occurrence bradyrhizobium sp. (lupinus) their spp. ornithopus … · dyrhizobium sp. (lupinus) from...

Plant Physiol. (1989) 89, 78-850032-0889/89/89/0078/08/$01 .00/0

Received for publication April 11, 1988and in revised form August 1, 1988

Occurrence of H2-Uptake Hydrogenases in Bradyrhizobiumsp. (Lupinus) and Their Expression in Nodules of Lupinus

spp. and Ornithopus compressus'

Jesus Murillo, Ana Villa, Manuel Chamber, and Tomas Ruiz-Argueso*Departamento de Microbiologia, E. T.S. Ingenieros Agr6nomos, Universidad Politecnica, 28040 Madrid, Spain(J.M., A.V., T.R.); and Servicio de Investigaciones Agrarias, Consejeria de Agricultura y Pesca, San Jos6 de la

Rinconada, Sevilla, Spain (M.C.)

ABSTRACT

Fifty-four strains of Bradyrhizobium sp. (Lupinus) from world-wide collections were screened by a colony hybridization methodfor the presence of DNA sequences homologous to the structuralgenes of the Bradyrhizobium japonicum hydrogenase. Twelvestrains exhibited strong colony hybridization signals, and subse-quent Southem blot hybridization experiments showed that theyfell into two different groups on the basis of the pattem of EcoRIfragments containing the homology to the hup probe. All strainsin the first group (UPM860, UPM861, and 750) expressed uptakehydrogenase activity in symbiosis with Lupinus albus, Lupinusangustifoiius, Lupinus luteus, and Ornithopus compressus, butboth the rate of H2 uptake by bacteroids and the relative efficiencyof N2 fixation (RE = 1 - [H2 evolved in air/acetylene reduced)) bynodules were markedly affected by the legume host. L. angusti-folius was the less permissive host for hydrogenase expressionin symbiosis with the three strains (average RE = 0.76), and 0.compressus was the more permissive (average RE = 1.0). Noneof the strains in the second group expressed hydrogenase activityin lupine nodules, and only one exhibited low H2-uptake activityin symbiosis with 0. compressus. The inability of these putativeHup+ strains to induce hydrogenase activity in lupine nodules isdiscussed on the basis of the legume host effect. Among the 42strains showing no homology to the B. japonicum hup-specificprobe in the colony hybridization assay, 10 were examined insymbiosis with L. angustffolius. The average RE for these strainswas 0.51. However, one strain, IM43B, exhibited high RE values(higher than 0.80) and high levels of hydrogenase activity insymbiosis with L. angustifolius, L. albus, and L. Iuteus. In Southemblot hybridization experiments, no homology was detected be-tween the B. japonicum hup-specific DNA probe and total DNAfrom vegetative cells or bacteroids from strain IM43B even underlow stringency hybridization conditions. We conclude from theseresults that strain IM43B contains hup DNA sequences differentfrom those in B. japonicum and in other lupine rhizobia strains.

Lupines (Lupinus sp.) are herbaceous annual or perenniallegumes grown for centuries in light acid soils of the semi-arid zones of Mediterranean countries (Lupinus albus, Lupi-nus angustifolius, Lupinus luteus) and in South America

' This work was supported by grants from Comisi6n Asesora deInvestigaci6n Cientifica y Tecnica (project 2462/83), Ministerio deAgricultura, Pesca y Alimentaci6n (project 02.43), and INIA (Project5585).

(Lupinus mutabilis) for grain, forage, or soil improvement(8). Because of their high seed protein content (30-40%),lupines have a potential significance as source of protein foranimal fodder. The presence of poisonous alkaloids in theseeds have negatively affected the spread oflupine cultivation.However, the development of low-alkaloid content cultivarshas stimulated the expansion of lupine production for graininto new areas of Australia and South America (30).

Lupines are nodulated by slow-growing rhizobia that alsoinfect Ornithopus sp. These rhizobia have been included inthe genus Bradyrhizobium and designated as Bradyrhizobiumsp. (Lupinus) (14). To extend the cultivation of lupines tonew areas, there is a need for inoculation of seeds withappropriate rhizobial strains (1, 24). The possession of H2uptake hydrogenase has been considered as a desirable char-acteristic of rhizobial strains to be used as legume inoculants.This assessment is based mainly on experiments with strainsof Bradyrhizobium japonicum containing H2 uptake hydrog-enase (Hup+ strains), which recycle the H2 generated bynitrogenase as an obligate by-product of the N2 fixationprocess. The recycling of H2 by these Hup+ strains is claimedto increase N2 fixation and crop productivity in soybeans (9,10). Besides B japonicum, Hup+ strains have also been re-ported in Bradyrhizobium sp. (Vigna) and in Rhizobiumleguminosarum (5). To our knowledge, strains have not beenscreened with the express purpose of identifying Hup+ phe-notype within Bradyrhizobium sp. (Lupinus).Genes involved in the H2 uptake process (hup genes) were

