MICROBIAL TRANSFORMATIONS OF THE TOBACCO ALKALOIDS I. CULTURAL AND MORPHOLOGICAL CHARACTERISTICS OF A NICOTINOPHILE PETER L. SGUROS Biochemical Division of the Research Department, R. J. Reynolds Tobacco Company, Winston-Salem, North Carolina Received for publication June 7, 1954 Only a few articles in the past ten years have attempted to define the relationships between autochthonous microflora and the alkaloid products of the tobacco plant. Almost from the moment of the germination of the tobacco seed the synthesis of nicotine begins in the root in significant quantities (Dawson, 1948). At maturity, the extent of total alkaloid production normally reaches 2 to 4 per cent of the dry weight of the plant tissue. Most of this percentage is represented by nicotine or structurally related compounds (figure 1). Although there are certain published conceptions, it is instinctive to rebel at the assumption that such materials are present without adequate reason. Furthermore, the number of known alkaloids in nature indi- cates that they represent another great category of natural materials which, for certain purposes of classification, may be aligned with such groups as amino acids, carbohydrates, and fatty acids. The significance of these materials in the life of the tobacco plant is unknown. While possibly fortuitous, it is interesting to note that such highly important compounds as pyridoxin and nicotinic acid bear close resemblance to many of the so-called "pyridine" alkaloids. To date, how- ever, the preponderance of questions concerning the metabolic pathways of the tobacco alkaloids in plant and animal tissue remains essentially unanswered. To such relatively new and po- tentially important fields of knowledge the bacteria may provide the key. This report describes an initial step in the study of the microorganism-alkaloid relation- ship: the isolation and characterization of the nicotinophilic bacterium, Arthrobacter oxydans (Sguros, 1954). Conn and Dimmick (1947) described in this journal a new genus of the family Corynebac- teriaceae which they named Arthrobacter (Fischer, 1895). The type species, A. globiforme, had been 28 initially described as Bacterium globiformis Conn (Conn, 1925, 1928) and further character- ized by Conn and Darrow (1930, 1935), Taylor and Lochhead (1937), Lochhead and Taylor (1938), Taylor (1938), Lochhead (1940, 1948), and Lochhead and Chase (1943). In addition to A. globiforme, Conn and Dimmick have trans- ferred two species of soil corynebacteria (Jensen, 1934) to the genus Arthrobacter as A. helvolum and A. tumescens. Lochhead and Burton (1953) have added A. pascene and A. terreens to the genus, and the senior author is presently prepar- ing the genus Arthrobacter for inclusion in the 7th edition of Bergey's Manual (Breed, 1954, personal communication). Detailed nutritional studies on A. pascens and A. terreens have been reported by Lochhead and Burton (1953) and Burton and Lochhead (1953). Sacks (1954) has recently isolated and characterized A. citreus. Nicotine decomposing bacteria have been described by Weber (1935), Wenusch (1942, 1943), Bucherer (1942, 1943), Frankenburg (1948), Abdel-Ghaffar et al. (1952), and Wada and Yamasaki (1953, 1954). Only in the case of Bucherer was a thorough cultural study at- tempted, but little is presented concerning metabolic implications. The Japanese workers present the rudiments of a possible degradation pathway but do not actually characterize the responsible culture. EXPERIMENTAL METHODS Seven strains of A. oxydans were isolated from various places in the southeastern section of the United States and studied in parallel (table 1). The isolation medium consisted of nicotine 0.4, monobasic potassium phosphate 0.2, potassium chloride 0.5, and yeast extract (Difco) 0.01 per cent in tap water at pH 6.8. Magnesium and ferrous sulfates (0.0025 per cent) were added routinely. The addition of 1.5 per cent agar did on March 25, 2020 by guest http://jb.asm.org/ Downloaded from
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MICROBIAL TRANSFORMATIONS OF THE TOBACCO ALKALOIDS
I. CULTURAL AND MORPHOLOGICAL CHARACTERISTICS OF A NICOTINOPHILE
PETER L. SGUROSBiochemical Division of the Research Department, R. J. Reynolds Tobacco Company, Winston-Salem,
North Carolina
Received for publication June 7, 1954
