THE BACTEROIDS OF THE GENUS RHIZOBIUM'

D. C. JORDANDepartment of Microbiology, Ontario Agricultural College, Guelph, Ontario, Canada

I. Introduction ............................... 119II. General Considerations................................................................... 120

A. Definitions ........................................ 120B. Production and Occurrence within Nodule ........................... 120C. Reproduction ........................................ 122D. Role and Fate......................................................................... 122

III. Cytology................................................................................. 123IV. Biochemical Properties........................................ 127

A. Chemical Composition ..................................... 127B. Respiration and Cytochrome System .............................. 128C. Porphyrin and Heme Synthesis ................................ 131D. General Metabolism ............................ 132

1. Hydrogenase........................................................................ 1322. Vitamin B12......................................................................... 1343. Nitrate reductase ........................................ 1344. Activation of ammonia.............................................................. 1355. Glutamine synthetase........................................ 1356. Transamination........................................ 135

V. Summary and Concluding Remarks ........................ 136VI. Acknowledgments......................................................................... 137VII. Literature Cited .............................. 137

I. INTRODUCTION

One of the most significant microbiologicalprocesses in agriculture is the production ofnodules on the roots of leguminous plants bybacteria of the genus Rhizobium (generally knownas the rhizobia), since this results normally in theconversion of atmospheric nitrogen into uti-lizable forms. These small rodlike bacteria enterthe plant roots via the root hairs, become en-cased in a tubular infection thread, and progressinto the cortex. The liberation of the rhizobiainto the cortical cells of the host often initiatesnodule development. If the intimate associationbetween the bacteria and the host cells is destinedto result in effective nitrogen-fixing tissue, thenthe bacterial cells undergo a drastic morpho-logical change, becoming enlarged, vacuolated,and in some cases distinctly branched. At theend of the active life of the nodule these aberrantrhizobial cells, the so-called bacteroids, apparentlyundergo disintegration.

After Ward (111) and Beijerinck (15) firmly1 The major portion of this review was written

at the University of Nottingham and Rotham-sted Experimental Station while the author wasthe holder of a Nuffield Fellowship.

established the bacterial nature of the causativeagent of nodulation a great bulk of literaturebegan to accumulate on the morphological,cultural, and general biochemical properties ofthe rhizobia. The mass of data was such that in1932 Fred, Baldwin, and McCoy (48) were ableto devote over 80 pages of their publication tothe properties of these particular bacteria, andthe recent steady flow of information in thisfield has occasioned the appearance of a mono-graph (115) and a number of important generalreviews, including those of Wilson (112) andAllen and Allen (2, 3). As might be expected,the greatest portion of this research has beendevoted to the free-living rhizobia, possibly be-cause of a considerable interest in their cultiva-tion for the artificial inoculation of leguminousseeds and the previous lack of published methodsfor the extraction of intact bacteroids fromnodule tissue. This has resulted in a relativepaucity of information regarding the bacteroids.Indeed, in the older literature mention is madeof only their shape and general appearance, theirproduction and ability or lack of ability to re-produce, and the identity of certain intracellulargranules. Since the bacteroids usually represent

119

D. C. JORDAN

the dominant bacterial cell type within thenodules during the active period of nitrogenfixation (a process which in the symbiotic sys-tem cannot be carried out by plant or bacteriumacting separately) their physiology assumes con-siderable importance. Therefore, the presentarticle constitutes a brief review of pertinentreports appearing subsequent to the year 1932,in the hope that delineation of some of theunanswered problems will stimulate furtherresearch in a field still filled with rather fascinat-ing possibilities.

II. GENERAL CONSIDERATIONSA. Definitions

The identitv of the bizarre bodies in the innercells of legume root nodules was obscure to theearlier workers, who believed them to be prod-ucts of plant metabolism. Consequently, theywere referred to by such indefinite names asSprnsszellen or vibrio-ahnlichen Korper (48).Brunchorst (29), who believed them to be pro-tein bodies, introduced the word "bacteroid"(bacteria-like) which, although inapt, is in com-mon use today. Forms morphologically similarto nodule bacteroids can be produced in culturedstrains of rhizobia by the addition to the growthmedium of plant extracts, glycerol, organicacids, and a number of other biochemical agents,as well as by bacteriophage attack and bymodification of the physical environment (48).Thus the general bacteroid shape represents oneof the end results of a syndrome initiated by awide variety of physiological stresses.

Unfortunately the term "bacteroid" has beenused in various senses. According to Fred et al.(48), Dangeard considered only branched formsunder this name, thus excluding those linearforms found in nodules such as those producedon the soybean or cowpea. Other authors (18,81) regarded the unbranched bacteria fromsoybean nodules as morphologically similar tocultured cells, although I have found theseformer cells, after negative staining with nigrosin,to be definitely enlarged as compared with thefree-living forms. Fred et al. (48) defined bac-teroids as "the enlarged, frequently club-shapedor branched, vacuolated or banded forms of theroot nodule bacteria, both as they occur in thenodule and in culture media." The small bandedcells normally appearing in older cultures onsolid or in liquid media were excluded. This

definition, based as it is on morphological con-siderations only, will be accepted for the purposesof this particular review, with the qualificationthat the bacteroids produced in laboratory mediawill be designated as being artificially produced.This may not be merely an academic point.It is not known if naturally and artificiallyproduced bacteroids are exactly equivalent bio-chemically. Morphologically similar bacteroidscould be produced by a blockage at any one ofa number of points in a sequence of enzymaticsteps, such as might lead to cell-wall synthesis,for example. Blockage at one particular pointin this sequence, however, might be a necessaryprerequisite to the commencement of nitrogenfixation in nodule tissue. Thus it becomes con-ceivable that many types of bacteroids producedartificially might not be able to function in thenitrogen-fixing system, even if all the otherrequirements for the system were met. Thispoint cannot be checked experimentally as yet,but until the hypothesis is proved untenableattempts to integrate data obtained from thesetwo types of bacteroids should be viewed withsome degree of caution.

B. Production and Occurrence within Nodule

The formation of bacteroids from normal cellswas described most simply by Lewis (69) whostated that "the cells appear to lose the capacityfor cellular division while retaining the capacityfor growth and deposition of fat bodies and be-come transformed into . . . swollen bacteroids."The nodular agents causing these changes arenot known and the phenomenon can be beststudied in the laboratory. In fact, the artificialproduction of bacteroids by the growth of rhizobiain various media received considerable earlyattention. Since that time bacteroids have beenobserved during the growth of cowpea rhizobiain asparagus-mannitol broth (32), and formerreports that swollen cells are produced by thegrowth of rhizobia on caffeine-containing mediahave been confirmed (89). The high alkaloidcontent of several plant-extract media employedfor the growth of rhizobia has been implicated asa cause of bacteroid formation and has stimu-lated studies on the influence of caffeine,strychnine, and other alkaloids on culturedrhizobia isolated from species of Phaseolus,Trifolium, and Astragalus (58, 59). In general,the distorted forms were produced in greater

120 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

numbers under conditions of poor growth.Lojtjanskaja (71) was convinced that a directcorrelation existed between alkaloid content oflupine nodules and number of bacteroids. Bac-teroids also have been observed in a mediumdegraded by previous growth of rhizobia (41),in media containing blood (54) or large amountsof thiamine (77-79) and during the associativegrowth of the root-nodule bacteria with eitherazotobacter (75, 76) or an unknown facultativeanaerobe (55). However, the basic mechanismshave not been elucidated.The factors initiating bacteroid formation

within the nodule do not exert their activityagainst those bacteria which remain within theconfines of the infection thread (86, 97).In some cases the genetic constitution of the

host plant exerts a considerable influence onbacteroid development. This was emphasized byBergersen and Nutman (23) who examined thestructure of nodules produced by strains ofrhizobia on red clover homozygous for two in-dependent recessive genes, il and ie, both ofwhich caused failure in nitrogen fixation. Theineffectiveness due to the host factor ii was foundonly in a symbiosis involving the normallyeffective strain A of rhizobia used in this work,whereas ineffectiveness associated with ie wasnot bacterial strain specific. Both of these in-effective responses were correlated with theinability of the invading bacteria to producebacteroids, so that under these conditions bac-teroid formation was dependent not solely oneither bacterium or host, but upon a complexinteraction between the two entities.

Longitudinal sections of mature nodules revealfour zones of tissue differentiation: a distalmeristematic zone, a central bacteroid zone, aperipheral cortical laver, and a vascular system.The central bacteroid region represents from 16%to just over 50% of the total dry weight of thenodule (28, 81, 101) and its mean relative volumeis much greater in effective nodules than inineffective nodules (34). In some instances thevolume of the bacteroid region reaches a maxi-mum early in the life of the nodule and there-after remains relatively constant, so that as thenodule enlarges the ratio of this volume to thetotal nodule mass decreases (6). With regard tothe intranodular distribution of the bacteroids,Heuman (52) pointed out that the maturebranched forms were found only in the central

cells of the bacteroid region of pea nodules. In afurther study (54) he considered four differentzones in longitudinal sections of pea nodules(tip, transitional zone, central zone, and base)and estimated the percentage of bacteroids in thetotal bacterial population of each of these zonesas 0, 70, 93, and 45%, respectively, the remain-ing cells being rod forms. Characteristically,this distribution is true of most, if not all, legumenodules since the infection proceeds outwardfrom the bacteroid zone by extension of theinfection thread, which contains only the smallrod forms of rhizobia, or by division of recentlyinfected cortical cells whose bacterial componenthas not yet become "bacteroidal." Only smallrods are present in young nodules which havenot burst the root epidermis (52), indicating thatnodule formation commences before bacteroidinitiation.The deoxyribonucleic acid (DNA) of cells

vitally stained with acridine orange and ex-amined by ultraviolet microscopy fluorescesgreen or yellow depending upon its concentra-tion. Depolymerization of the DNA results in acopper-red color. Consequently, by this methodliving bacterial cells appear predominantly greenor yellow, whereas dead cells appear red. Usingultraviolet techniques Heuman (54) gave thepercentages of living bacteroids in the transi-tional zone, central zone, and base of pea nodulesas 60, 35, and 20%, respectively, so that underthe conditions employed the distribution ofliving bacteroids within these nodules did notexactly parallel that of the total number ofbacteroids. However, the understandable in-fluence of nodule age on the percentage of livingbacteria in nodules has been shown by similarwork involving Lupinus luteus, Ornithopus sativus,and Trifolium pratense (92). During the entirevegetative period the percentage of living bac-teria was the same, amounting to 97 or 98% ofthe total bacterial population, but when thenodules turned brown at the end of their produc-tive period increasing numbers of dead bacteriawere noticed. As a matter of interest, the bac-teroid contents of the nodules of L. luteus and0. sativus were quite small, amounting to 1.7to 4.5% and 0.30 to 0.62%, in comparison withthe rod forms present, whereas in the nodules ofT. pratense the bacteroids were present inlarger numbers.We are on relatively firm ground with respect

1962] 121

D. C.JORDANvL

to the viability and location of bacteroids withinthe nodule, and the statement that the majorityof these forms are initially living and locatedin a mass of host cells centrally located withinthe nodule is not likely to be disputed.