first cloned and isolated in B. japonicum strain 122DES (6).The existence of homology between hup specific DNA fromR. japonicum 122DES and DNA from Hup+ strains of R.leguminosarum biovar viceae has been demonstrated (18, 22)and used to isolate hup genes from this rhizobial species (17).We report here on the energy efficiency of N2 fixation by

lupine nodules and on the identification of Bradyrhizobiumsp. (Lupinus) strains that contain hup genes, whose expressionin nodules is markedly affected by the host legume. We alsoshow that two different types ofDNA sequences determiningthe Hup+ phenotype occur within Bradyrhizobium sp. (Lu-pinus) strains, only one of which is homologous to B. japon-icum hup DNA.

MATERIALS AND METHODS

Bacterial Strains, Sources, and Maintenance of Cultures

The Bradyrhizobium sp. (Lupinus) strains used in this workwere obtained from bacterial collections worldwide. Strains

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H2 METABOLISM IN BRADYRHIZOBIUM SP. (LUPINUS)

359a, 366a, 368a, and 370a were provided by 0. A. Berestet-sky (ARIAM, Leningrad, USSR); WU8, WU 143, and WU425by C. A. Parker (UWA, Nedlands, Australia); A89 and Al 12by G. Jager (IBG, Groningen, The Netherlands); 3040, 3045,3046, 3053, and 3061 by H. H. Keyser (USDA, Belts-ville,MD); 96A 10, 96B9, 96E7, and 96N1 by J. Burton (Ni-tragin Co., Milwaukee, WI); 465, 466, 470, and 471 by D. C.Jordan (DMG, Guelph, Canada); 6.0 and 6.4 by D. Cornet(GB, Gemblaux, Belgium); 623, 624, 625, and 750 by C.Rydin (DMAC, Uppsala, Sweden); 107, 118, 119, and 121 byE. B. Roslycky (RIO, Ontario, Canada); IS68, ISlll, LlAl,L4.1, L5.2, L9.2, LO0.1, L12.1, L13.2, L13.4, L15.2, L16.2,L17.3, L18C2, L20.2, L21.1, OR14, OR21, and IM43B by F.Temprano (SIA, Sevilla, Spain). Strains UPM860, UPM861and UPM862 were isolated from nodules of Ornithopus sati-vus in this study. Rhizobia were routinely grown in yeastextract-mannitol (YEM) medium (29) and maintained inYEM agar slants at 4°C.

Plant Material and Growth

Seeds of white lupine (Lupinus albus L. cv Multolupa),blue lupine (Lupinus angustifolius L. cv Uniharvest), yellowlupine (Lupinus luteus L. cv Aurea) and Ornithopus compres-

sus L. cv Pitman were surface-disinfected, germinated on

water-agar plates and planted in vermiculite in sterile Leonardjar-type assemblies containing N-free solution as described byVincent (29). Four lupine seedlings or six Ornithopus seed-lings were planted in each Leonard jar culture unit and were

inoculated with 10 mL of a 5-d-old culture of the appropriateBradyrhizobium sp. (Lupinus) strain. Four replicates were

used per each legume-strain combination together with fouruninoculated controls per legume. Plants were grown in a

controlled environmental chamber with a 16/8 light/darkcycle at 25/18°C and a mean irradiance of 400 uE m-2*s-provided by Sylvania cool-white fluorescent and incandescentlamps. Plants were harvested after 30 d (lupines) or 35 d(Ornithopus) of growth. The nodulated root system was usedfor nodule and bacteroid assays, and plant shoots were driedfor 48 h at 80°C and weighed.

Nodule and Bacteroid Assays

Nitrogenase acetylene (C2H2) reduction and H2 evolutionrates were determined in separate samples of nodules (0.2-0.4 g) attached to pieces of roots (2-5 cm). Ethylene (C2H4)and H2 were quantified by gas chromatography using flameionization and thermal conductivity detectors, respectively,with columns and conditions previously described (19). Allmeasurements were completed within 30 min after the rootswere excised from the plants. Rates ofC2H2 reduction and H2evolution were linear for at least 45 min. Bacteroids were

prepared aerobically from nodules collected from three or

four Leonard jar culture units and assayed for H2 uptakehydrogenase activity by the amperometric method as previ-ously described (19). Oxygen or methylene blue were used as

terminal electron acceptors for H2 uptake.