Only a few articles in the past ten years haveattempted to define the relationships betweenautochthonous microflora and the alkaloidproducts of the tobacco plant. Almost from themoment of the germination of the tobacco seedthe synthesis of nicotine begins in the root insignificant quantities (Dawson, 1948). Atmaturity, the extent of total alkaloid productionnormally reaches 2 to 4 per cent of the dry weightof the plant tissue. Most of this percentage isrepresented by nicotine or structurally relatedcompounds (figure 1). Although there are certainpublished conceptions, it is instinctive to rebelat the assumption that such materials arepresent without adequate reason. Furthermore,the number of known alkaloids in nature indi-cates that they represent another great categoryof natural materials which, for certain purposesof classification, may be aligned with such groupsas amino acids, carbohydrates, and fatty acids.The significance of these materials in the life ofthe tobacco plant is unknown. While possiblyfortuitous, it is interesting to note that suchhighly important compounds as pyridoxin andnicotinic acid bear close resemblance to many ofthe so-called "pyridine" alkaloids. To date, how-ever, the preponderance of questions concerningthe metabolic pathways of the tobacco alkaloidsin plant and animal tissue remains essentiallyunanswered. To such relatively new and po-tentially important fields of knowledge thebacteria may provide the key.This report describes an initial step in the
study of the microorganism-alkaloid relation-ship: the isolation and characterization of thenicotinophilic bacterium, Arthrobacter oxydans(Sguros, 1954).Conn and Dimmick (1947) described in this
journal a new genus of the family Corynebac-teriaceae which they named Arthrobacter (Fischer,1895). The type species, A. globiforme, had been
28
initially described as Bacterium globiformisConn (Conn, 1925, 1928) and further character-ized by Conn and Darrow (1930, 1935), Taylorand Lochhead (1937), Lochhead and Taylor(1938), Taylor (1938), Lochhead (1940, 1948),and Lochhead and Chase (1943). In addition toA. globiforme, Conn and Dimmick have trans-ferred two species of soil corynebacteria (Jensen,1934) to the genus Arthrobacter as A. helvolumand A. tumescens. Lochhead and Burton (1953)have added A. pascene and A. terreens to thegenus, and the senior author is presently prepar-ing the genus Arthrobacter for inclusion in the7th edition of Bergey's Manual (Breed, 1954,personal communication). Detailed nutritionalstudies on A. pascens and A. terreens have beenreported by Lochhead and Burton (1953) andBurton and Lochhead (1953). Sacks (1954) hasrecently isolated and characterized A. citreus.
Nicotine decomposing bacteria have beendescribed by Weber (1935), Wenusch (1942,1943), Bucherer (1942, 1943), Frankenburg(1948), Abdel-Ghaffar et al. (1952), and Wadaand Yamasaki (1953, 1954). Only in the case ofBucherer was a thorough cultural study at-tempted, but little is presented concerningmetabolic implications. The Japanese workerspresent the rudiments of a possible degradationpathway but do not actually characterize theresponsible culture.
EXPERIMENTAL METHODS
Seven strains of A. oxydans were isolated fromvarious places in the southeastern section of theUnited States and studied in parallel (table 1).The isolation medium consisted of nicotine 0.4,monobasic potassium phosphate 0.2, potassiumchloride 0.5, and yeast extract (Difco) 0.01 percent in tap water at pH 6.8. Magnesium andferrous sulfates (0.0025 per cent) were addedroutinely. The addition of 1.5 per cent agar did
not detract from the isolating capacity. Themedium was regularly sterilized in the autoclavefor 20 minutes at 121 C.
Cultural studies were controlled through theparallel use of A. globiforme, ATCC 8010, and A.tumescens, ATCC 6947. Other comparisons weretaken from the literature or personal communi-cation.Taxonomic characteristics were studied for the
most part according to the Manual of Methodsforthe Pure Culture Study of Bacteria (1953). Mor-phological studies were performed by means ofthe Leitz Ortholux microscope (1,125 X and1,425 X for both dark field and stained prepa-rations) and the Leitz phase microscope. In thelatter case, a glass cell 2.5 by 5.5 by 0.3 mm wasfabricated to afford the indispensable air andmoisture to the organisms in a drop of semisolidnutrient agar suspended from a coverslip. Thiswas the only feasible method found for followingthe growth of a given cell through its life cycle athigh magnification. The ordinary light micro-scope was used to examine the organisms for gramreaction, metachromatic granulation, flagella,spores, fat, acid-fastness, and capsulation.Motility and cell formations were investigated bydark field illumination.