C. ReproductionAlthough many of the early investigators con-

sidered bacteroids to be degenerate or involutionforms incapable of multiplying, others believedthat such cells were capable of reproduction byprocesses such as budding, or the release ofspores or coccus-like cells. The best attempt tosettle the controversy was made by Almon (11)who employed single-cell isolation techniquestogether with eight different media. Of 411bacteroids isolated from suspensions preparedfrom the nodules of pea, red clover, and alfalfa,only 1 cell multiplied, and this multiplicationwas attributed to faulty technique. Of 163"normal" rods isolated from similar suspensions,25 grew. Only 1 cell multiplied of a total of 10artificially produced bacteroids isolated from abroth culture, whereas 22 of 83 rod forms iso-lated from laboratory media underwent division.In addition, 75 bacteroids and 44 rods from clovernodules were located on the numbered squares ofa special cover slip previously spread with agarmedium and a dilute cell suspension. After 4 days,4 of the rod forms, but none of the bacteroids,had produced colonies. Almon concluded justi-fiably that if bacteroids reproduce they do soonly rarely. The fact that many of the rod formsdid not grow suggests that many of these cellsalso are incapable of growth or, more probably,that optimal growth conditions were not present.Single cells of many bacterial species are oftenquite difficult to grow, even in rich media. Sincemore success was obtained in the growth of therods isolated from laboratory media rather thanfrom nodules, this might imply that a differencein viability existed between the rod forms fromthe two sources, but the inability to distinguishbetween nodular rod forms and immature bac-teroids conceivably could account for this dif-ference.

Almon's work usually is considered to be thefinal word on the reproductive ability of bac-teroids, although her viewpoint still is not sharedby all.2 The most recent claim for bacteroid mul-

2 De Fonbrune (46a) mentions that he success-fully grew isolated bacteroids in an oil chamber,

tiplication has been put forth by Heuman (54),but the photographic evidence is not entirelyconvincing.

D. Role and Fate

The growth of legumes under appropriateconditions can be quantitatively related to themass and duration of the bacteroid-containingtissue formed within the nodules (34), suggestinga relationship between this tissue and nitrogenfixation. Virtanen (105) considered that thepresence of bacteroids in the nodule was essentialfor fixation and this view was partially sub-stantiated by the findings relating failures innitrogen fixation in certain nodules to the in-ability of the rhizobia to produce bacteroids(23). Furthermore, a direct relationship has beenestablished between the initiation of bacteroidformation and a rapid increase in the amount ofnitrogen fixed by soybean nodules (19). Furthercircumstantial evidence is provided by the find-ing that bacteroids are only present in thosehost cells which contain a type of hemoglobin,the concentration of which bears a direct rela-tionship with nitrogen fixation (63, 108, 109), andby the observation that certain cytologicalchanges in the bacteroids, such as occur uponcontinuous illumination of the host plant (46),can be correlated with reduced nitrogen-fixingability (17). Bacteroids can be found in someineffective nodules, but this does not invalidatethe relationship postulated between these bac-terial forms and nitrogen fixation because in-effective nodules often fix nitrogen initially,although the ability soon is lost because of thepremature decomposition of the bacteroid area(34, 97). Moreover, there is no reason to inferthat if bacteroids are necessary for fixation thisprocess must occur when bacteroids are present.Some other necessary component in the systemmay be lacking.A positive relationship between the numbers

of bacteroids and fixation is not always found.Although an apparent connection has been notedbetween increasing numbers of bacteroids inthe nodules of Lupinus and Ornithopus spp.

using a medium partially degraded by the previ-ous metabolism of such cells. However, the neces-sary details are lacking and I have been unable toestablish either the source of these cells, theirmorphological characteristics, or what the authorimplied in his use of the word "bacteroids."

122 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIU.M1

and progressive fixation of nitrogen, no suchrelationship has been detected in the nodules ofT. pratense (92).There is some reason to believe that the bac-

teroids are implicated only indirectly in nitrogenfixation. Bergersen (21) has hypothesized thatthe function of the bacteroids is threefoldin that they induce the production of nitrogen-fixing structures by the plant tissue, providereducing power for the production of ammoniafrom free nitrogen, and provide ammonia-accept-ing compounds.

In the past the fate of the bacteroids within thenodule has been a matter of considerable interest,particularly because of a postulate, no longertenable, that fixed nitrogen is transferred to thehost plant only through the decomposition ofthe bacteroids. Bacteroid breakdown occurs atthe termination of the active life of the nodule,usually coincident with the nodule necrosisoccurring upon flowering of the host plant. Thisnecrosis commences in the innermost cells ofthe bacteroid region (51) or near the base of thenodule (98) and progresses distally. Eventually,at least in certain nodules, the rod forms of therhizobia attack the host cell walls and pass intothe middle lamellae, which consequently swell(96, 98). Finally the nuclei of the host cellsdisintegrate, the cell walls collapse, and theentire central area of the nodule is destroyed.One of the first indications of nodule break-

down is a mottled condition in the inner cells ofthe bacteroid area, presumably due to a clump-ing of the rhizobia (5). The bacteroids then losemuch of their strongly staining cytoplasm,apparently by an internal digestive process,and are transformed into lightly staining vesicles,containing granules which initially stain deeply,but which soon either lose their staining abilityor disappear (96). The final result is death anddissolution. Schaede (84) studied this digestionof bacteroids by observing their release of"albuminous" substances and reported that theprocess as it occurred in the nodules of Lupinusalbus was similar to that just described, but thatin Vicia faba nodules the older rhizobia prema-turely lost their stainability toward fuchsin be-fore any definite evidence of internal digestionoccurred. The cells then shrunk and at thetermination of the digestive process all thatremained were the lightly stained and crumpled

cell membranes, which agglutinated to formflocculent masses.A premature dissolution of the branched bac-

teroids of pea nodules has been described afterthe plant roots were placed in an atmosphereconsisting of a 2:1 mixture of hydrogen andoxygen, a process which also destroyed thenodule hemoglobin (53). These changes were notreversed when nitrogen was supplied, but newnodules formed were normal.

Although the reason for the breakdown ofbacteroids is largely conjectural, several processes,acting alone or in combination, have been im-plicated. Vacuolation and ultimate disintegrationof bacteroids by bacteriophage were observed byDemolon and Dunez (40) and others (104) inthe nodules of several legumes. This process hasbeen considered to be the cause of the diseaseknown as alfalfa fatigue (la fatigue des luzernieres)(39). Alternatively, the degenerative phenomenonmay be simply a form of autolysis following thedeath of the cells by reason of carbohydratestarvation. Such a limitation in energy supply hasbeen offered as the reason for the invasion of thehost cell walls by the rod forms of the rhizobiaduring nodule senescence (98). Another possibilityis that the breakdown is the result of the activityof proteolytic enzymes secreted by old host cells.This potentiality might be clarified by thepreparation of bacteria- and bacteriophage-freejuice from carefully crushed senile nodules anda study of its ability to digest bacteroids fromyoung healthy nodules.

III. CYTOLOGYA knowledge of normal cytology is essential

for the understanding of abnormal cytology,but unhappily our concept of the "normal"cytology of the rhizobia is clouded by uncorrelatedand often conflicting data. Controversies oversome of the aspects have extended over a periodof 40 years.Young cultured cells of the rhizobia are small

gram-negative rods (but compare (26)), oftenmotile, and staining uniformly with simplebasic stains. Their DNA has been described aseither nonlocalized and diffuse (73, 85, 87) orlocalized as discrete nucleoids (14, 16, 110).It is believed also (38) that both conditions mayresult in young cells, the cells bearing thenucleoids being rather rare. Baylor et al. (14)were uncertain whether the granules they ob-

1962] 123

D. C. JORDAN

served in soybean rhizobia were lipoid or nu-clear material, although treatment with 1.0 Msodium chloride, which solubilizes nucleoprotein,produced cleared areas in the cytoplasm andsuggested that some type of nuclear substancewas present. In older cultures, rhizobia becomeswollen and exhibit a banded condition uponsimple staining. Lewis (69) considered thesebasophilic bands to be cytoplasm compressedinto strands by lipids accumulating in the cyto-plasm as unstained refractile globules. However,Bisset and Hale (27) considered the bands to becross walls modified by the deposition of stain-able material and thought that in certain strainsof rhizobia the unstained areas representedsites for the production of coccoid swarmers.Reports on the nuclear structure in the barredrhizobia are scanty, but what appear to bepaired nucleoids have been discovered in theindividual cells contained within the large mul-ticellular swarmer-producing cells (26). Theunusual report (26) of heat-resistant endosporesin the swarmer-producing rhizobia has not beenconfirmed by others (J. Kleczkowska, unpub-lished data).

In contrast to cultured rod forms, bacteroidsare devoid of flagella (27) and although generallydescribed as gram-negative they often containintracellular granules and bars which stain gram-positive. Some bacteroids have been depicted aspossessing cross walls (26), although they usuallyappear unicellular. It has been known for sometime that the shape of these cells is characteristicfor certain strains or species of rhizobia and atleast three major morphological groups can bedistinguished, even by the casual observer. Thebacteroids from Trifolium spp. are predominantlypearshaped. Those from cowpea, soybean, lupine,and related legumes are only slightly swollenrods, whereas those from many other commonlegumes constitute a mixture of swollen andelongated rods with varying numbers of branchedforms. These branched cells have led some authorsto suggest a relationship between the rhizobiaand the corynebacteria (60, 61). Curiouslyenough, the nodule bacteria of Sesbania gran-diflora are typical rods regardless of the age ofthe nodule (51), and there must be some doubtwhether such cells should be regarded as truebacteroids.The initial work on the intracellular cytology

of bacteroids was confined almost exclusively

to the highly refractile granules observed insome cells, which have been considered to belipid deposits (4, 69). Fred et al. (48) believedthat the fatty material extracted from suchcells by chloroform was a polymer of hydroxy-butyric or hydroxyvaleric acid. This was con-firmed recently by Forsyth, Hayward, andRoberts (47) who discovered that all the cul-tured rhizobia they examined possessed largeamounts of intracellular /3-hydroxybutyric acidpolymer. Since crushed nodules of Mimosa pudicaand a species of Pueraria also contained thismaterial, the authors concluded that the sudan-ophilic granules observed in the bacteroids ofthese plants were composed of this polymer,although they did not separate the bacteroidsfrom the host plant tissue to ascertain theamount present in the bacteria alone. They be-lieved that the polymer had been confused pre-viously with "true lipids" in some bacteria andwith stages in the supposed life cycle of rhizobia.These granules, if analogous to those present inBacillus megaterium (93), represent energy-richstorage material and possess no respiratory ac-tivity. But bacteroids do contain granules func-tional in oxidation-reduction processes. In thisconnection it was shown (16) that particles withinthe bacteroids from nodules of Trifolium sub-terraneum reduced Janus green B and tetrazoliumchloride and gave a positive Nadi reaction atpH 5.7, but only occasionally at higher pH values.These same bodies yielded a positive Gomori testfor alkaline phosphatase and were few in num-ber in the bacteroids from very young nodulesand from mature ineffective nodules. The reduc-ing sites in soybean bacteroids were confined tothe poles where they could be stained by osmicacid and Sudan black B (22), intimating thepresence of a lipoid component. In ultrathinsections of these cells the polar sites appeared asempty spaces, the contents evidently beingextracted during embedding. The exact relation-ship between the poly-13-hydroxybutyrate gran-ules and those granules functional in redox reac-tions is not known, but presumably both typesof bodies occur.

Electron microscopy of intact bacterial cellsusually reveals very little of their internalstructure. Nevertheless by this means two prin-cipal types of soybean bacteroids have beendistinguished (14). The first type comprised30% of the bacteroids in the cell suspensions

124 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

examined and consisted of cells having two denseinternal bodies connected by a band, thus form-ing a dumbbell-like structure, whereas thesecond type was comprised of cells with electron-dense ends, probably the reducing granules, andconsiderable internal vacuolation. After stainingwith hematoxylin, such cells showed three darkbands under the optical microscope, but notunder the electron microscope. The internalstructure of sweet clover bacteroids was not aseasily revealed as that of the cultured rods. Novacuolation was encountered but exposure toosmium tetroxide yielded an internal spiraleffect similar to that observed in cultured cells.Most of these data are difficult to interpret, butit should be noted that these recorded observa-tions were limited to rather small cells, suggestiveof "pre-bacteroidal" rods and it is regrettablethat the larger forms of the sweet-cloverbacteroids were not studied.