DNA Techniques

Total DNA was isolated from free-living cells and bacter-oids as previously described (17). The 5.9 kb HindIII and 2.9

kb EcoRI DNA fragments from pHU52 (16) were used ashup-specific hybridization probes. These DNA fragments wereisolated from low-melting point agarose gels and labeled with[a-32P]dATP by nick-translation (21). Restriction enzymedigestions, agarose gel electrophoresis, and Southern transferofDNA to nitrocellulose were by standard techniques (21).

Filters for colony hybridization were prepared by themethod of Maas (20). Hybridization of filters and Southernblots were carried out at 42°C in 50% formamide, 5 x SSC(1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1 xDenhardt's, 0.2% SDS, and 200 ug/mL herring sperm DNA.After 24 h, blots and filters were washed three times, 30 mineach, in 200 mL of 2 x SSC and 0.1% SDS at 42°C, thendried and exposed to Kodak-S x-ray film at -70°C withCronex intensifying screens. For low stringency conditions,formamide was lowered to 35%, and the temperature forhybridization and washes was 37°C.

RESULTS

Screening for Hup Sequences

To investigate the existence of hydrogenase-positive (Hup+)strains of rhizobia that nodulate lupines, 54 strains of Bra-dyrhizobium sp. (Lupinus) from collections worldwide werescreened for DNA sequences homologous to B. japonicumhup-specific DNA by the colony hybridization method. Weexpected the existence of homology between hup DNA fromB. japonicum and hup DNA from Bradyrhizobium sp. (Lu-pinus) since both types of rhizobia are taxonomically related(14). The 5.9 kb HindIlI DNA fragment from pHU52 con-taining the structural genes of the hydrogenase of B. japoni-cum strain 122DES (25) was used as the hup probe. Anexample ofthe type of hybridization signals obtained is shownin Figure 1. The same relative intensity of hybridizationsignals among colonies was not reproducible from filter tofilter. This could be attributable to variations in binding ofDNA to the filters or to the size of the colonies. TwelveBradyrhizobium sp. (Lupinus) strains (UPM860, UPM861,750, 359a, 366a, 368a, 623, 624, 625, A89, A112, and 466)showed strong hybridization signals in at least one filter outof three replicate filters examined and were considered ascandidates to contain hup specific sequences. A strong hybrid-ization signal was always associated with colonies from B.japonicum 122DES, included as positive control. Weak or nohybridization signals were consistently observed in the re-maining strains analyzed, including the Hup- strain 3.15B3of B. japonicum (19).

Relative Energy Efficiency and Hydrogenase Activity inNodules of L. angustifolius as Affected by the RhizobialStrains

To evaluate the magnitude ofenergy losses by H2 evolutionin lupine nodules and to check the validity of the colonyhybridization test as a screen for a Hup+ phenotype, all strainsshown to contain homology with B. japonicum hup DNA and10 other strains randomly chosen among the strains exhibitingweak or no homology were examined in symbiosis with plantsof L. angustifolius grown under bacteriologically controlledconditions.

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Figure 1. Autoradiogram showing colony hybridization of Bradyrhi-zobium sp. (Lupinus) strains to B. japonicum 122DES hup specificDNA. The 5.9 kb Hindlil fragment from pHU52, containing the struc-tural genes of the hydrogenase of B. japonicum 122DES, was usedas hybridization probe. B. japonicum 1 22DES (positions 5A, 9F, and41) and B. japonicum 3.155B3 (positions 8C, 4E, and 7G) were usedas positive and negative (19) control strains, respectively.

To calculate the RE,2 the formula (1-H2 evolved in air!

total nitrogenase activity]) (26) was applied. Total nitrogenaseactivity was routinely estimated by measuring C2H4 formationby nodules exposed to an atmosphere of air containing 10%C2H2. However, because in these conditions H2 evolution wasnot completely suppressed during the C2H2 assay in lupinenodules, the C2H4 formation rate was not considered a validmeasurement of the total nitrogenase activity. Nodule hydro-gen evolution was not suppressed even by raising the C2H2concentration to 15% in the atmosphere. The average rate ofH2 evolution in the presence of 10% C2H2 was calculated tobe about 10% ofthe C2H2 reduction rate from determinationscarried out in lupine nodules produced by six strains ofBrady-rhizobium sp. (Lupinus) (Table I). Consequently, henceforthvalues ofC2H2 reduction were multiplied by 1.10 to calculatetotal nitrogenase activity by all the strains tested.The nitrogenase activity, H2 evolution and RE of nodules,