Life cycle observations were made by almostcontinuous observation while gram staining re-
Figure 1. Certain of the known tobacco al-kaloids (Py symbolizes the pyridine ring).
TABLE 1Culture sources
Strin Source Source Type Location
1 Air North Carolina4 Air North Carolina
22 Air North Carolina117 Tobacco leaf Flue-cured North Carolina122 Tobacco leaf Flue-cured South Carolina127 Tobacco leaf Flue-cured North Carolina131 Tobacco leaf Burley Kentucky
actions were studied at hourly intervals for thefirst 24 hours and every 24 hours following formore than one week. Other staining reactionswere observed at 24 hour intervals. Dark fieldobservations were made on cultures sampled asfor gram staining above.
Semisolid, nutrient agar (0.7 per cent) wasemployed for the continuous study of the growingorganism. Nutrient and nicotine-salts broths andagars were employed for the growth of organismsdestined for staining procedures. Broth smearswere flame-fixed while Bouin fixation was em-ployed on plated agar cut-outs (Welshimer andRobinow, 1949).Growth curve-morphology correlations were
made during the growth of A. oxydans in shakeculture. A washed suspension of resting cystiteswas used to inoculate 20 ml quantities of liquidmedia contained in 25 by 250 mm tubes. Thetubes were then mounted in special racks on theplatform of a rotary shaker and agitated at 270rpm and 25 C. Both the nicotine isolatingmedium and nutrient broth were employed inthese studies. The tubes were removed from theshaker at intervals and the turbidities determinedon a Lumetron photoelectric colorimeter. Thenicotine medium required the use of a 420 filterwhile a 620 filter was employed for the nutrientbroth culture. Immediately following eachreading, smears were made from the media andgram stained for microscopic observation.
RESULTS
While all strains of A. oxydane were verysimilar in cultural behavior pattem, it seemsfeasible to divide them into biotypes. Two of theseven strains are chrome yellow on all mediatested while the rest are pearl gray or white. Theformer produce a viscid growth on agar while
the latter retain a butyrous consistency. It isalso of interest to note that the yellow culturesgrow very scantily upon potato glucose agar foralmost a week and then seem to abruptly burstinto luxuriant growth. The white cultures do notshow this lag. Accordingly, the biotypes are calledA. oxydams biotype xanthum and A. oxydans bio-type album.
Morphology. Old broth and agar cultures,especially in synthetic media, are invariablycoccus and coccoid in appearance, the cells vary-ing in diameter from 1 to 3 ,u (figure 2). Arrange-ments vary from single cells and pairs to chainsand masses. Within 3 hours, following inocu-lation into fresh nutrient broth or semisolid agar,germination begins (figure 3). The surface of agiven arthrospore or "cystite" (Jensen, 1934) isinterrupted by one or two-infrequently three-protrusions which rapidly assume the shape oftapered, tubular appendages 1 to 4 ,& in lengthand 0.5 to 1 ,u in width. In many cases, the rod-likestructure of the nascent tube is sharply dif-ferentiated from the spheroid structure of thecystite. Frequently such differentiations are moresubtle and the cystite-tube combination appearssimply as a clavate or tapered, uneven-sided rod.The germ tube may be straight- or curvedalthough graceful curvature is a more generalcharacter. It seems important to note that in thisspecies the germ tubes are not commonly indiametric opposition with regard to their pointsof issuance from the cystite. Rather, theseprojections commonly appear at an obtuseangle. When the germ tubes are more mature,they appear swollen to the extent that theterminal cystite is no longer a distinct structure.This results in the germ tube-cystite-germ tubecombination assuming the shape of a boot orboomerang, a form commonly observed inmicroscopic preparations. Another distinguishingfeature of this species is the strong tendencyfor the cystites to remain attached in pairs andshort chains regardless of the number of germtubes emerging from them. This results inbizarre arrangements of myceloid nature (figure4). Branching per se is actually not a prominentcharacteristic. Within 8 to 12 hours, a process offragmentation of the myceloid appears to begin(figure 5). Initially, this may be detected by theseparation of a rod form from the tip of themature, elongated germ tube. Within 15 hoursthe myceloids appear to disintegrate into un-