Bacterial suspensions prepared from crushednodules generally reveal a variety of cell typesunder the electron microscope. Bacteria fromT. subterraneum were shown (62) to range fromsmall electron-dense rods, through elongatedrods with a low degree of vacuolation, to large,swollen cells which were 50% or more electrontransparent (Fig. 1 and 2). The scatteredremnants of electron-dense material in thesecells gave the impression of banding and granula-tion under the optical microscope. The entireeffect was strongly suggestive of a gradualinternal digestion of the protoplasm. Bac-teriophage could be demonstrated in several ofthe cell suspensions and what appeared to beintracellular or surface-adsorbed phage particleswere observed in some instances (Fig. 3).The nuclear apparatus of bacteroids has re-

ceived some attention since Milovidov (73)demonstrated that the bacteria from clover andlupine nodules gave strongly diffuse DNA reac-tions after acid hydrolysis. Uher (103) stainedsmear preparations and sections of nodules ofT. pratense with iron hematoxylin and the Feulgenmethod and described a wide variety of nucleatedcells, including "sporangia," "symplasm," bi-and multinucleated types, and small rods. Thespindle-shaped nuclear bodies reminded Uherof mitosis in higher plants, whereas other typeswere reminiscent of biskuiitformige amitosis. Themajority of the branched forms were devoid ofnuclei. One might expect, however, that confu-

sion would occur in such work in attempts todistinguish bacterial forms from plant cell ma-terial, especially in the smear preparations.Schaede (87) remarked that the bacteroids ofV. faba and Pisum sativum were not stained bythe Feulgen technique, whereas those fromlupine nodules gave a diffuse reaction whichdiminished as the cells underwent digestion.On the other hand Heuman (54) observed thatan average of 42% of the bacteroids from peanodules contained Feulgen-positive nucleoids,the remainder yielding a diffuse reaction. Usingfluorescence microscopy, an age influence wasuncovered when it was noted (38) that thenuclear material was diffuse in bacteroids fromyoung active nodules and inactive nodules andthat nucleoids appeared only later. These nu-cleoids were described as being 0.3 to 0.5 ,.in diameter and situated at the base of the cellbranches. Their number varied between oneand four per cell, depending upon the degreeof cell branching, and they fluoresced light greenin a yellow-green protoplasm when acridinewas employed. The scarcity of discrete nuclearbodies in cultured cells perhaps accounts for amention (2) that Feulgen-positive reactions wereobtained more readily in bacteroids from peanutand clover than in smears of young and oldcultured cells.

Nuclear material in the form of axial strands,which branch in branched cells, has been demon-strated in the bacteroids of T. subterraneum(16). These strands, which eventually broke upinto discrete bodies, developed from pairednucleoids present in the small rod forms duringbacteroid formation. Alkaline phosphatase, pres-ent in plant and mammalian nuclei, could notbe demonstrated in the chromatin and this factmay have a bearing on the absence of reproductiveability in bacteroids. There formed ultimately acurious perinuclear area, unstainable by theacid-Giemsa method, but stainable by Giemsaalone. Bacteroids from mature ineffective nodulesof Trifolium ambiguum were identical to thosefrom very young effective nodules in that theyshowed no such areas (17). Either the age of thebacteroids or the difference in the species ofhost plant might be responsible for the findingthat the nuclear material in soybean bacteroidsexisted in two configurations: as spherical bodieswith unstained perinuclear areas and as elongatedstructures without such areas (22). Electron-

1962] 125

1D. C. JORDAN

1A

2

IA4

5fiAi'. 6

FIG. 1-6. Bacterial cell types from nodules of Trifolium subterraneum L.FIG. 1 and 2. Small rods and bacteroids from a suspension prepared from crushed nodules. Osmium-fixed.FIG. 3. Cells showing what appears to be intracellular or surface-adsorbed phage. Phosphotungstic-acid-

negative stain.FIG. 4. Bacteroids, one of which shows a bleb in the outer membrane. Osmium-fixed. The bleb may be a

weakened elastic section of the cell wall forced out by internal turgor pressure or it may be a portion of theprotoplast protruding through an opening in the wall. %

FIG. 5. Spherical form. Phosphotungstic-acid-negative stain. At least one extracellular phage particle ispresent also. The flagella belong to small rod forms present in the suspension.

FIG. 6. Development of a spherical form from an'elongated cell. Phosphotungstic-acid-negative stain. Theflagella belong to small rod forms present in the suspension.

micrographs of ultrathin sections of these soy- without outer membranes. The perinuclearbean organisms showed the nuclei as typical regions, when present, were diffuse and electronelectron transparent matrices containing small dense.osmiophilic granules and intertwined filaments, Differences in the degree of dispersion or

126 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

agglomeration of bacterial DNA have beenattributed to such factors as accumulation ofintracellular inclusions, increasing cell age, andvariations in the concentration of inorganicsalts in the suspending medium. A comparison ofthe effects on the nuclear morphology of therhizobia of the preparatory techniques used bythe various workers might be revealing. Atpresent the over-all image formed from the pub-lished data is that of small rod forms with nuclearmaterial dispersed throughout the cytoplasmunder some conditions but condensed to formdiscrete nucleoids under other conditions. Duringbacteroid formation there is an apparent inhibi-tion of cell division and a continued synthesisof chromatin, which condenses, elongates, andeventually breaks up into small masses. The"maturation" of some bacteroids is associatedwith the appearance and enlargement of peri-nuclear areas, perhaps representing acid-extract-able decomposition products of the chromatin.This supposition is supported by the disclosure(16, 87) that the amount of nuclear materialdiminishes as the cells age. In old bacteroids,just before complete dissolution, the bulk of thenuclear substance is probably entirely digested,accounting for the several reports of no Feulgen-positive material in mature cells of this type.

Recently, the rather unusual report was madethat no cell walls could be demonstrated in intactcells or ultrathin sections of isolated soybeanbacteroids, although walls were present on thebacteroids within the nodules (22). The formerobservation was modified subsequently to in-clude about 70% of the bacteroids (20), but thisfinding is still in contradistinction to resultsobtained from studies on the bacteroids fromsubterranean clover (16, 62). If many of theisolated bacteroids from soybean are truly devoidof a cell wall then it becomes difficult to visualizewhy there is no rounding up of the resultingprotoplasts, which seem to maintain their usualrod shape. Surely some rigid remnant of the wallmust be present, temporarily at least, even if itwas not detected by the methods employed.The cell walls supposedly left behind in one of thecentrifugal fractions obtained from nodule debrisafter removal of the bacteroids (22) could havebeen derived from bacteroids which had under-gone normal degeneration or had been disruptedduring the nodule-crushing procedure used toprepare the cell suspensions. Still, there is

evidence of localized weaknesses in the cell wallsof many mature bacteroids (62), as shown bythe presence of blebs (Fig. 4), and progressiveweakness of the wall could account for thespherical protoplast- or spheroplast-like entitieswhich have been observed in ineffective red clovernodules produced by the Coryn strain of rhizobia(34). Such forms also have been seen among thecells of Rhizobium trifolii growing in a mineral-salts medium containing 0.5% yeast extract andin suspensions prepared after dialyzing bacteroidsfrom crushed nodules of soybeans and sub-terranean clover against distilled water (62)(Fig. 5 and 6). The swelling and eventual ruptureof these osmotically sensitive structures couldaccount for the changes in optical density oftenobserved when freshly isolated bacteroids aresuspended in various hypotonic fluids.

IV. BIOCHEMICAL PROPERTIES

A. Chemical CompositionIn one of the more intensive studies on the

chemical composition of rhizobia, Rautanenand Saubert (81) analyzed bacteroids obtainedby homogenizing the interiors of nodules in0.5 M sucrose, removing the host cell debris bylow speed centrifugation, and collecting thesediment formed in 30 min at 20,000 X g. Theprocedure was similar to that previously employedby other workers (95). Soybean bacteroids con-tained 64.9% of the total dry weight of thewater-insoluble portion of the nodule, 47.3%of the total nodule nitrogen, 44.1% of the totalprotein, 45.1% of the total phosphorus, and41.5% of the total iron. The content of nucleicacid, nitrogen, and phosphorus of each of threefractions (hot and cold trichloroacetic acid ex-tracts and remaining insoluble residue) of soy-bean and cowpea bacteroids then were compared.The major difference lay in the elevated nitrogencontent of the insoluble proteinaceous residue ofthe cowpea organisms as compared to that de-rived from the bacteroids of soybean. When thebacteroid compositions were compared with thoseof 4-day-old cultured cells, the latter were foundto contain more phosphate, a result attributed tothe high phosphate content of the growth mediumemployed. Various other minor differences wererecorded, but the authors emphasized the greaternucleic acid contents of the cultured cells con-trasted with those of their respective bacteroids,particularly with the soybean rhizobia. Such a

1962] 127

D. C. JORDAN

difference in nucleic acid content would appearto confirm the cytological observations relatedto the gradual disappearance of nuclear materialduring bacteroid aging. Unfortunately, suchdifferences are difficult to evaluate because thecomposition of laboratory-grown rhizobia isknown to vary with age, as well as with the com-position of the growth medium (57). Because ofsuch fluctuations it is extremely unlikely thatanalytical values derived from cells in any onestage of their growth can describe adequatelyconditions within the cells over the entire growthcurve. It is true that the chemical compositionsof different species of microorganisms can becompared when the cells have reached the samephase of growth in similar media, but the dif-ficulties to be overcome to synchronize both theages and nutritional backgrounds of bacteroidsand cultured cells can be appreciated.

Bergersen (19) showed that changes occurredin both the dry weight and nucleic acid contentof soybean bacteroids as the nodules aged. Duringthe first 2 weeks of nodule development, bac-terial proliferation occurred and the dry weightof the bacteria decreased, possibly due to de-creasing cell size. After the cessation of growth,bacteroid formation took place, after which timethe dry weight increased for a period of 1 week,and thereafter declined until an unexpected riseensued at a nodule age of 5 weeks. Since the"total nitrogen" content of the bacteroids re-mained relatively constant throughout the 6-week observation period, it was suggested thatthe changes in dry weight represented changes innon-nitrogenous cellular constituents. The nucelicacid content ranged from 3% of the cell dryweight at a nodule age of 1 week and again at6 weeks to 10.5% at an age of 4 weeks. Thelower figure is similar to that of Rautanen andSaubert (81). These fluctuations in nucleic acidcontent were somewhat similar in magnitude tothose which took place in cells of the sameorganism grown in fluid culture. Further studyshowed that the ratio of acid-soluble nucleotidesto nucleic acid in the bacteroids was 0.12 to 0.13during nitrogen fixation and resembled valuesobtained for cultured cells in the mid-logarithmicstage of growth.

Consequently, no very definite changes inchemical composition have been observed thusfar during the conversion of the rod forms ofrhizobia into bacteroids. During this transitional

period, the bacteroids increase in dry weight andnucleic acid content (19, 118), suggesting thatthere is at least a temporary continuation ofsome types of cellular syntheses.Although 16 to 17% of the high content of

riboflavin in soybean nodules is present in thebacteroids (101), it is not known how this valuecompares to the average riboflavin content ofcultured cells. Such a comparison might beinteresting in view of the role of riboflavin in thesynthesis of certain respiratory enzymes.

