THE GENERA LEUCOTHRIX AND THIOTHRIX · THE GENERALEUCOTHRIXANDTHIOTHRIX RUTHHAROLDAND R. Y. STANIER...

THE GENERA LEUCOTHRIX AND THIOTHRIX

RUTH HAROLD AND R. Y. STANIER

Department of Bacteriology, University of California, Berkeley, California

We have isolated a remarkable filamentous siderable number of infusions were prepared frommarine organism, Leucothrix mucor, which was algal thalli and incubated in diffuse daylight atfirst described in 1844 by Oersted (1). Leucothrix 25 C. Seven species of algae were used, of whichmay be characterized succinctly as a chemohet- one belonged to the Chlorophyta, two to theerotrophic counterpart of the colorless sulfur- Phaeophyta, and four to the Rhodophyta. Onlyoxidizing organism Thiothrix. It has been ob- infusions prepared from the green alga, Ulvaserved on a few occasions (2, 3) in the century lactuca Linn., gave rise to the development ofsince its original description, but the existing Leucothrix, which appeared without fail in everyaccounts of its morphology and development, flask containing Ulva. The enrichment procedurebased entirely on the examination of crude cul- is, of course, far from specific. Within a few daystures, are either incomplete or inaccurate. Thanks a pellicle of bacteria and protozoa forms on theto the ease with which it can be grown in pure surface of the liquid, and the characteristic fila-culture, we have been able to determine its com- ments of Leucothrix generally become evidentplete cycle of development, which includes a upon microscopic examination of this pellicleunique and hitherto undescribed process of about the eighth day. Between the eighth andgonidial aggregation to form many-celled ro- twelfth days, Leucothrix has multiplied suffi-settes. Such an aggregation stage has never been ciently to permit its isolation via streaked plates.observed in Thiothrix, but the descriptions of It persists for a long time in such infusions; twothis organism provide a good deal of circumstan- strains were isolated from enrichment culturestial evidence for its occurrence, and we believe almost 3 months old in which it was the dominantthat the technical difficulties of cultivating organism, its filaments forming a complex net-Thiothrix have been responsible for the failure to work throughout the culture.detect it. Accordingly, we have decided to com- For the isolation and maintenance of pure cul-bine the description of Leucothrix with a critical tures we have used a dilute medium similar to thatreview and reinterpretation of the scanty and recommended by Pringsheim (4) for the cultiva-scattered work on the morphology and develop- tion of members of the Vitreoscillaceae. It con-ment of Thiothrix. sists of: tryptone 0.4 g, yeast extract 0.4 g, beef

extract 0.2 g, Na acetate 0.2 g, synthetic seaEnrichment and Isolation of Leucothrix water (5) 1 liter. After autoclaving, the pH is

A few years ago, one of us observed that marine 8.0-8.3. For solid media, 2.0 per cent agar isspirilla can be regularly enriched by placing added.pieces of the thalli of marine algae in flasks almost As a rule the colonies of Leucothrix comprisefilled with sea water and incubating these in- a small minority of those developing on platesfusions for several days at 25 C. When a repeti- streaked from enrichment flasks. However, thetion of such enrichments was undertaken re- morphology is so characteristic that its coloniescently, we observed that some of the flasks can be detected with ease when plates are exam-contained long, colorless, immotile filaments with ined with the aid of a dissecting microscope,the characteristic cell structure of blue-green and pure cultures can be readily obtained byalgae. Plates were being streaked at regular picking young colonies from the first plates underintervals from the infusions in order to isolate the microscope and restreaking them. In all, 6spirilla, and eventually colonies of the filamentous strains were isolated.organism were discovered on one of these plates.It proved on further study to be Leucothrix mucor. General Description of LeucothrixA repetition of the enrichment experiments was In liquid cultures, Leucothrix grows in the form

undertaken to discover, if possible, what factor of long, colorless, unbranched, tapering threadsdetermined the appearance of Leucothrix. A con- which are attached basally to the wall of the cul-