even, lesser masses of short rods, coccoids andcocci. Within one to three weeks the cultureassumes a homogeneous appearance of cocci insingle, paired, chain, or grape-like clusters.The whole cycle is schematically represented infigure 6. It will be noted that fragmentation ofthe myceloid may proceed to completion withouta separation of the components by significantdistances; hence, the appearance of chains andclusters. As has been mentioned above, thetenacious nature of this post-fragmentationassociation assumes determinative importance.The picture is essentially the same on 1.5 per centnutrient agar although the myceloids are lessexaggerated. Nicotine agar and broth mediaappear to reduce the complexity of the myceloidscausing a more subtle transition through thecycle. An important characteristic of this species(Conn and Dimmick, 1947) is the gram reactionobserved during the life cycle period. Cystitesare gram negative although they usually containa gram positive granule. The granules appearviolet or reddish purple with alkaline methyleneblue stain. During germination these granulesmay increase enormously in size almost fillingtheir cystites. The emerging germ tubes are verywealdy gram negative at first but rapidly increasein counterstain affinity. Soon hereafter the verytips of the germ tubes develop their own, smaller,gram positive granule. The number of granules ofthe elongating germ tube appear to increase untilfragmentation into cystites occurs leaving eachwith its own granule. The cycle is completed. Inmost cases, not disallowing procedure-inducedartifact, the granule appears at the surface regionof the mature, resting cystite as a dot or an oval,deeply stained body. During intermediate stages,rod forms appear to have the gram positivematerial at the surface region although it may begreatly stretched longitudinally. Seldom are cellsobserved which are totally gram positive, andeven in such cases the intensity is very delicate.It appears doubtful that more than a singlegranule is normally present in each, nondividingcell (Clark and Mitchell, 1942) or that the largeparental granule of the arthrospore is functionallydifferent from the minute granule of the germtube end. It is logical, due to the greater age of thedistal portion of the germ tube, that this regionshould be the first to synthesize the new granule.The cystite granule is undoubtedly that describedby Conn and Dimmick (1947) as occurring at the
"nodes" of the "branches". When a cystitegerminates in two or three directions, theoriginal granule remains at the apices. Finally,the granule may, within the germinating cystite,assume the almost exact shape and size of thecell; or, filling half of it, appear ovoid or fusiform;or remain as a small globule; or be seen as anelliptical form. In this species, the cystite isusually not filled entirely. It is distinctive thatthe granule favors only one edge of the cell,ordinarily being coincident with it. This follows,of course, from the above described granulationof the resting cystite. It is obvious that cystitegermination and the size of the granule are relatedin a manner probably more than superficial. Noattempt will be made at this time to discuss thenature of this relationship or the detailed se-quence of the development of the granule. Clarkand Mitchell (1942) have determined thisgranule in A. globiforme to be volutin.No "tails" were observed as have been re-
ported by Clark and Mitchell (1942) as well asby Taylor and Lochhead (1937). In this connec-tion it is interesting to note that the "tailingsequence", as described in this early work, maybe taken as a corroboration of the writer's ob-servations if the "sequence" is merely reversed.It is assumed, of course, that the "tail" wasactually a germ-tube. In this event the sequencecould be developed as follows: (a) coccal cystite,(b) emergence of a faintly staining, young germ-tube, (c) mature germ-tube, swelling and withstrong affinity for stain, (d) uneven-sided "rodform" with arthrospore structure no longerdistinguished from the enlarged germ-tube(figures 4 and 6). Neither true motility norflagella were ever observed. While this is con-trary to the recent observations of Sacks (1953,1954), it conforms well with descriptions of allother valid species known to this author. Nega-tive results were also obtained in tests forcapsules, spores, and acid-fastness. Fat stainsgave positive results. AMetachromatic granuleswere observed.