B. Respiration and Cytochrome SystemThe nodule bacteria are believed to utilize

only a small proportion, estimated at 3 to 6%,of the total carbohydrate synthesized by thehost plant (7, 10). Using data relative to therelease of CO2 from intact soybean nodules, oneworker calculated that 19 mg of carbohydratewere consumed within the nodule for eachmilligram of nitrogen fixed, and that of thisamount 15 mg were utilized by the bacteria (28).This oxidation of carbohydrate probably playsan important role in nitrogen fixation, possiblysupplying reducing power for the conversion ofnitrogen into ammonia (21, 45).

Studies on the oxidative capacity of rhizobiawere initiated by the finding that both bacteroidsand cultured cells of pea, soybean, and cowpearhizobia could utilize glucose, arabinose, sodiumfumarate, and sodium pyruvate (94) and thatcultured cells possessed higher Qo2 values thanbacteroids. Later it was indicated (114) thatbacteroids possessed a higher endogenous respira-tion than cultured cells and, moreover, that bothcells types of soybean and cowpea rhizobiashowed increased respiration upon the additionof lactate, malonate, and maleate. The equivalentcell types of the pea rhizobia, however, showedno particular responses to these three carbonsources.With those bacteria which utilized malonate

and maleate, both known inhibitors of succinicdehydrogenase in other cells, neither of thesecompounds, even at a concentration of 0.1 M,inhibited glucose oxidation, whereas the respira-tion on glucose in the presence of lower concentra-tions of these substances was additive. This lackof inhibition is compatible with the assumptionthat under certain conditions the glucose isoxidized via a C-1 preferential pathway, ratherthan by the tricarboxylic acid cycle.

128 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

Succinate is considered to be one of the bestsubstrates for oxidation by both cultured cellsand bacteroids (19, 95, 115). Burris and Wilson(30) found that pea and vetch bacteroids respiredon this substrate at higher rates than culturedcells, although the reverse was true for soybeanrhizobia. They pointed out that the rapid oxida-tion of succinate might suggest that a four-carbondicarboxylic acid cycle operates in rhizobia,but that this is doubtful because of the failureof the four-carbon dicarboxylic acids to show anincrease in oxygen uptake on glucose above thatexpected from their direct oxidation. The utiliza-tion of succinate implies the presence of succinicdehydrogenase, thus accounting for the observedcompetitive inhibition of succinate oxidation inrhizobia by malonate and maleate (114). Theparticipation of the tricarboxylic acid cycle, inaddition to a pentose phosphate shunt, in theintermediary metabolism of the rhizobia mightbe suspected, but few specific facts are available.The oxidation of the sodium salts of succinic,

fumaric, malic, acetic, citric, and pyruvic acidsby bacteria obtained from soybean nodules ofdifferent ages has been followed by Bergersen(19). The depression occurring in the oxygen up-take 2 weeks after the nodules first appeared wasfollowed by a sharp rise corresponding to theonset of bacteroid development and rapid nitro-gen fixation; then after a plateau a further riseoccurred 1 week before nodule degeneration.This respiratory activity varied somewhat, de-pending upon the growth environment of thehost plant. The bacteroids failed to oxidize theacids of the tricarboxylic acid cycle to completion,but the number of moles of oxygen consumedper mole of substrate did not change appreciablyduring the life of the nodule and was similarto that found for the cultured rhizobia. Twoimportant conclusions were drawn from thiswork, namely, that changes in bacteroid respira-tion were not connected always directly withthe nitrogen-fixing activity in the nodules andthat there was seemingly little or no differencebetween pathways of oxidation in bacteroids andin cultured cells.

In a study of the effects of different partialpressures of oxygen on the respiration of rhizobia,soybean bacteroids behaved like cultured cellsin that they respired at a maximal rate at lowtensions (30). On the other hand, bacteroidsfrom pea and vetch nodules produced by the

same strain of bacteria attained their maximalrespiration at a lower P°2, amounting to 0.03atm, than that required for resting cells of thecultured organisms. These latter cells requireda P02 of 0.15 for comparable respiratory rates.The capacity of bacteroids to respire at highrates at low oxygen pressures has been confirmed(100) and constitutes an important metabolicfeature, since the nodule interior is probablyunder partially anaerobic conditions (9, 10, 42).The ability of bacteroids to obtain energy by

the anaerobic decomposition of carbohydratealso would be of potential importance underthe low oxygen tension within the nodule. Initialexperiments along these lines indicated thatglucose could be fermented by crushed nodulesand pure cultures of rhizobia with the productionof lactic, butyric, and acetic acids, ethyl alcohol,CO2, and H2 (107). Later it was discovered thatsuspensions of bacteroids or cultured cellsliberated CO2 from glucose in an atmosphere ofhydrogen, and that the cultured cells were moreactive in this respect than the bacteroids (30).The anaerobic Qco2 values were 50% or less ofthe Qo2 values of the same cells. Moreover,Bond (28) presented evidence to show that theliberation of CO2 per unit dry weight of intactsoybean nodules was about three times that fromthe root tissues of water-cultured plants. Thebacterial region of the nodules was estimated tocontribute three-quarters of the total CO2 formedfrom the nodules. That the release of this gas bynodules is in excess of that released by the roots isnot supported by data obtained by the use ofroots and detached nodules from soybean (99),but is substantiated by results from smallerlegumes (8). Actually it is not known for certainthat rhizobia can carry out true glycolysis, for anumber of inhibitors known to prevent theoperation of the Embden-Meyerhof system donot exhibit any effect on the anaerobic productionof CO2 from glucose by these cells (115). Rela-tive to this, Thorne and Burris (95) found thatfluoride greatly inhibited the respiration ofcultured cowpea rhizobia on glucose, but stronglysimulated bacteroid respiration. Fluoride is rela-tively nonspecific in its action, but this differencein respiratory activity may be related to anabsence of enolase in the bacteroids. No seriousefforts have been made to detect those enzymesunique to the glycolytic pathway or to followthe distribution of radioactive carbon after

1291962]

D. C. JORDAN

fermentation of specifically labeled C14-glucose.However, since there is some evidence thatpyruvate can be produced via the Entner-Duodoroff pathway in cultured rhizobia (64),there is reason to suspect that this scheme mayoperate in bacteroids as well.No very distinct differences have been found

between cultured cells and bacteroids in theirreactions to the inhibitors ethyl urethane,iodoacetate, and pyrophosphate (30, 95). Butin one investigation (95) sodium azide stimulatedthe oxygen uptake on glucose by vetch and peabacteroids, whereas the cultured cells were in-hibited. Contrary to these results, the bacteroidsfrom soybean and cowpea nodules were inhibitedby azide and to a greater degree than the cul-tured bacteria, pointing to a distinct differencein the metabolism of these bacteroids as com-pared to those from vetch and pea. However,the lack of specificity of azide makes it hazardousto offer any simple explanation for its differentialeffect on intact cells of rhizobia.

Further work with inhibitors (30, 94) indicatedthat cyanide caused a 90% inhibition of theoxygen uptake on glucose or suceinate by bothbacteroids and cultured cells of pea, soybean,and vetch rhizobia. This cyanide sensitivity wasconfirmed elsewhere (100) and has been taken toindicate that cytochrome oxidase is present inthese bacteria. Although cyanide does inhibitcytochrome oxidase it inhibits many otherenzymes as well, including some which do notinvolve any metal catalysis whatsoever. Cyto-chrome oxidase is associated usually with thetype a cytochromes, particularly a3 althoughcytochromes a, or a2 may take over this functionin certain bacteria. Using low temperaturespectroscopy, cytochromes b and c, but notcytochrome a, were detected in effective soybeanand clover bacteroids (12, 45). The same rhizobiagrown on an agar medium seemingly containedall three pigments, confirming an observationpreviously made by others (74, 91). In a per-sonal communication, C. A. Appleby mentionedthat repeated spectroscopic examinations ofrhizobia reduced with various substrates ordithionite have failed to indicate the presence ofcytochromes a, or a2 in either cultured cells orbacteroids. Since the apparent loss of cytochromea from the bacteroids could occur under thelow oxygen tension present in nodules, studieswere made of the effect of low concentrations

of oxygen on the cytochrome content of cul-tured cells and washed soybean bacteroids (12).At a partial pressure of 0.02 atm, increases incytochromes b and c were demonstrated in bothtypes of organisms. The absence of cytochromea in the bacteroids was confirmed, whereas thissame respiratory pigment has decreased inamount, but was still present, in the free-livingcells. However, the low oxygen pressure withinnodules probably does not explain adequately theabsence of cytochrome a in effective soybean bac-teroids, since bacteria from ineffective soybeannodules still contained small amounts of thismaterial.A more sensitive test for cytochrome oxidase is

the light-reversible inhibition of its activity bycarbon monoxide. Partial inhibition by CO of theoxygen uptake on succinate in the dark wasdemonstrated by Falk, Appleby, and Porra (45)in cultured cells and bacteroids of soybean rhizo-bia. The reversibility of this inhibition by lightwas low in the cultured cells and bacteroids of anineffective strain, very low in the cultured cellsof an effective strain, and entirely absent in theeffective bacteroids. These results implied that"cytochrome oxidase," together with a light-insensitive oxidase capable of binding CO, waspresent in all cells but the effective bacteroids,which contained only the light-insensitive en-zyme. Such conclusions are not entirely supportedby the report that the respiration of rhizobia isCO resistant (94) or by the data of Tuzimura(100, 102) who found no CO inhibition of therespiration of soybean or vetch bacteroids onglucose or succinate, although an inhibition wasdetected in the case of cultured cells of pea, vetch,and red clover rhizobia on glucose (100). Allthese results do support the conclusion that thestrong inhibition of nitrogen fixation in intactnodules by CO is not a result of an inhibition ofbacteroid respiration. Combination of this gaswith nodule hemoglobin is more likely.

Recent investigations involving the use of asplit-beam spectrophotometer for the determina-tion of the difference spectra of anaerobic cellsuspensions, with and without CO equilibration,have yielded provocative results (45). Using cul-tured cells and bacteroids of both effective andineffective soybean rhizobia, no absorption peakswere observed near 427 or 430 myi, correspondingto cytochromes a, or a3. The principal absorptionpeak was at 416 m~t, so that the main CO-binding

130 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

constituent in all the cells was similar to the "CO-binding pigment" described by Chance, Smith,and Castor (33). Such a pigment, showing a light-irreversible inhibition by CO, has been implicatedas the terminal oxidase in a number of bacteriaand may serve the same purpose in rhizobia,although perhaps in conjunction with the cyto-chrome a in cultured cells. The identity in cul-tured cells and ineffective bacteroids of the com-ponent exhibiting a light-sensitive CO inhibitionis a matter for future study. C. A. Appleby hasinformed me that the CO complex of this com-ponent in cultured cells has absorption peaks at592, 550, and 429 m~i, and may not be identicalwith the cytochrome a.

There is a small amount of evidence againstthe presence of a polyphenol oxidase in bac-teroids (62). Catechol was not oxidized by washedbacteroids from nodules of subterranean cloverpreviously grown in media containing two levelsof copper and, in addition, the oxygen uptake bythese bacteroids on tyrosine was not inhibited by0.0001 M phenylthiourea, an inhibitor of poly-phenol oxidase.An indication of the site of cytochrome c has

been obtained by an inquiry into its release frommechanically disrupted cultured cells and bac-teroids (45). About 20% of the total cytochrome cwas released into solution upon cell rupture andwas not sedimented in 10 min at 173,000 X g.Most of the remaining cytochromes appeared in afraction which sedimented below 10,000 X g.Upon continued disruption of this residue, nomore cytochrome c was released, but more smallparticles, each containing all the cytochromes,were sedimented between 10,000 and 170,000 Xg. These results suggested that most of the cyto-chrome c was present on the cytoplasmic mem-brane, a fact which could ensure a direct trans-ference of electrons between the respiratory chainof rhizobia and extracellular leghemoglobin.