and the hydrogenase activity of bacteroids produced by the22 strains examined are shown in Table II. The data demon-strated that all strains evolved H2 in nodules of L. angustifol-ius. However, the RE values varied from 0.17 to 0.89 depend-ing on the strain. When bacteroids were examined for theircapacity to oxidize externally supplied H2 with 02 as a finalelectron acceptor, only the bacteroids from strains UPM860,UPM86 1, and IM43B exhibited a detectable rate of H2-uptake. This H2-uptake capacity was consistent with the highrelative efficiencies exhibited by these strains. StrainsUPM860 and UPM861, but not IM43B, showed homologywith the B. japonicum hup-specific probe in the colony hy-

2 Abbreviations: RE, relative efficiency of N2 fixation; kb, kilobasepairs.

bridization assay. No detectable rate of H2-uptake was ob-served in bacteroids produced by any of the other strainsexhibiting either strong or no colony hybridization signals.

Plant top dry weights were also determined for each strain(data not shown), and average values ranged from 0.34 ± 0.02to 0.54 ± 0.03 g per plant. The average value for noninocu-lated plants used as controls was 0.28 ± 0.05. No correlationwas found between RE and plant dry weight (correlationcoefficient of 0.22).

Host Effect on Hydrogenase Activity

Since several Bradyrhizobium sp. (Lupinus) strains whichstrongly hybridized to the B. japonicum hup DNA probeinduced no detectable hydrogenase activity in symbiosis withL. angustifolius, we examined the host effect on the expressionof hydrogenases from lupine rhizobia. With this aim, all thestrains showing the positive hybridization trait, and strainIM43B, were tested for their capacity to induce hydrogenaseactivity and to recycle H2 in symbiosis with two other Lupinusspecies and 0. compressus.

Strains UPM86 1, UPM860, IM43B, and 750 expressedhydrogenase activity in nodules from all the four legume hoststested as indicated by the capacity of the bacteroids to oxidizeexternally supplied H2 with O2 or methylene blue as terminalelectron acceptors (Table III). However, both the rate of H2oxidation and the RE were markedly affected by the legumehost. Bacteroids from L. angustifolius showed the lowest ratesof H2-uptake with the four strains, and bacteroids from 0.compressus showed the highest rates with three of the strains.The host effect on the expression of hydrogenase activity wasalso evident in strain 624, which only derepressed the hupgenes in symbiosis with 0. compressus but not in lupine hosts.In most cases, the expression of hydrogenase activity in bac-teroids was associated with high RE values (>0.80). No de-tectable hydrogenase activity was observed in bacteroids pro-duced by strains 366a. A89, and 625 (Table III) or by strains368a, Al 12, 359a, 623, and 466 (data not shown) in any ofthe four legume hosts tested. Nodules produced by all thesestrains evolved large amounts of H2 in air and had low RE(<0.70).When methylene blue was used instead of 02 as a terminal

electron acceptor for H2 oxidation by bacteroids, higher H2-uptake rates were observed in most cases (Table III). This isparticularly remarkable in nodules produced in L. angustifol-ius by all clearly Hup+ strains. The lower H2-uptake ratesobserved with O2 may be due to some damage ofthe electrontransport chain during preparation of bacteroids or, alterna-tively, may reflect the existence of a limiting step in thetransport of electrons to O2 in intact bacteroids, since H2 issupposed to donate electrons directly to methylene blue (2).

Conservation of Presumptive Hup Sequences

Total DNA from each ofthe Bradyrhizobium sp. (Lupinus)strains suspected to contain hup specific sequences on thebasis of colony hybridization and/or the bacteroid H2-uptakeassays was digested with EcoRI, electrophoresed, and hybrid-ized to B. japonicum hup-specific probes. The probes werethe 5.9 kb HindlIl fragment used in the colony hybridizationassay and an additional 2.9 kb EcoRI fragment from pHU52

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Table I. Hydrogen Evolution in an Atmosphere of Air Containing 10% Acetylene by Nodules Producedin L. angustifolius by Bradyrhizobium sp. (Lupinus) Strains

Strains Acetylene Reduction Hydrogen Evolution H2 EvolutionC2H2 Reduction

pumol/h g nodule fresh wt %

121 28.1 ± 2.Oa 3.1 ± 0.08 11.03053 21.3 ± 3.8 1.7 ± 0.02 8.2OR21 24.1 ± 0.6 2.1 ± 0.04 8.8A112 15.2 ± 1.1 1.5 ± 0.02 9.9L13.4 20.8 ± 1.8 2.5 ± 0.04 12.1623 22.0 ± 1.6 2.9 ± 0.05 13.2

a Figures are means of determinations made in four replicate cultureunits ± SE.