Cultural characteristics. Nutrient agar platecolonies may be punctiform or circular. They aresmooth, entire or slightly undulate, convex,glistening, viscid, and opaque. One varietytypically exhibits a bright yellow, insoluble pig-ment while the other is pearl gray or white.
Gelatin plate colonies are punctiform, convex,smooth, entire, opaque, and slowly liquefying.
Agar stroke growth is luxuriant, filiform,glistening, viscid, and yellow. Achromogenicstrains are butyrous and pearl gray. A glassy,metallic sheen is acquired with age.
Nutrient broth growth typically exhibits asurface ring, strong turbidity, and viscid sed-iment. Achromogenic strains are slower inproducing viscidity.Acid production in fermentation tests is con-
sistent among these strains only in fructose andsucrose. Acid in glucose is weak and transient.No gas is observed in any case. All of the con-ventionally employed phenol red broths con-taining mono-, di-, and polysaccharides as well assugar alcohols and glucosides permit good growthand turn basic.
Gelatin stabs exhibit crateriform liquefactionwhich begins in 2 days and slowly reaches 50 percent to 75 per cent completion without pro-gressing significantly further.Growth on nicotine-salts-yeast extract plates
and slants is somewhat more abundant thandescribed above but otherwise quite identical.In these cases, however, a deep blue, diffusiblepigmentation characteristically augments theyellow or pearl white, nondiffusible colors. Intime, the blue pigment turns reddish or yellow-brown. This blue material is formed during theoxidation of nicotine and is practically insolublein all of the common immiscible solvents with theexception of benzyl alcohol. It is both pH andredox potential sensitive. In shaken brothcultures the color change to brown is quitegradual, but if these cultures are removed fromthe shaker in the "blue stage", the organismsreduce their own product to a yellow-brownwithin a few hours. Shaking again restores theblue color.No growth can occur in the absence of oxygen
on Brewer's medium in a hydrogen atmosphere.If this deprivation is allowed to continue for afew days, the organisms appear irreparablydamaged. None but surface growth has ever beenobserved in stab cultures of various media.
Litmus milk turns basic with reduction of thelitmus. This is usually followed by slow clearing.
Nitrates are strongly reduced.Hydrogen sulfide is not produced.Indole is not produced. Methyl red and Voges-
Proskauer tests are negative, but citrate supportsgrowth.
Figure 6. Schematic representation of the lifecycle as observed in a single field by phasemicroscopy. Organism in semisolid agar.
Growth does not occur in the presence ofphenol or cresol but is fair in the presence ofnaphthalene and good on ethanol agar. Am-monium salts and amino acids can serve as
nitrogen sources.
Starch is hydrolyzed.Potato glucose agar. Growth is luxurious, but the
yellow variety exhibits a lag of about one week.In nutrient media, growth is favored by pH 7
while in nicotine-salts media pH 6 gives an optimalresponse. In either case, 25 C is the optimalgrowth temperature.The sources (table 1) are air and tobacco leaf
although the habitat is probably soil. The or-
ganisms appear hardy, and their characteristicsare stable in stock culture storage.
It is interesting to note that upon one occasionafter plating a suspension from a "high alkaloid"burley sample on nicotine-salts agar media, a
large population appeared that was almost a pure
culture of A. oxydans in both chromogenicvarieties. The white variety predominated by a
large margin in this instance.The control cultures, A. globiforme and A.