Because of the lack of control over the composi-tion of the growth substrate and cell age in bac-teroid studies, many of the respiratory differencesfound between nodular and cultured rhizobiamay be of small significance. Apparently this hasled several authors (95, 114, 115) to suggestthat the respiratory responses of these cells aresimilar and that the same enzymes exist in bothcases, although the extent of their activities differ.This opinion, although undoubtedly correct inmany instances, should not negate the need for

further research since relatively few enxymes havebeen directly compared. Furthermore, the workon the cytochrome system has revealed one casein which a biochemical change has occurred in thetransition of free-living rhizobia into effectivebacteroids. This change, associated with an ap-parent loss of cytochrome a, has yet to be clarifiedcompletely, especially with respect to those fac-tors known to influence the cytochrome content ofbacteria, but certainly it is worthy of continuedresearch.

C. Porphyrin and Heme SynthesisMost of the literature pertinent to the red

hemoglobin-like pigment in effective root noduleshas been reviewed elsewhere (3, 106). It is syn-thesized only during the effective symbioticassociation between bacterium and host and it ispresent only in the cytoplasm of the bacteroid-containing nodule cells, where its appearanceaccompanies bacteroid formation (52).While investigating a possible mode of action

of the pigment it was shown (70) that hog hemo-gfolobin increased the rate of oxygen uptake byresting cells of several microorganisms, includingthat of soybean bacteroids on succinate, at lowoxygen tensions, but not at the PO2 of air. A simi-lar effect was observed when the nodule pigmentreplaced the mammalian one, but the stimula-tion was less than half as great. Heating eitherpigment destroyed its activity. In later experi-ments on cultured rhizobia (31) it was decidedthat the observed stimulation was not associatedwith the oxygen-carrying power of hemoglobin,but with its ability to convert resting-cell metabo-lism into the more active state characteristic ofcells either growing or about to undergo growth.It is uncertain how this interpretation can be ap-plied to bacteroids.

Although the discovery of a relationship be-tween nodule hemoglobin and nitrogen fixationcreated considerable interest, it was not until1954 that serious attempts were made to studythe synthesis of heme and related compounds innodule tissue. In that year Richmond, Salomen,and Caplin (83) reported that, like bone marrow,homogenates of root nodules from 6-week-oldsoybean plants incorporated the second carbonof glycine and both carbon atoms of acetate intohemin, which was isolated and purified by columnchromatography. The incorporation of the num-ber one carbon of glycine was low. In striking con-

1962] 131

D. C. JORDAN

trast to bone marrow, in which incorporation ofthe second carbon of glycine was inhibited 50% bysodium fluoride, there was a 190% stimulation byfluoride of the incorporation of this carbon atominto hemin by the nodule homogenates. In addi-tion there was a 400% increase in the incorpora-tion of the number one carbon. However, no at-tempt was made to study the bacteroidsseparately from the plant tissue.The existence of a free porphyrin-hemoglobin

system in the nodules of red kidney bean and soy-bean was uncovered by Kluver (65). Theporphyrin he isolated from kidney bean noduleshad the spectroscopic characteristics of a copro-porphyrin, but behaved in other respects like atype of uroporphyrin. Following this, an investi-gation was made on the porphyrins and hemespresent in the root nodules and bacteroids of soy-bean (45). Crushed effective nodule tissue, butnot ineffective tissue, contained heme and con-siderable quantities of protoporphyrin, copro-porphyrin, and a compound resembling uropor-phyrin; the latter predominated. A possible bio-chemical reason for ineffectiveness is revealed bythe finding that homogenates of ineffective nod-ules from 8-week-old plants were unable to formporphyrins from 6-aminolevulinic acid (ALA) orporphobilinogen (PBG), although a synthesis ofproto-, copro-, and uroporphyrins took placeupon the addition of an unknown heat-stable ma-terial present in a Kochsaft of yeast or of effectivenodules.

Further evidence established that isolated bac-teroids contained no protoporphyrin and verylittle of the other porphyrins. In fact, the smallamounts of these pigments found in the bac-teroids could be attributed to adsorption onto thecells during the nodule-crushing procedure. Bac-teria from effective and ineffective soybeannodules possessed the enzymatic capacity to syn-thesize uroporphyrin only and were unable tosynthesize PBG from ALA, in contrast to cul-tured cells and nodule homogenates. The cul-tured cells could also convert ALA to both proto-and coproporphyrin. Thus, the synthetic abilitiesof cultured rhizobia with regard to porphyrin syn-thesis were altered drastically by their conversionto bacteroids, but since young effective nodules(from 3-week-old plants) could synthesize porphy-rin to a greater extent than older nodules it be-comes important to study synthetic reactions inbacteroids over a wide range of nodule ages.

The major role of porphyrin synthesis withinthe nodule appears to be played by the planttissue, but the activity of the bacteroids stillseems essential since nodule hemoglobin is formedonly during effective symbiosis. One hypothesissuggested from the published data is that thehost cells of effective nodules possess the entiremechanism for the synthesis of heme from ALA orits precursors, with the exception of a factorcatalyzing the formation of uroporphyrin fromporphobilinogen. This factor could be suppliedby the bacteroids.A possible function of the hemoglobin emerged

from work wherin sonic extracts of effective soy-bean nodules oxidized the heme pigment to hemi-globin in the presence of nitrogen (50). Afterconfirmation it was demonstrated that soybeanbacteroids were capable of reversing this oxi-dation (25), suggesting that the reducing power ofhemoglobin is required for nitrogen fixation andthat the resulting oxidized form, hemiglobin, isre-reduced by the bacteroids that use this com-pound as a terminal respiratory pigment (21).Bauer and Mortimer (13) hypothesized that asecondary attachment of nitrogen to the hemegroup of nodular hemoglobin is the first step insymbiotic nitrogen fixation. Hence the actualsite of fixation is probably not within the bac-teroids themselves and, indeed, Bergersen andWilson (24) found no detectable N'5 in thesecells until soybean nodules were exposed to thelabel for over 1 hr. Possible fixation sites are thosedouble membranes found enclosing small groupsof bacteroids within soybean nodules (22). Theseunusual membranes appear to originate from thehost cells and their developmental stages corre-spond to the onset and the maximal rate of fixa-tion. Their presence in soybean nodules has beenconfirmed by I. Grinyer and myself at Guelph,but we have not yet observed such structures inultrathin sections of young alfalfa nodules (Fig.7).

D. General Metabolism1. Hydrogenase. The search for hydrogenase in

symbiotic nitrogen-fixing cells is of interest be-cause of a possible connection between this en-zyme and the fixation process itself (116). In 1941it was reported (80) that hydrogenase was absentfrom cultured cells of pea rhizobia, but that bac-teroids from nodules produced by this organismactively reduced methylene blue in the presence

132 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

_Wt offs..FIG. 7. Ultrathin section of the bacteroid zone of an effective 4-week-old alfalfa nodule. For sectioning,

small blocks of tissue were osmium fixed, dehydrated, impregnated with a partially polymerized mixture of10% methyl methacrylate and 90% butyl methacrylate for 48 hr at 4 C, warmed to room temperature, and im-bedded in fresh methacrylate subsequently polymerized overnight at 60 C. The membrane (m) enclosing eachbacteroid section is probably the bacteroid cell wall, separated from the rest of the cell during preparation.Electron-transparent regions (r) containing osmiophilic granules (g) and filaments (f) can be seen in thegranular cytoplasm of the bacteroids and may represent nuclear material. Membranes, similar to those en-closing groups of bacteroids in soybean nodules, are not present.

1331962]

D. C. JORDAN

of hydrogen. Attempts to find such an enzyme incultured cells and bacteroids of soybean rhizobiafailed. Since the high endogenous respiration ofbacteroids makes the demonstration of hydro-genase difficult, hydroxylamine was employed tostop this activity (113) and the uptake of gas bycells incubated in a 96% H2 and 4% 02 mixturewas studied manometrically. Suitable controlswere carried out in air or in a helium-oxygenmixture. No hydrogenase activity was found; sus-pensions of pea and cowpea bacteroids showedthe same total gas uptake in all the gaseous mix-tures. This apparent lack of hydrogenase, whichcontradicted the earlier findings, was verifiedby additional tests (90, 117). Later, some evi-dence was found for hydrogenase activity innodule tissue in that a preparation of disinte-grated soybean nodules showed spectral shifts inthe presence of hydrogen but not in its absence(116). This evidence was strengthened by the ob-servation that hydrogen was evolved from de-tached soybean nodules (56). The liberation ofhydrogen was greatly increased in the presence ofoxygen, drastically reduced when the noduleswere sliced, and eliminated when the noduleswere ground or when the hemoglobin was con-verted into green "legeholeglobin." With regardto washed bacteroids, however, the negative re-sults formerly reported remain undisputedand any activity hydrogenase may have in nodulemetabolism (21, 88) is probably confined to theplant tissue.

2. Vitamin B12. Large concentrations of B12are present in leguminous root nodules, in somecases amounting to 310 to 330 mug per g of tissue(66). The work of Levin, Funk, and Tendler (68)revealed that the concentration of this vitaminin the pink effective nodules of alfalfa, clover,and peas was from 3 to 43 times greater than thatin root tissue and that with alfalfa the concentra-tion was 400% greater than that in the white in-effective nodules. Bacteroids isolated from pinknodules gave different ratios of B12 excreted toB12 retained as compared with cultured cells. Theratios were 14:1 for alfalfa-sweet clover bac-teroids and 5:1 for clover bacteroids, whereasthose for the cultured cells were 1:2 and 3:1, re-spectively. Bacteroids, particularly those fromclover nodules, synthesized more molecules ofB12 per cell than did the cultured bacteria.The synthesis of this cobalt-containing vitamin

is particularly significant in view of the recent

implication of cobalt in nitrogen fixation (1, 49,82). Relative to this, S. B. Wilson, G. Norton, andmyself have shown (unpublished data) that morethan 85% of the cobalt accumulating in effectivenodules of 11-week-old plants of subterraneanclover and 8-week-old soybean plants aftervarious exposures to CoI0 is associated with thesoluble nonproteinaceous fraction of the nodulesrather than with rapidly washed bacteroids.

3. Nitrate reductase. A high specific activityof DPN-dependent nitrate reductase, catalyzingthe conversion of nitrate to nitrite, has beendetected in washed soybean kacteroids, but not inthat fraction of homogenized nodules which con-tained the hemoglobin (44). The reductase couldbe extracted by grinding the cells in alkalinebuffer and the extracted enzyme appeared similarto that present in neurospora and soybean leaves.The bacteroids contained a two or three timesgreater concentration of molybdenum per mgprotein than the hemoglobin-containing fractionof the nodules, suggesting that this element maybe as important in nitrate reductase in these cellsas it is in other systems.

