. In BiologicalNitrogen Fixation in Natural and AgriculturalHabitats ed. Lie, TA. & Mulder, E.G. pp. 85-104.The Hague: Martinus Nijhoff.

EVA s, W.R. & KEISTER, D.L. 1976 Reduction ofacetylene by stationary cultures of free-livingRhizobium sp. under atmospheric oxygen levels.Canadian Journal oj Microbiology 22, 949-952.

JOSEY, D.P., BEYNO , J.L., JOH STON, A.W.B. &·BERI GER, J.E. 1979 Strain identification inRhizobium using intrinsic antibiotic resistance.Journal oj Applied Bacteriology 46, 343-350.

KANESHIRO, T, CROWELL, CD. & HA RAHA ,R.F., JR1978 Acetylene reduction activity in free-livingcultures of rhizobia. International Journal oj Syste­matic Bacteriology 28, 27-31.

KA ESHIRO, T, NEWTO , J.W., SELKE, E. & SLODKI,M.E. 1980 Dinitrogen e5 2) fixation and acetylenereduction in free-living strains of Rhizobium. CurrentMicrobiology 3, 279-281.

KEELE, B.B., JR, HAMILTO ,P.B. & ELKAN, G.H. 1970Gluconate catabolism in Rhizobium japonicum.Journal oj Bacteriology 101, 698-704.

KEISTER, D.L. & RANGA RAO, V. 1977 The physiologyof acetylene reduction in pure cultures of rhizobia.In Recent Developments in Nitrogen Fixation ed.Newton, W., Postgate, J.R. & Rodriguez-Barrueco,C pp. 419-430. London: Academic Press.

KEYSER, RH. & WEBER, D.F. 1979 USDA-BeltsvilleRhizobium culture collection catalogue. PPH I ReportNo. 16 Agricultural Research Center, Beltsville.

KUYKENDALL, L.D. & ELKAN, G.H. 1976 Rhizobiumjaponicum derivatives differing in nitrogen-fixingefficiency and carbohydrate utilization. Applied andEnvironmental Microbiology 32, 511-519.

LOWRY, a.H., ROSEBROUGH, .1., FARR, AL. &RA DALL, R.J. 1951 Protein measurement with theFolin phenol reagent. Journal ojBiological Chemistry193, 265-275.

LUDWIG, R.A 1978 Control of ammonium assimila­tion in Rhizobium 32H 1. Journal ojBacteriology 135,114-123.

O'GARA, F. & SHA MUGAM, K.T. 1976 Control ofsymbiotic nitrogen fixation in rhizobia regulation of

H4 assimilation. Biochimica et Biophysica Acta451, 342-352.

PAIN, A.N. 1979 Symbiotic properties of antibiotic­resistant and auxotrophic mutants of Rhizobiumleguminosarum. Journal oj Applied Bacteriology 47,53-64.

PANKHURST, CE. & CRAIG, AS. 1978 Effect of oxygenconcentration, temperature and combined nitrogenon the morphology and nitrogenase activity ofRhizobium sp. 32H 1in agar culture. Journal ojGeneralMicrobiology 106, 107-219.

SCOTT, D.B., HE ECKE, H. & LIM, ST. 1979 Thebiosynthesis of nitrogenase MoFe protein polypep­tides in free-living cultures of Rhizobium japonicum.Biochimica et Biophysica Acta 565, 365-378.

SHAPIRO, B.M. & STADTMA , E.R. 1970 Glutaminesynthetase (Escherichia coli). Methods in Enzymology17A,910-922.

TRI ICK, M.J. 1980 Relationships amongst the fast­growing rhizobia of Lablab purpureus, Leucaenaleucocephala, Mimosa spp., Acacia Jarnesiana andSesbania grandifiora and their affinities with otherrhizobial groups. Journal oj Applied Bacteriology49,39-53.

WERNER, D. & BERGHAUSER, K. 1976 Discriminationof Rhizobiumjaponicum, Rhizobium lupini, Rhizobiumtrifolii, Rhizobium leguminosarum and of bacteroidsby uptake of 2-ketoglutaric acid, glutamic acid andphosphate. Archives oj Microbiology 107, 257-262.