Table II. Physiological Functions of Root Nodules and Bacteroids Produced in L. angustifolius cvUnicrop by Strains of Bradyrhizobium sp. (Lupinus) with or without Homology to B. japonicum hupSpecific DNA

The 5.9 kb Hindlil fragment of pHU52 (16) was used as probe to screen Bradyrhizobium sp. (Lupinus)strains for DNA sequences homologous to hup specific DNA from B. japonicum 1 22DES by the colonyhybridization method. Relative efficiency of nodules was calculated as 1 -rate of H2 evolution in air/rate of C2H2 reduction. The C2H2 reduction values have been corrected as indicated in the text to reflectthe total nitrogenase activity of nodules. H2-uptake by bacteroids was determined amperometricallywith 02 as a terminal electron acceptor.

Strain Evolution c2H2 elaive Bacteroid H2-UptakeHomology Evolution Reduction Efficiency

pumol/h.g nodule fresh wt

4.9 ± 0.3 46.2 ± 8.6 0.89 ± 0.024.8 ± 0.8 35.0 ± 6.2 0.86 ± 0.014.8 ± 0.7 32.7 ± 2.50.85 ± 0.058.4 ± 0.7 31.3 ± 1.50.73 ± 0.01

16.6 ± 1.5 58.0 ± 3.20.70 ± 0.048.9 ± 0.4 28.4 ± 2.50.68 ± 0.049.8 ± 0.8 24.9 ± 1.60.62 ± 0.029.7 ± 0.5 23.3 ± 0.80.59 ± 0.01

10.2 ± 0.710.5 ± 0.610.7 ± 2.613.9 ± 0.77.5 ± 0.88.1 ± 0.4

13.9 ± 0.69.0 ± 0.4

18.1 ± 0.912.8 ± 0.414.2 ± 0.519.9 ± 0.110.8 ± 0.616.2 ± 0.2

22.9 ± 1.7 0.54 ± 0.0422.6 ± 1.70.53 ± 0.0323.5 ± 3.40.53 ± 0.0329.1 ± 1.50.53 ± 0.0516.4 ± 0.60.53 ± 0.0717.8 ± 2.6 0.50 ± 0.0426.5 ± 0.50.48 ± 0.0416.7 ± 1.00.46 ± 0.0630.9 ± 0.1 0.41 ± 0.0321.1 ± 1.1 0.40 ± 0.0422.3 ± 1.00.38 ± 0.0124.3 ± 0.7 0.19 ± 0.0113.2 ± 0.9 0.18 ± 0.0719.7 ± 0.9 0.17 ± 0.04

nmol/h .mg protein

256030

<10<10<10

I <10<10<10<10

I <10<10<10<10<10<10

I <10I <10

<10<10<10

I <10a Results are means of four replicates ± SE.

also containing DNA essential for Hup+ phenotype in B.japonicum 122DES (16). Two strains not suspected to beHup+ (121, L9.2) were also examined.

All strains which had shown strong hybridization signals tothe 5.9 kb HindIlI probe in the colony assay contained EcoRIfragments with homology to both hup DNA probes. Twodifferent hybridization patterns were observed. In one pattern,strains UPM860, UPM861, and 750 (lanes a, b, and c, Fig.2) contained a single EcoRI fragment (9.0 kb) and two EcoRIfragments (9.4 kb and 1.7 kb) which hybridized to the 5.9 kbHindIII and 2.9 kb EcoRI hup DNA probes, respectively. In

the other pattern, strains 359a, 366a, 624, 625, A89, Al 12,and 466 (lanes f-l, Fig. 2) and strains 368a and 623 (data notshown) hybridized to the same two probes in a single EcoRIfragment (16 kb) and in two EcoRI fragments (5.0 and 1.7kb), respectively. No hybridization to any of the two hupprobes was detected with DNA from the strains IM43B, 121,and L9.2 (lanes d, m, and n, Fig. 2).On the basis of the total DNA digestion patterns (Fig. 2),

strains 359a, 366a, 368a, 623, 624, 625, A89, and Al 12 were

indistinguishable. This result suggests an apparent identity ofthese strains, but it was unexpected considering the different

UPM861IM43BUPM860121366a368a623624L13.4L18C23053L20.2750466OR21A112A89WU425L17.3359a96A10625

+

+

++++

++

++

+

+

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Table Ill. Effect of the Legume Host on Relative Efficiencies and Hydrogenase Activities ofBradyrhizobum sp. (Lupinus) Strains in Symbiosis with Lupinus spp. and Ornithopus Compressus

Relative efficiency values were calculated from H2 evolution and C2H2 reduction rates by nodules asdescribed in Table II. H2 uptake rates were determined aerobically prepared bacteroids by using 02 ormethylene blue (figures in parentheses) as terminal electron acceptors.