Cheniae and Evans (35) discovered that theformation of nitrate reductase in cultured cellsof soybean rhizobia was induced by nitrate, butthat reductase activity could be detected in theexcised nodules of plants grown in a nutrientsolution containing less than 1 ppm of nitrate asa contaminant. No nitrate or nitrite was found inthe root juice of plants grown for 3 weeks in asterile medium containing ammonium sulfate, orin the medium itself, so ruling out the possibilityof nitrate production by the plant. The authorsthereby concluded that the bacteroids within thenodule were being continuously supplied withnitrate or some other compound producing theadaptive formation of nitrate reductase. Ofcourse, in the bacteroids this enzyme could beconstitutive, in contrast to that in cultured cells.3

3 Subsequent to the preparation of the presentmanuscript, Bergersen (21) reported that thelevel of nitrate reductase intact soybean bac-teroids was maximum sev ral days before thecommencement of nitrogen fixation, and there-after declined. Such results would tend to dis-credit the hypothesis that reductase activity isinduced by products of nitrogen fixation. How-ever, the reductase activity of crushed bacteroidsincreased rather than decreased with nodule age.To explain this noncomformity the author sug-gested that aging either decreased the permeabil-

134 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

The nitrate reductase from soybean noduleshas been purified about 15-fold (36), but themethods used for extracting the enzyme from thebacteroids have resulted in a particulate prepa-ration and attempts to solubilize the reductasewith surface-active agents have resulted in inac-tivation. The Km for the enzyme with respect tonitrate is 2.5 X 10-, a value lower than that re-ported for nitrate reductases from other sources.Either succinate or reduced DPN can serve ashydrogen donor and the reaction sequence ineach case has been followed by the use of in-hibitors (37). The sequence from reduced DPNinvolves an unidentified factor (replaceable bymenadione), a cytochrome, the Slator factor,nitrate reductase, and nitrate. The transfer fromsuccinate is similar except that succinic dehydro-genase replaces the unidentified component.Lowe and Evans (72a) found that the particu-

late reductase fraction from soybean bacteroidsalso catalyzed a nitrate-inhibited transfer ofelectrons from succinate to oxygen. In addition,the fraction possessed the capacity to transferelectrons from succinate to oxyhemoglobin, thusforming hemoglobin, although efforts to find asuccinate-dependent reduction of nodule hemi-globin to hemoglobin were unsuccessful. Sinceoxyhemoglobin could be produced by combi-nation of hemoglobin with any oxygen present inthe nodules, its reduction by this enzyme fractionmight make hemoglobin available to fulfill apossible role in transporting electrons to atmos-pheric nitrogen (21). Such a postulation couldexplain the apparent positive correlation foundby Cheniae and Evans (36a, 37a) betweennitrate reductase activity and nitrogen-fixingefficiency of nodules (but see footnote 3).

4. Activation of ammonia. "Cell-free" extractsprepared from soybean bacteroids possess theability to activate ammonia enzymatically bymeans of adenosine triphosphate (ATP), withthe resulting formation of adenylamidate (43).

ity of intact cells to nitrate or else resulted in a

gradual breakdown of the electron transport sys-tem in these intact cells. The latter effect, if itoccurred, would not have been evident withcrushed bacteroids because in the assays usingthis particular material a reduced diphosphopyri-dine nucleotide (DPNH)-generating system was

added. Regrettably, the effect of added DPNH on

the enzymatic activity of intact bacteroids was

not studied.

The reaction corresponds to:

ATP + NH3 AMP NH2 + P P

and although it is absent from soybean root ex-tracts, it occurs in a number of microorganisms.Its significance in the formation of ammonia,now recognized as the key intermediate in nitro-gen fixation, is conjectural and it is probably em-ployed by the bacteroids to produce amino acidsfrom the ammonia produced by another mecha-nism.

5. Glutamine synthetase. This enzyme catalyzesthe reaction:

Glutamate + ATP + NH3

glutamine + ADP + inorganic phosphate

Reasons for believing that glutamine may be oneof the first organic nitrogenous compounds formedduring nitrogen fixation have been given by Leaf(67). Therefore, ineffectiveness in fixation couldresult from a failure to carry out glutamine syn-thesis, although all the legume nodules tested byLoomis (72) possessed a high synthetase activityand no differences were noted between the ac-tivities of cultured cells of an effective and in-effective strain of clover rhizobia. Cooper andJordan (62) separated crushed soybean nodulesinto a bacteroid fraction, a supernatant fluid anda fraction sedimenting in 10 min at 30,000, X gsupposedly containing the membranes enclosinggroups of bacteroids in the intact nodules. Allthree fractions readily synthesized glutaminefrom added glutamate and ammonia in the pres-ence of ATP. The synthesis per mg of protein wasgreater in the supernatant than in the membranefraction, but unfortunately the quantitativeestimation of synthesis by the bacteroids washindered by technical difficulties.

6. Transamination. For at least some timeafter their formation, bacteroids retain the parentcells' capacity for synthesizing amino acids bytransamination. Bacteroids from subterraneanclover nodules are known to synthesize glutamatefrom a-ketoglutaric acid and various amino-groupdonors, including aspartic acid, alanine, isoleu-cine, histidine, and valine (62). Previously it hadbeen mentioned (30) that aspartate could be pro-duced from amino acids and oxalacetate inmacerated nodules, but this or similar reactionswith oxalacetate have not been studied in iso-lated bacteroids.

1962] 135

D. C. JORDAN

V. SUMMARY AND CONCLUDING REMARKSFrom the accumulated information a gen-

eralized picture of bacteroid formation and physi-ology is beginning to emerge. Under natural con-ditions these cells are produced from the smallrodlike rhizobia shortly after their release fromthe infection thread into the cytoplasm of thehost cells. The bacteroid-producing factors in thenodule are unknown as yet, but the mode of ac-tion may be similar to those of the wide range ofinimical agents causing corresponding effects incultured cells. However, there seems to be no easyway of telling if the nodule bacteroids are exactlyequivalent biochemically with the bacteroidsproduced in laboratory media.An attractive hypothesis regarding bacteroid

development is that it represents a selective inter-ference in the normal synthesis of cell wall, ofcytoplasmic membrane, or of both these struc-tures. Under such a condition cross-wall forma-tion would be abortive, cell division would cease,and continued cytoplasmic growth would resultin the formation of abnormal cells. Fortunately,this hypothesis is amenable to experimentalattack with the techniques currently in use for thestudy of the synthesis of cell walls and protein inother microorganisms. A study of the uptake ofvarious substances into the internal pools ofcultured cells and bacteroids might also be profit-able, since any drastic change in the permeabilityof the cell barrier would suggest membrane dam-age. However, the reason for the alteration of cellpolarity in the case of the branched bacteroidsrepresents still another problem in cellular physi-ology, but the phenomenon probably has a closeconnection with the postulated abnormal syn-thesis of cell-wall material, thus making thebranching somewhat analogous to that observedwhen certain gram-negative bacteria are exposedto penicillin, an antibiotic known to prevent theproper development of the bacterial cell wall.4The degeneration of bacteroids is accompanied

by a weakening of the existing cell-wall structure,clumping (possibly related to a change in thechemical properties of the cell wall), a loss of thebasophilic properties of the cytoplasm, the forma-

4 Vincent and Colburn (104a) have relatedbranching of cultured cells of Rhizobium trifolii tomagnesium deficiency. Although such a deficiencycould be a cause of branching in bacteroids, theactual mechanism which might be involved re-mains obscure.

tion of large spaces or vacuoles in the cell interior,and nuclear breakdown. Eventually dissolutionoccurs. The exact reason for the "lytic" phe-nomenon is obscure, but presumably it could bethe result of any one of several factors includingbacteriophage attack or the action of host-cellenzymes. On the other hand there seems to be noreason to suspect that it is anything more thannormal autolysis resulting from a restricted carbo-hydrate supply or as a result of a protracted deathdue to the postulated primary inhibition of mem-brane synthesis. As the degeneration advances agradual loss of enzymatic capacity would be ex-pected although, surprisingly enough, a numberof the physiological reactions of cultured cells re-main relatively intact in the bacteroids for anappreciable length of time. The only very definitechanges are associated with an apparent loss ofcytochrome a, a decreased ability to synthesizecertain porphyrin compounds, and a decreasedsensitivity to respiratory inhibition by fluoride.The first two changes conceivably could be re-lated.

Difficulties are inherent in any attempts tocompare the physiology of bacteroids with that oflaboratory-grown cells. The primary difficulty liesin the inability to synchronize the ages and nutri-tional backgrounds of these two cell types. Atpresent the only way to evaluate age influences isto examine cultured cells at various stages intheir growth curve and to study bacteroids fromnodules of different ages. Another factor is theextreme heterogeneity of bacteroid suspensionsprepared from crushed nodules, in which there isan intermixture of rod forms and bacteroids inseveral stages of "maturity." Results from suchpreparations may be useful, but can onlyrepresent averages of a mixed population. Oneway to control this variable to some extent duringstudies on older bacteroids would be to isolatethese cells only from the central tissue of thebacteroid area, thus discarding about four-fifthsof the nodule mass. This technique would befeasible only with the larger types of nodules,such as those of soybean. Since bacteroids becomeprogressively less dense during their degeneration,there is a possibility of separating them intodifferent age groups by employing density-gradi-ent techniques. Still another difficulty, en-countered in electron microscopy and respiratorystudies, is caused by a starchy gum which adheresto bacteroids after their separation from certain

136 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

nodules and which is not entirely removed byseveral washings in buffer or distilled water (62).A rather neglected area of investigation is con-

cerned with the presence in nodules of substancesnot seen in the normal host tissue, or else presentin very small amounts. Such substances includethe proteinaceous granules seen in the bacteroid-containing cells of peanut nodules (4) and thewater-soluble polysaccharide found in the infectedcells of soybean nodules (18). Also in this categorywould be the increased catalase activity of red,effective nodules compared with that of oldergreen nodules, and the increased ascorbic acidcontent of nodules compared with root tissue(106). Only further study will establish why suchcompounds accumulate and any possible connec-tion this may have with bacteroid metabolism.Although the evidence for a relationship

between bacteroid formation and nitrogen fixa-tion is substantial, the bacteroids are unable tofix nitrogen after separation from the nodule.Consequently it is not known whether the bac-teroids represent a cause oran effect phenomenon.Perhaps the processes set in motion by the plantin an effort to isolate and destroy the invadingrhizobia may themselves give rise to the nitrogen-fixing mechanism. But it is more probable thatthe bacteroids do function in the fixation se-quence. In this respect their potential ability totransfer hydrogen to nitrogen via nodule hemo-globin (21) is worthy of consideration. Throughthe mediation of the nitrogen-fixing enzyme,"nitrogenase," nitrogen would then act as aterminal hydrogen acceptor, with the resultingformation of ammonia. The ability to transferhydrogen from reduced substrate to the hemo-globin may be connected with the aforemen-tioned change in the terminal respiration of rhizo-bia during their conversion into bacteroids.Even if the bacteroids play only a secondary

role in symbiotic nitrogen fixation they will stillpresent intriguing biochemical and cytologicalproblems, so that continued interest in these cellscan be anticipated.

VI. ACKNOWLEDGMENTS

The author wishes to acknowledge the financialhelp of the Nuffield Foundation and the kindassistance of the library staffs at the Universityof Nottingham School of Agriculture andRothamsted Experimental Station. Valuablehelp with respect to the electron microscopy was

received from R. Horne and Margaret Pleasance,University of Cambridge, and D. J. Craik, BootsPure Drug Company, Nottinghamshire.

VII. LITERATURE CITED1. AHMED, S., AND H. J. EVANS. 1959. Effect of

cobalt on the growth of soybeans in theabsence of supplied nitrogen. Biochem.Biophys. Research Commun. 1:271-275.

2. ALLEN, E. K., AND O. N. ALLEN. 1950. Bio-chemical and symbiotic properties of rhizo-bia. Bacteriol. Rev. 14:273-330.

3. ALLEN, E. K., AND O. N. ALLEN. 1958. Bio-logical aspects of symbiotic nitrogen fixa-tion, p. 48-118. In W. Ruhland, [ed.], Hand-buch der Pflanzenphysiologie, VIII.Springer-Verlag, Berlin.

4. ALLEN, 0. N., AND E. K. ALLEN. 1940. Re-sponse of the peanut plant to inoculationwith rhizobia, with special reference tomorphological development of the nodules.Botan. Gaz. 102:121-142.

5. ALLEN, 0. N., AND E. K. ALLEN. 1954. Mor-phogenesis of the leguminous root nodule.Brookhaven Symposia in Biol. 6:209-234.