tumescens, behaved in a manner which left no
doubt that A. oxydans is a different species ofthe genus. While morphologies and gram staincharacteristics are similar, there is sufficientdifference to warrant a separate speciation. Thisis most apparent in A. tumescens which is of
smaller cell size in most respects, rendering itslife cycle transformations much more subtle.Culturally, this organism grows very scantilyupon the nicotine medium and does not produceany pigmentation. While its growth rate on mostcommon media is much slower than that of A.oxydans, it nevertheless liquefies gelatin withfar greater rapidity. Growth in broth is weakwithout surface phenomena. A. globiforme,while morphologically more similar to A. oxydans,fails to grow upon the nicotine medium. Thetype species grows moderately well on mostcommon media but with a cream chromogenesis.Of the three species, its rate of gelatin liquefactionis the slowest. No acid is observed in sucrose orfructose. Nitrate reduction, in this strain atleast, is very restricted or absent. Starch is nothydrolyzed. In general, the growth rates of thesecontrol cultures are less rapid than in the case ofA. oxydans. In spite of these differences, how-ever, the total pattern of observations shows thatthese three species are generically related. A finalcase in point is that of A. citreus (Sacks, 1954).The morphology of this organism, in so far asthe publication reveals it, shows it to be verysimilar to A. oxydans. Routine taxonomic tests inthe writer's laboratory demonstrate that, unlikeoxydans, citreus does not hydrolyze starch, changelitmus milk, enjoy the same pH optima, producesurface growth in liquid media, grow well onpotato, potato-glucose, nicotine, nicotine-glucose,glycerol, or asparagine agars. In addition, citreusis claimed to have a motile stage.
Growth morphology correlations. The nicotinemedium served to provide the best correlatingexperiment from a time standpoint (figure 7).Within 33 hours, the entire life cycle is completedhomogeneously. From the figure it would appearthat the greatest period of cell synthesis occursduring the myceloid stage. Since the methodemployed measures "total cell substance" andsince the shaking process augurs against extensivemyceloid formation, there is little tendency forsudden changes in the curve due to fragmenta-tion. It is noteworthy that during the lag period,germination proceeds through its initial stages ina manner detectable only by microscopic ob-servation. On the other hand, nutrient brothshake cultures require more than 300 hours beforethe cystite stage becomes prominent. Even then,homogeneous cultural morphology is lacking.Fragmentafion proceeds very slowly. The lag
period is demonstrably shorter tithe nicotine medium, but the logthe same rate. Static nutrient brcthe same short lag period, but tiis much reduced; maximumreached only after some 200nutrient broth 32 hours; shakei48 hours). However, at 300 hounculture of cystites is obtainedallows more extensive myceloid fithe curve is irregular with a sud'the point where the myceloids beThe total fragmentation phas4quires many hours for completio:
In the various growth cycle sttbeen partly depicted in figuresgeneous cystite formation hasprimarily in three situations: (1)4the semisynthetic nicotine medgrowth in the static nutrient(3) during growth in the phchamber. It will be noted that in tthe organism was forced to relysynthesizing mechanisms; in t]
1 ~/c/CO#RRE
xP~-,o /l j%,/#s l
e
0 0 20HR&
Figure 7. Correlation of mcgression with turbidimetric growArthrobacter oxydans in nicotine
han in the case of;phase maintains)th cultures showhe log phase ratetransmission iShours (shaken
n nicotine broths a homogeneousL. Static growthormation; hence,len, short rise at)gin to fragment.e, however, re-1n.idies which have6 and 7, homo-
growth proceeded under conditions of probablyless than adequate aeration (unpublished re-sults); in the third circumstance, the chamberbeing a closed system, inadequate aeration and anaccumulation of metabolic gas were probablyboth operative. While the significance of cystiteformation is not known, it has been implied, bythe use of the synonym "arthrospore", to be amore resistant phase in the life of the organism.Regarding the aforementioned observationsspeculatively, it seems not remiss to suggest thatthe above implication may have some justi-fication. Unfortunately, the results of directexperimentation on this important point arelacking at present.