Strain Plant Host Evolution Reduction Efficiency

Amoll/h-g nodule fresh wtUPM861 L. albus

L. angustifoliusL. luteus0. compressus

IM43B L. albusL. angustifoliusL. luteus0. compressus

UPM860 L. albusL. angustifoliusL. /uteus0. compressus

a Results are means of four replicates ± SE. b Not determined.

0.96 ± 0.030.90 ± 0.041.001.00

0.85 ± 0.050.88 ± 0.030.86 ± 0.040.63 ± 0.03

0.95 ± 0.050.85 ± 0.040.97 ± 0.071.00

0.64 ± 0.070.59 ± 0.060.90 ± 0.031.00

0.49 ± 0.020.54 ± 0.030.49 ± 0.040.61 ± 0.06

0.38 ± 0.060.68 ± 0.050.57 ± 0.070.65 ± 0.06

0.46 ± 0.020.48 ± 0.040.37 ± 0.020.59 ± 0.04

0.32 ± 0.120.29 ± 0.050.23 ± 0.030.49 ± 0.06

H2Uptake

nmol/h .mg protein130 (1052)35 (291)

500 (ND)b445 (426)640 (1430)72 (110)170 (1000)162 (170)150 (900)30 (150)

400 (580)560 (610)230 (323)<10 (56)25 (126)

407 (285)<10 (<10)<10 (<10)<10 (<10)69 (417)<10 (<10)<10 (<10)<10 (<10)<10 (<10)<10 (<10)<10 (<10)<10 (<10)<10 (<10)<10 (<10)<10 (<10)<10 (<10)<10 (<10)

geographical origin of the strains. All the other strains exam-ined in Figure 2 were clearly distinct strains on the basis ofthe restriction fragments resulting from digestion of totalDNA.

Since strain IM43B clearly exhibited a Hup+ phenotype(Table III) and showed no detectable homology to B. japoni-cum hup DNA probes, we repeated the hybridization experi-ments under low stringency conditions. Again, no hybridiza-tion signals were observed. Also, to exclude the possibilitythat the IM43B culture used was a mixture of cells containingonly a few authentic lupine rhizobial cells, additional hybrid-ization experiments were conducted with genomic DNA iso-lated directly from bacteroids and from a culture obtainedfrom nodules of L. albus inoculated with IM43B. No se-

quences homologous to B. japonicum hup probes were de-tected in either DNA from the bacteroids or the reisolate. Thepattern of EcoRI-restricted total DNA was in both cases

identical to that of IM43B culture used as inoculum. Finally,EcoRI-restricted DNA from strains IM43B, UPM86 1, and750, all ofwhich showed a clearly Hup+ phenotype, and fromB. japonicum 122DES, were hybridized to a R. meliloti niHIDprobe. In the three cases, a similar degree ofhomology to thisprobe was observed (data not shown), indicating that IM43Bwas not different from 750, UPM86 1, and 122DES withregard to nifHD gene conservation. We concluded from theseresults that strain IM43B contains a type of hup sequencesdifferent from strains 750, UPM86 1, and 122DES.

DISCUSSION

The evolution of H2 as a by-product of the N2 fixationprocess is a general feature of most nodulated legumes. Theonly nodules in which evolution of H2 has not been detectedare those elicited by certain rhizobial strains capable ofinduc-ing a H2-uptake (Hup) hydrogenase. Lupine nodules are not