6. ALLEN, E. K., K. F. GREGORY, AND O. N.ALLEN. 1955. Morphological developmentof nodules on Caragana arborescens Lam.Can. J. Botany 33:139-148.

7. ALLISON, F. E. 1935. Carbohydrate supply asa primary factor in legume symbiosis. SoilSc. 34:123-143.

8. ALLISON, F. E., C. A. LUDWIG, S. R. HOOVER,AND F. W. MINOR. 1939. Legume nodulemetabolism and nitrogen fixation. Nature164:711.

9. ALLISON, F. E., C. A. LUDWIG, S. R. HOOVER,AND F. W. MINOR. 1940. Biochemical nitro-gen fixation studies. I. Evidence for limitedoxygen supply within the nodule. Botan.Gaz. 101:513-533.

10. ALLISON, F. E., C. A. LUDWIG, F. W. MINOR,AND S. R. HOOVER. 1940. Biochemicalnitrogen fixation studies. II. Comparativerespiration of nodules and roots, includingnon-legume roots. Botan. Gaz. 101:534-549.

11. ALMON, L. 1933. Concerning the reproductionof bacteroids. Z. Bakteriol. Parasitenk.Abt. II, 87:289-297.

12. APPLEBY, C. A., AND F. J. BERGERSEN. 1958.Cytochromes of Rhizobium. Nature 182:1174.

13. BAUER, N., AND R. G. MORTIMER. 1960. Sec-ondary gasation of heme proteins and bio-logical N2-fixation. Biochim. Biophys.Acta 40:170-171.

14. BAYLOR, M. B., M. D. APPLEMAN, 0. H.

1371962]

D. C. JORDAN

SEARS, AND G. L. CLARK. 1945. Somemorphological characteristics of nodulebacteria as shown by the electron micro-scope. J. Bacteriol. 50:249-256.

15. BEIJERINCK, M. W. 1888. Die Bakterien derPapilionaceenknollchen. Botan. Ztg. 46:726-735, 741-750, 757-771, 781-790, 797-804.

16. BERGERSEN, F. J. 1955. The cytology of bac-teroids from root nodules of subterraneanclover (Trifolium subterraneum L.). J.Gen. Microbiol. 13:411-419.

17. BERGERSEN, F. J. 1957. The occurrence of apreviously unobserved polysaccharide inimmature infected cells of root nodules ofTrifolium ambiguum M. Bieb. and othermembers of the Trifolieae. Australian J.Biol. Sci. 10:15-24.

18. BERGERSEN, F. J. 1957. The structure of in-effective root nodules of legumes: an un-usual new type of ineffectiveness and anappraisal of present knowledge. AustralianJ. Biol. Sci. 10:233-242.

19. BERGERSEN, F. J. 1958. The bacterial com-ponent of soybean root nodules; changes inrespiratory activity, cell dry weight andnucleic acid content with increasing noduleage. J. Gen. Microbiol. 19:312-323.

20. BERGERSEN, F. J. 1960. Incorporation of15N2 into various fractions of soybean rootnodules. J. Gen. Microbiol. 22:671-677.

21. BERGERSEN, F. J. 1960. Biochemical path-ways in legume root nodule nitrogen fixa-tion. Bacteriol. Rev. 24:246-250.

21a. BERGERSEN, F. J. 1961. Nitrate reductase insoybean root nodules. Biochim. Biophys.Acta 52:206-207.

22. BERGERSEN, F. J., AND M. J. BRIGGS. 1958.Studies on the bacterial component ofsoybean root nodules: cytology and organ-ization in the host tissue. J. Gen. Micro-biol. 19:482-490.

23. BERGERSEN, F. J., AND P. S. NUTMAN. 1957.Symbiotic effectiveness in nodulated redclover. IV. The influence of the host factorsii and ie upon nodule structure and cytol-ogy. Heredity 11:175-184.

24. BERGERSEN, F. J., AND P. W. WILSON. 1959.Location of newly-fixed nitrogen in soynodules. Bacteriol. Proc., p. 25.

25. BERGERSEN, F. J., AND P. W. WILSON. 1959.Spectrophotometric studies of the effectsof nitrogen on soybean nodule extracts.Proc. Natl. Acad. Sci. U. S. 45:1641-1646.

26. BISSET, K. A. 1952. Complete and reducedlife cycles in Rhizobium. J. Gen. Microbiol.7:233-242.

27. BISSET, K. A., AND C. M. F. HALE. 1951. The

production of swarmers in Rhizobium spp.J. Gen. Microbiol. 5:592-595.

28. BOND, G. 1941. Symbiosis of leguminousplants and nodule bacteria. I. Observationson respiration and on the extent of utiliza-tion of host carbohydrates by the nodulebacteria. Ann. Botany (N.S.) 5:313-337.

29. BRUNCHORST, J. 1885. Vber die Knollchen anden Leguminosenwurzeln. Ber. deut. botan.Ges. 3:241-257.

30. BURRIS, R. H., AND P. W. WILSON. 1939. Re-spiratory enzyme systems in symbioticnitrogen fixation. Cold Spring HarborSymposia Quant. Biol. 7:349-361.

31. BURRIS, R. H., AND P. W. WILSON. 1952.Effect of haemoglobin and other nitroge-nous compounds on the respiration of therhizobia. Biochem. J. 51:90-96.

32. CARROLL, W. R. 1394. A study of Rhizobiumspecies in relation to nodule formation onthe roots of Florida legumes. II. Soil Sci.37:227-241.

33. CHANCE, B., L. SMITH, AND L. CASTOR. 1953.New methods for the study of the carbonmonoxide compounds of respiratory en-zymes. Biochim. Biophys. Acta 12:289-298.

34. CHEN, H. K., AND H. G. THORNTON. 1940.The structure of 'ineffective' nodules andits influence on nitrogen fixation. Proc.Roy. Soc. (London), Ser. B, 129:208-229.

35. CHENIAE, G., AND H. J. EVANS. 1956. Nitratereductase from the nodules of leguminousplants. p. 184-188. In W. D. McElroy, [ed.],Symposium on inorganic nitrogen metabo-lism. Johns Hopkins Press, Baltimore.

36. CHENIAE, G. M., AND H. J. EVANS. 1956.Studies on nodule nitrate reductase. PlantPhysiol. (Proc. Suppl.), 31:10.

36a. CHENIAE, G., AND H. J. EVANS. 1957. On therelation between nitrogen fixation and nod-ule nitrate reductase of soybean root nod-ules. Biochim. Biophys. Acta 26:654-655.

37. CHENIAE, G., AND H. J. EVANS. 1959. Proper-ties of a particulate nitrate reductase fromthe nodules of the soybean plant. Biochim.Biophys. Acta 35:140-153.

37a. CHENIAE, G., AND H. J. EVANS. 1960, Physi-ological studies on nodule-nitrate reduc-tase. Plant Physiol. 35:454-462.

38. CHIZHIK, G. YA. 1959. The structure of theroot-nodule bacteria. (In Russian, Englishsummary.) Mikrobiologiya 28:28-33.

39. DEMOLON, A., AND A. DUNEZ. 1935. Recher-ches sur le role du bacteriophage dans lafatigue des luzernibres. Ann. agron. 5:89-112.

40. DEMOLON, A., AND A. DUNEZ. 1938. Symbiose

138 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

bacterienne et culture des legumineuses.Ann. agron. 8:220-237.

41. DEMOLON, A., R. RozowsKA, AND G. JACO-BELLI. 1950. Observations biochimique surle developpement du Bacterium radicicola(Rhizobium leguminosarum). Compt. rend.230:1015-1018.

42. EBERTOVA, H. 1959. Redox potentials in soy-bean nodules during the vegetative period.Nature 184:1046-1047.

43. ELLFOLK, N., AND N. KATUNUMA. 1959. Theoccurrence of ammonia-activating enzymein various organisms. Arch. Biochem.Biophys. 81:521-522.

44. EVANS, H. J. 1954. Diphosphopyridine nucleo-tide-nitrate reductase from soybean nod-ules. Plant Physiol. 29:298-301.

45. FALK, J. E., C. A. APPLEBY, AND R. J. PORRA.1959. The nature, function and biosyn-thesis of the haem compounds and porphy-rins of legume root nodules. Symposia Soc.Exptl. Biol. 13:73-86.

46. FEDOROV, M. V., AND V. USPENSKAIA. 1955.The influence of continuous illuminationof leguminous plants (peas and soybeans)on nitrogen-fixing activity of club-shapedbacteria in the nodules. (In Russian.)Mikrobiologiya 24:291-302.

46a. FONBRUNE, P. DE. 1949. Technique demicromanipulation. Masson et Cie. Paris.204 p.

47. FORSYTH, W. G. C., A. C. HAYWARD, ANDJ. B. ROBERTS. 1958. Occurrence of poly-fl-hydroxybutyric acid in aerobic gram-nega-tive bacteria. Nature 182:800-801.

48. FRED, E. B., I. L. BALDWIN, AND E. McCoy.1932. The root nodule bacteria and legumi-nous plants. Univ. Wisconsin Studies inSci. Madison, Wisc. 343 p.

49. HALLSWORTH, E. G., S. B. WILSON, ANDE. A. N. GREENWOOD. 1960. Copper andcobalt in nitrogen fixation. Nature 187:79-80.

50. HAMILTON, P. B., A. L. SHUG, AND P. W.WILSON. 1957. Spectrophotometric exam-ination of hydrogenase and nitrogenase insoybean nodules and azotobacter. Proc.Natl. Acad. Sci. U. S. 43:297-304.

51. HARRIS, J. 0., E. K. ALLEN, AND 0. N.ALLEN. 1949. Morphological developmentof nodules on Sesbania grandiflora Poir.with reference to the origin of nodule root-lets. Am. J. Botany 36:651-661.

52. HEUMAN, W. 1952. Uber Wesen and Bedeu-tung der Bakteroide in den Wurzelknoll-chen der Erbse. Naturwissenschaften 39:66.

53. HEUMAN, W. 1952. Uber das Abhangigkeit-

verhaltnis zwischen Hamoglobin-, Starke-,Bakteroidvorkommen und Stickstoff-bin-dung in den Wurzelknollchen der Erbse.Naturwissenschaften 39:66-67.

54. HEUMAN, W. 1952. Physiologische und mor-phologische Studien an Rhizobium legumi-nosarum in Knollchen und auf verschie-denen Nahrboden. Ber. deut. botan. Ges.65:229-233.

55. HEUMAN, W. 1954. Die Bakteroidbildung vonRhizobium leguminosarum in Mischkulturmit einem anderen Bakterium. Naturwis-senschaften 41:192.

56. HOCK, G. E., H. N. LITTLE, AND R. H. BUR-RIS. 1957. Hydrogen evolution from soy-bean root nodules. Nature 179:430-431.

57. HOPKINS, E. W., W. H. PETERSON, ANDE. B. FRED. 1929. The composition of thecells of certain bacteria with special refer-ence to their carbon and their nitrogencontent. J. Biol. Chem. 85:21-27.

58. ITANO, A., AND A. MATSUURA. 1936. Studieson the nodule bacteria. VII. Influence ofthe extract of nodules on the growth ofnodule bacteria. Ber. Ohara Inst. land-wirtsch Forsch. Kurashiki Japan 7:379-401.

59. ITANO, A., AND A. MATSUURA. 1938. Studieson the nodule bacteria. X. Influence ofsome stimulating chemicals with specialreference to the alkaloids upon the growthand morphology of the nodule bacteria.Ber. Ohara Inst. landwirtsch. Forsch. Kur-ashiki Japan 8:53-68.