been obtained DISCUSSIONduring growth in Growth on nicotine would not appear to be aLium, (2) during common characteristic among microorganisms.t medium, and Many bacteria are inhibited by it even in thetase microscopy presence of acceptable substrates. In the author'sthe first instance, laboratory it has been strikingly apparent thatheavily upon its few, if any, soil or plant molds will grow evenhe second case, suboptimally on nicotine as the source of carbon
and nitrogen-strange behavior indeed for thisnotoriously omnivorous group. On the other hand,there seems to be a far greater tendency for
< ',d ;.t } . microorganisms, generally, to grow where glucose; / . ee4 is the carbon source and nicotine the source of
_ * nitrogen. It is not, therefore, possible to speculatethat microorganisms have difficulty in openingthe ring structure to obtain carbon since, inorder to obtain nitrogen, the ring rupture mustundoubtedly be accomplished anyway (figure 1).In any event, it is only possible to hypothesizethat some of those organisms reported thus faras oxidizing nicotine may fall into a taxonomi-cally feasible group within the genus Arthrobacter.The tenuous nature of the above statement re-
LASIONH flects the inadequacy of earlier reports which in-r/lvLraMW completely describe the cultures involved. Buch-
erer (1942, 1943) succeeded in describing fourspecies of nicotine decomposing bacteria whichhe named Bacterium nicotinobacter, B. nico-tinophagum, B. nicotinovorum, and B. nicotianum.While all species grew on nicotine with the pro-duction of a blue pigment, only B. nicotinovorumappears to resemble A. oxydanm. However, while
Jo 40ordinarily complete descriptions were given in all
)rphological pro- cases, no attempt was made to determine a liferth expression for cycle in the case of B. nicotinovorum. The onlyshake culture. morphological clue given is that the culture was
oval and round forms. It would seem that wherea life cycle exists, other cultural characteristicsdecrease in importance from a determinativestandpoint overshadowed as they are by mor-
phological criteria. Bucherer (1942) unqualifiedlystates that B. nicotinovorum is gram positiveand that it grows as well aerobically as anaero-
bically. The last feature especially serves todifferentiate this organism from A. oxydans.Whether there are morphological similaritiesmust be left to conjecture. Another organism,designated unofficially as Corynebacterium nico-tinovorum, has also been described (Abdel-Ghaffar et al., 1952). It was reported as a grampositive, "coccoid bacillus" which was capableof "destroying" nicotine with the production of ablue pigment. It was further characterized as
citrate positive, nitrate reducing, and unable toferment the common sugars. From the briefmorphological description, it is very doubtfulthat the culture is related to A. oxydans. Finally,a soil organism, designated as probably belongingto Pseudomonas (Wada and Yamasaki, 1953),has been reported to oxidize nicotine with bluepigment production. No cultural description isgiven. While the taxonomic significance of theproduction of a blue pigment from nicotine is as
yet unknown, it is evident that this is primarilya characteristic of species importance at thepresent time. Cultures of other Arthrobacter and ofCellulomona, Mycobacterium, and Corynebac-terium have failed even to grow in the presenceof nicotine in the author's laboratory (unpub-lished results). Lochhead (1954, per8onal com-munication) has stated that A. terregens, A.pascen, and A. helvolum grew poorly on thenicotine, isolating medium. No chromogenesiswas observed. In addition to these facts, theresults of nutritional studies on A. oxydans (tobe published) provide further areas of differencebetween the nicotinophile and these Arthrobacter.Lesser cultural differences are also prevalent.
ACKNOWLEDGMENT
The author gratefully wishes to recognize theconsiderate and helpful correspondence of Dr.R. S. Breed, Chairman of the Bergey's ManualEditorial Board, and of Dr. A. G. Lochhead,Chief of the Bacteriology Division, CanadianDepartment of Agriculture. Technical assistance
was provided by Mr. Randall Jones and MissTrudy Truell.
SUMMARY
Seven strains of a microorganism isolated fromtobacco leaves as well as from the air of tobaccoenvirons and recently named Arthrobacter oxydanshave been fully described taxonomically. Twobiotypes, recognized as yellow-viscid and pearl-white-butyrous, have been called A. oxydansbiotype xanthum and A. oxydans biotype album,respectively. The organisms proceed through alife cycle which involves germination from spher-ical arthrospores or cystites, myceloid formationdue to the multiple germination of cystite groups,fragmentation of the myceloids into rods andcocci, and the return of the homogeneous coccoidappearance. The cyclic progression is accom-panied by the development and transformationof a gram positive granule in each cell. The or-ganism is gram negative, obligately aerobic, non-motile, metachromatically granulated, nitratereducing; indole, methyl red, Voges-Proskauer,and hydrogen sulfide negative but citrate posi-tive; hydrolytic on starch, casein, and gelatin;nonfermentative; catalase positive. Nicotine isvigorously utilized as a sole C and N source withthe production of a diffusible, blue pigment. Tur-bidimetric expressions of growth in various mediahave been correlated with cultural metamor-phosis.
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