750

624

366a

A89

1.0 ± 0.6a4.2 ± 0.40.00.02.5 ± 0.73.9 ± 1.03.8 ± 0.8

13.6 ± 0.6

1.3 ± 0.35.2 ± 0.70.8 ± 0.30.0

7.2 ± 1.514.6 ± 1.31.8 ± 0.50.0

8.2 ± 1.211.6 ± 0.712.0 ± 0.710.3 ± 2.5

12.7 ± 0.911.5 ± 0.910.4 ± 2.110.9 ± 0.7

15.4 ± 1.017.2 ± 0.821.2 ± 1.418.9 ± 1.0

13.7 ± 2.014.8 ± 0.420.5 ± 2.118.6 ± 1.4

25.9 ± 4.043.1 ± 5.829.8 ± 1.935.6 ± 5.017.9 ± 1.631.0 ± 5.027.7 ± 2.136.0 ± 2.0

26.1 ±2.035.0 ± 3.527.1 ± 1.233.6 ± 3.2

20.7 ± 0.236.5 ± 2.620.6 ± 3.034.1 ± 3.1

16.1 ± 2.925.5 ± 0.624.2 ± 2.926.1 ± 4.2

20.7 ± 5.336.3 ± 4.026.7 ± 7.333.5 ± 6.5

28.9 ± 1.933.5 ± 0.233.8 ± 0.948.0 ± 4.6

20.5 ± 1.121.3 ± 1.526.8 ± 1.437.7 ± 3.7

L. albusL. angustifoliusL. /uteus0. compressus

L. albusL. angustifoliusL. luteus0. compressus

L. albusL. angustifoliusL. luteus0. compressus

L. albusL. angustifoliusL. /uteus0. compressus

625 L. albusL. angustifoliusL. /uteus0. compressus

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a b c d e f q h i k m n

160 _ _ __a12.9

9.0w m

9.4-

5.0- _ .

2.9-

1.7- -_ -__mm _ _

Figure 2. Identification of sequences homologous to B. japonicumhup specific DNA in Bradyrhizobium sp. (Lupinus) strains. A, Agarosegel electrophoresis of EcoRI-digested total DNA. B and C, Autoradi-ograms of Southem blots from gels as in panel A hybridized to the5.9 kb HindlIl (B) or 2.9 kb EcoRI (C) DNA fragments from pHU52which contain hup specific DNA from B. japonicum 1 22DES. Numbersin margins indicate the molecular sizes (kb) of the hybridizing frag-ments. Sources of DNA: Bradyrhizobium sp. (Lupinus) strainsUPM860 (lane a), UPM861 (lane b), 750 (lane c), IM43B (lane d), 359a(lane f), 366a (lane g), 624 (lane h), 625 (lane i), A89 (lane j), Al 12(lane k), 466 (lane 1), 121 (lane m), L9.2 (lane n), and B. japonicumstrain 1 22DES (lane e) used as control.

an exception to this phenomenon, since most Bradyrhizo-bium sp. (Lupinus) strains examined in this study in symbiosiswith three species of Lupinus evolved large amounts of H2.The average RE of N2 fixation (percentage of electron fluxthrough nitrogenase that is allocated to N2) for the 22 strainstested in symbiosis with L. angustifolius was 0.53, which issimilar to average values reported in other legume-rhizobiacombinations (11). Unlike nodules from other legumes (26),lupine nodules still evolved H2 in an atmosphere with 10%C2H2 at rates representing an average of 10% of the acetylenereduction rates. This result corroborates the observationsmade by Gibson et al. (13) and may reflect an incompletesaturation of the nitrogenase enzyme under the C2H2 reduc-tion assay conditions.

Rhizobia have been assayed for the presence of uptakehydrogenases by different criteria, including high RE valuesin nodules, H2 uptake by bacteroids, or hydrogenase inductionin free-living cells. Most surveys indicate that the occurrence

of Hup+ strains is rather sporadic within endosymbiotic bac-teria (5, 9). In this study we report the identification of four

strains ofBradyrhizobium sp. (Lupinus) (UPM860, UPM86 1,750, and IM43B) which clearly induced H2 uptake (Hup)hydrogenase activity in symbiosis with lupines and 0. com-pressus. The three first strains were identified by screeninglupine rhizobia for DNA sequences homologous to a hupprobe containing the hydrogenase structural genes from B.japonicum. The Hup+ phenotype of strain IM43B, whichshowed no homology to B. japonicum hup DNA, was sus-pected on the basis of its high RE in symbiosis with L.angustifolius. To our knowledge, this is the first time a Hup+phenotype has been ascribed to a particular strain of Bradyr-hizobium sp. (Lupinus). Previously, Suzuki and Maruyama(27) reported that two hydrogenase activities measured by 3H2uptake under anaerobic conditions were detected in cell-freeextracts oflupine nodule bacteroids produced by an unnamedlupine rhizobial strain. One of these hydrogenase activitieswas ATP-independent and could be due to a H2-oxidizingtype of hydrogenase.Out of the 54 Bradyrhizobium sp. (Lupinus) strains exam-