60. JACOBS, S. E. 1949. The relationship ofCorynebacteriumfasciens (Tilford) Dowson,to the bacteria causing gall and noduleformation. Proc. Intern. Congr. Microbiol.,4th Congr. (Copenhagen, 1947), Sect. IV,p. 425.

61. JENSEN, H. L. 1952. The coryneform bacteriaAnn. Rev. Microbiol. 6:77-90.

62. JORDAN, D. C. 1960. Preliminary investiga-tions on Rhizobium bacteroids, p. 35-38.Univ. of Nottingham School of AgricultureReport. Sutton Bonington, Nottingham-,hire.

63. JORDAN, D. C., AND E. H. GARRARD. 1951.Studies on the legume root nodule bacteria.I. Detection of effective and ineffectivestrains. Can. J. Microbiol. 29:360-372.

64. KATZNELSON, H. 1955. Production of pyru-vate from 6-phosphogluconate by bacterialplant pathogens and legume bacteria.Nature 175:551.

65. KLUVER, H. 1948. On a possible use of theroot nodules of leguminous plants for re-search in neurology and psychiatry (pre-

1391962]

D. C. JORDAN

liminary report on a free porphyrin-hemo-globin system). J. Psychol. 25:331-356.

66. KON, S. K. 1955. Other factors related to B12.Biochem. Soc. Symposia 13:17-35.

67. LEAF, G. 1959. Biochemical aspects of nitro-gen fixation. Advance. of Sci. 60: :386-392.

68. LEVIN, A. P., H. B. FUNK, AND M. D. TEND-LER. 1954. Vitamin B12, rhizobia and legu-minous plants. Science 120:784.

69. LEWIS, I. M. 1938. Cell inclusions and thelife cycle of rhizobia. J. Bacteriol. 35:573-587.

70. LITTLE, H. N., AND R. H. BURRIS. 1947.Activity of the red pigment from legumin-ous root nodules. J. Am. Chem. Soc. 69:838-841.

71. LOJTJANSKAJA, M. S. 1941. On the develop-ment of nodule bacteria in the roots ofalkaloid-free lupine. (In Russian, Englishsummary.) Mikrobiologiya 10:15-32.

72. LooMIs, W. D. 1959. Amide metabolism inhigher plants. III. Distribution of glutamyltransferase and glutamine synthetaseactivity. Plant Physiol. 34:541-546.

72a. LOWE, R. H., AND H. J. Evans. 1961. Furtherstudies on a particulate enzyme prepara-tion from nodules of soybean plants. PlantPhysiol. 36:545-549.

73. MILOVIDOV, P. F. 1935. Ergebnisse der Nu-clealfarbung bei den Myxobakterien undeinigen anderen Bakterien. Arch. Mikro-biol. 6:475-509.

74. Moss, F. J., AND Y. T. TCHAN. 1958. Studieson N-fixing bacteria. VII. Cytochromes ofAzotobacteriaceae. Proc. Linnean Soc.N. S. Wales 83:161-164.

75. NAUNDORF, G., AND R. NILSSON. 1942. tberformbildende Wirkstoffe bei Azotobacterchroococcum und der Einfluss dieser forma-tiven Wirkstoffe auf die Bakteroiden-bil-dung von Bacterium radicicola. Naturwiss-enschaften 30:753.

76. NAUNDORF, G., AND R. NILSSON. 1943. tVberformbildende Wirkstoffe bei Azotobacterchroococcum und der Einfluss dieser forma-tiven Wirkstoffe auf die Bildung vonGigasformen bei Bacterium radicicola.Naturwissenschaften 31:346.

77. NILSSON, R., G. BJXLFVE, AND D. BURSTROM.1938. Vitamin B, als Zuwachsfaktor furBact. radicicola. I. Naturwissenschaften26:284.

78. NILSSON, R., G. BJXLFVE, AND D. BURSTROM.1938. Vitamin B, als Zuwachsfaktor furBact. radicicola. II. Naturwissenschaften26:661.

79. NILSSON, R., G. BJXLFVE, AND D. BURSTR6M.1939. tUber Zuwachsfaktoren bei Bact.

radicicola. III. Lantbrukshogsk. Ann.Chem. Liebigs 7:51-61.

80. PHELPS, A. S., AND P. W. WILSON. 1941.Occurrence of hydrogenase in nitrogen-fixing organisms. Proc. Soc. Exptl. Biol.Med. 47:473-476.

81. RAUTANEN, N., AND S. SAUBERT. 1955. Rootnodules of leguminous plants. A chemicalstudy. Suomen Kemistilehti. 28B:66-70.

82. REISENAUER, H. M. 1960. Cobalt in nitrogenfixation by a legume. Nature 186:375-376.

83. RICHMOND, J. E., K. SALOMEN, AND S. CAP-LIN. 1954. Biosynthesis of haemin in soy-bean nodule homogenates. Nature 174:34-35.

84. SCHAEDE, R. 1932. Das Schicksal der Bak-terien in den Knollchen von Lupinus albusnebst cytologischen Untersuchungen. Zentr.Bakteriol. Parasitenk., Abt. II, 85:416-425.

85. SCHAEDE, R. 1939. Zum Problem des Vorkom-mens von chromatischer Substanz beiBakterien und Aktinomyceten. Arch.Mikrobiol. 10:473-507.

86. SCHAEDE, R. 1940. Die Knollchen der adven-tiven Wasserwurzeln von Neptunia oleraceaund ihre Bakteriensymbiose. Planta 31:1-21.

87. SCHAEDE, R. 1941. Untersuchungen in denWurzelknollchen von Viciafaba und Pisumsativum. Beitr. Biol. Pflanz., II Heft, 27:165-188.

88. SCHNEIDER, K. C., G. E. HOCK, AND R. H.BURRIS. 1959. Hydrogen metabolism ofsoybean root nodules. Federation Proc. 18:318.

89. SEMBRAT, Z. 1934. Influence of caffeine onmorphological changes of the nodule bac-teria. (In Polish, English summary.) ActaSoc. Botan. Polon. 11:333-346.

90. SHUG, A. L., P. B. HAMILTON, AND P. W.WILSON. 1956. Hydrogenase and nitrogenfixation, p. 344-360. In W. D. McElroy,[ed.], Symposium on inorganic nitrogenmetabolism. Johns Hopkins Press, Balti-more.

91. SMITH, J. D. 1949. The concentration and dis-tribution of haemoglobin in the rootnodules of leguminous plants. Biochem. J.44:585-591.

92. SPICHER, G. 1954. Lebensdauer und Stick-stoffbindung der Knollchenbakterien vonLupine, Serradella und Klee in Abhangig-keit von ihrer Gestalt. Zentr. Bakteriol.Parasitenk., Abt. II, 107:383-419.

93. STORCK, R., AND J. T. WACHSMAN. 1957.Enzyme localization in Bacillus mega-terium. J. Bacteriol. 73:784-790.

94. THORNE, D. W., AND R. H. BURRIS. 1938.

140 [VOL. 26

BACTEROIDS OF GENUS RHIZOBIUM

Enzyme systems in nodules of leguminousplants. J. Bacteriol. 36:261-262.

95. THORNE, D. W., AND R. H. BURRIS. 1940.Respiratory enzyme systems in symbioticnitrogen fixation. II. Respiration of Rhizo-bium from legume nodules and laboratorycultures. J. Bacteriol. 39:187-196.

96. THORNTON, H. G. 1930. The influence of thehost plant in inducing parasitism in lucerneand clover nodules. Proc. Roy. Soc. (Lon-don), Ser. B, 106:110-122.

97. THORNTON, H. G. 1945. Effective and ineffec-tive strains of legume nodule bacteria.Nature 156:654.

98. THORNTON, H. G. 1947. The biological inter-actions of Rhizobium to its host legume.Antonie van Leeuwenhoek J. Microbiol.Serol. 12:85-96.

99. TUZIMURA, K. 1950. Studies on the respira-tion of the root-nodules of leguminousplants. I. (In Japanese, English summary.)J. Sci. Soil Manure (Japan) 21:111-115.

100. TUZIMURA, K. 1950. Studies on the respirationof the root-nodules of leguminous plants.II. (In Japanese, English summary.) J.Sci. Soil Manure, Japan 21:283-287.

101. TUZIMURA, K. 1950. Lactoflavin in the root-nodules of leguminous plants. (In Japa-nese, English summary.) J. Agr. Chem.Soc. Japan 24:97-100.

102. TUZIMURA, K. 1951. Studies on the hemopro-tein of the root-nodules of leguminousplants. II. Inhibition of respiration ofnodules by CO. (In Japanese, Englishsummary.) J. Agr. Chem. Soc. Japan 24:266-269.

103. UHER, M. 1937. Ein Beitrag zum Problemdes Bakterienkernes. Ann. acad. tchecos-lov. agr. 12:474-478.

104. VANDECAVEYE, S, C., W. H. FULLER, ANDH. KATZNELSON. 1940. Bacteriophage ofrhizobia in relation to symbiotic nitrogenfixation by alfalfa. Soil Sci. 50:15-23.

104a. VINCENT, J. M., AND J. R. COLBURN. 1961.Cytological abnormalities in Rhizobiumtrifolii due to deficiency of calcium or mag-nesium. Australian J. Sci. 147:269-270.

105. VIRTANEN, A. I. 1947. The biology and chem-

istry of nitrogen fixation by legume bac-teria. Biol. Rev. 22:239-269.

106. VIRTANEN, A. I. 1956. Biological nitrogenfixation. Proc. Intern. Congr. Biochem.,3rd Congr. (Brussels, 1955), p. 425-433.

107. VIRTANEN, A. I., M. NORDLUND, AND E.HOLLO. 1934. Fermentation of sugar bythe root nodule bacteria. Biochem. J. 28:796-802.

108. VIRTANEN, A. I., J. JORMA, H. LINKOLA, ANDA. LINNASALMI. 1947. On the relationshipbetween nitrogen fixation and leghaemo-globin content of leguminous root nodules.I. Acta Chem. Scand. 1:90-111.

109. VIRTANEN, A. I., J. ERKAMA, AND H. LINKOLA.1947. On the relationship between nitrogenfixation and leghaemoglobin content ofleguminous root nodules. II. Acta Chem.Scand. 1:861-870.

110. VOETS, J. 1949. Onderzoek over de nucleaireStructuur van Rhizobium. Mededel. Land-bouwhogeschool en Opzoekingssta. StaatGent 14:235-250.

111. WARD, H. M. 1887. On the tubercular swell-ings on the roots of Viciafaba. Phil. Trans.Roy. Soc. London, Ser. B, 178:539-562.

112. WILSON, J. K. 1939. Leguminous plants andtheir associative organisms. Cornell Univ.Agr. Expt. Sta., Mem. 221:1-48.

113. WILSON, J. B., AND P. W. WILSON. 1943. Theaction of inhibitors on hydrogenase inAzotobacter. J. Gen. Physiol. 26:277-286.

114. WILSON, P. W. 1939. Mechanism of symbioticnitrogen fixation. Ergeb. Enzymforsch.8:13-54.

115. WILSON, P. W. 1940. The biochemistry ofsymbiotic nitrogen fixation. Univ. Wis-consin Press, Madison. 302 p.

116. WILSON, P. W. 1956. Hydrogenase in biologi-cal nitrogen-fixing agents. Science 123:676.

117. WILSON, P. W., R. H. BURRIS, AND W. B.COFFEE. 1943. Hydrogenase and symbioticnitrogen fixation. J. Biol. Chem. 147:475-481.

118. YAKOVLEVA, Z. M. 1959. The isoelectricpoint of Rhizobium. (In Russian, Englishsummary.) Izvest. Akad. Nauk S. S. R.Ser. Biol. 4:595-598.

1962] 141