ined in a colony hybridization assay, 12 strains were found tocontain DNA sequences homologous to hup-specific DNAfrom B. japonicum. Subsequent Southern hybridization ex-periments revealed that these strains fell into two differentgroups on the basis of the pattern of EcoRI fragments withhomology to two hup DNA probes carrying essential genesfor hydrogenase activity in B. japonicum 122DES (16, 25).The organization of the presumptive hup sequences is highlyconserved within the strains of each group, which is remark-able considering the different geographical origin of thestrains. However, only strains in the first group (UPM860,UPM861, and 750) showed a clearly Hup+ phenotype. Nohydrogenase activity was induced by any of the nine strainsin the second group (359a, 366a, 368a, 623, 624, 625, A89,Al 12, and 466) in symbiosis with three species of Lupinus.The inability of these putative Hup+ strains of lupine rhizobiato induce hydrogenase activity in symbiosis with lupines canbe interpreted on the basis of the host effect observed on thesymbiotic expression of the hydrogenase (Table II). This isnot only evident for the clearly Hup+ strains but also forstrain 624, which exhibited a low but consistent hydrogenaseactivity in symbiosis with 0. compressus. Partial or completesuppression of hydrogenase expression by certain host leg-umes has also been reported in Hup+ strains of B. japonicum(15), Bradyrhizobium sp. ( Vigna) (12), and R. leguminosarum(3, 7, 19). The mechanism for the host effect on Hup pheno-type is unknown. Grafting experiments suggest that both rootand shoot plant factors control hydrogenase expression (4). Itis therefore conceivable that strains in the second group differfrom strains in the first group in regulation of hup genes,being more recalcitrant to their expression by the legumehosts tested than strains in the first group.An alternative explanation for the observed lack of hydrog-

enase activity in the lupine rhizobia strains which hybridizedto the B. japonicum hup probes is the absence of a completeset of functional hup genes in these strains. This interpretationis based on the idea that many genes other than the hydrog-enase structural genes are likely involved in H2-uptake. Infact, several transcriptional units that are essential for H2activity have been identified both in B. japonicum (16) andin R. leguminosarum (23). The gene products from these

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transcriptional units are unknown, except for the two subunitsof the hydrogenase in B. japonicum (25). Also, since the 5.9kb HindIII fragment used as hup probe contains more DNAthan is required to code for the two hydrogenase subunits,there is a possibility that the observed homology was due tonon-hup DNA sequences. However, the strong homologyexhibited also by the putative Hup+ strains to a second hupprobe (2.9 kb EcoRI fragment from pHU52) argues againstthis last explanation.We expected that hup DNA sequences were conserved

within endosymbiotic bacteria, since hup DNA from B. ja-ponicum has been shown to be homologous to hup DNAfrom R. leguminosarum (18, 22) and Azotobacter chroococ-cum (28). However, the lack of homology to B. japonicumhup-specific DNA observed in the Hup+ strain IM43B, dem-onstrates the existence of two different types of hup DNAsequences within Bradyrhizobium sp. (Lupinus). Although itshould be borne in mind that the degree and strength ofhybridization could vary with the stringency of the hybridi-zation reaction, unlike other Hup+ strains of lupine rhizobia,IM43B showed no homology to two different hup DNAprobes even under low stringency hybridization conditions.There is not an obvious explanation for this lack ofhomology.A different codon usage between strains IM43B and 122DESis unlikely since no differences in homology to a nif geneprobe were detected between those strains. Alternatively, thelack of homology may reflect different physical or catalyticproperties between the enzymes from these strains. This pos-sibility is currently under investigation. More trivial expla-nations like presence of only a few percentage of Hup+ cellsin our cultures of strain IM43B were eliminated, since no

homology to hup DNA was observed in either DNA frombacteriods or a reisolate from lupine nodules produced byIM43B.The occurrence of more than one type of hup sequence

within endosymbiotic bacteria obviously limits the generaluse of hup DNA probes to screen for Hup+ phenotype. Infact, we cannot eliminate the possibility that some strains,other than IM43B, are also hydrogenase positive within the42 strains showing no homology to the structural genes of theB. japonicum hydrogenase. Criteria based on hydrogenaseinduction tests in nodules may also underestimate the occur-

rence of Hup+ strains since the expression of hup genesdepends upon the appropriate choice of the test legume host.

ACKNOWLEDGMENT

We are grateful to J. C. Burton, 0. A. Berestetsky, C. A. Parker,G. Jager, H. H. Keyser, C. Rydin, D. C. Jordan, D. Cornet, E. B.Roslycky, and F. Temprano for providing Bradyrhizobium sp. (Lu-pinus) strains and to HJ Evans for cosmid pHU52.

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occurrence bradyrhizobium sp. (lupinus) their spp. ornithopus … · dyrhizobium sp. (lupinus) from...

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