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50 RUTH HAROLD AND R. Y. STANIER [VOL. 19

ture vessel at the liquid-air interface and hang 2.0 mm. The colony structure hence differsdown into the medium. The basal diameter is markedly from that of filamentous gliding organ-about 3 ,p and the apical diameter 1.5-2.0 ,. The isms belonging to the family' Vitreoscillaceae (7),length is extremely variable; it can attain well which can spread rapidly over agar surfaces as aover 5 mm. Each filament consists of many cells result of gliding movement.with a smooth common outer wall. Occasionally All the strains studied proved to be very simi-individual cells in a filament die and autolyze lar. On original isolation, they tended to fall intowith the production of empty compartments (the two groups which differed with respect to theso-called "necridia" of the phycologist) that length attained by the filaments prior to thereveal very clearly the multicellular structure. A formation of gonidia. In strains of one group thesheath cannot be detected, either by phase con- filaments broke up early, while in strains of thetrast microscopy of living filaments or by the other group the formation of gonidia was delayedexamination of preparations suspended in nigro- for a much longer period. This developmentalsin or India ink. The majority of the filaments in a difference was reflected by differences in colonialliquid culture are arranged characteristically in structure and in the macroscopic appearance ofthe form of rosettes, consisting of many threads growth in liquid media. However, the distinctionof varying lengths in close basal apposition. At did not prove to be constant, and after mainten-the base of each rosette or individual filament is ance for a month or two in pure culture all strainsan inconspicuous holdfast. As a filament matures, tend to reach a common condition, intermediatethe apical region develops a beaded appearance, between the two extremes.caused by constriction of the outer wall at thetransverse septa. This is followed by the abscis- The Developmental Cycle of Leucothrxsion of single cells or short chains. After 24 hours, The development of Leucothrix has been fol-a liquid culture contains large numbers of such lowed principally in slide cultures. These wereisolated cells, which show gliding motility in cog- prepared by sealing three edges of a coverslip to atact with a surface. The homologous structures slide with a vaseline-paraffin mixture so as toin Thiothrix were designated as gonidia by Wino- form a chamber of suitable depth and introducinggradsky (6), and we shall use the same term in a drop of inoculated medium from a capillarythis paper. pipette through the open side, which was thenOn solid media, very young colonies consist of a immediately sealed to prevent evaporation. If a

single filament (Plate I, figure 1). As it lengthens, sufficient volume of air is included in the sealedit is thrown into regular folds to form a "thumb- preparation, growth proceeds normally for manyprint" colony (Plate I, figures 1 and 2). When hours, and the complete cycle of development canexamined under high magnification, the filaments be observed under favorable optical conditions onshow the typical apical-basal differentiation. undisturbed material.Eventually the apical portions break up into We shall start the account of the life cycle withgonidia, and older colonies consist as a result of a the liberation of gonidia from the apex of amixture of filaments and gonidia (Plate I, figures mature filament, a process illustrated in Plate II,3 and 4). The gonidia are immobilized and remain figures 5 to 7. When a filament matures, con-in chains at the sites of formation. They are, strictions are formed between the terminal cellshowever, potentially motile, as can be shown by destined to be liberated as gonidia. Such a fila-flooding a colony with liquid medium and observ- ment is shown in figure 5; the sixteen pre-gonidialing the behavior of the gonidia through a cover- terminal cells are clearly distinguishable. Justglass placed over the flooded colony. On dry agar before the liberation of gonidia begins, the apexsurfaces, Leucothrix never forms rosettes. As will of the filament starts to wave back and forth in abe explained in the next section, this is a conse- rather jerky fashion. In figure 6 the eight terminalquence of the immobilization of the gonidia. cells have broken off as a unit. In figure 7 theSince the filaments themselves are permanently terminal portion of the filament has changed itsimmobile, the extension of a colony on solid media position, and the chain of gonidia has started tois a purely passive phenomenon, and the colonies glide away. As the gonidia break off and movealways remain compact and relatively restricted away, maturation progresses down the' filament,in size, seldom attaining diameters greater than which continues to produce gonidia over a period


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1955] GENERA LEUCOTHRIX AND THIOTHRIX 51

of several hours. As a rule, the gonidia are liber- gonidia are liberated from the apical tip. Figureated in short chains, rather than singly, but the 15 shows a typical field of rosettes at an earlychains mostly break up into individuals during stage of filament formation. The liberation oftheir subsequent movements. gonidia begins about 12 hours after rosetteThe gonidia are motile only in a liquid medium, formation.

and only in contact with a solid surface. Under In order to observe aggregation uncomplicatedthese conditions, they show slow, short, jerky, by continued liberation of gonidia, slide culturesinterrupted movements, sometimes swinging back seeded with pure gonidial suspensions are neces-and forth while remaining attached to the sub- sary. Such suspensions may be obtained verystrate by one end of the cell. Flagella could not be simply by filtering a young liquid culture throughdemonstrated by staining. Evidently these cells Whatman no. 1 filter paper; as a result of theirare capable of gliding movement, similar to that great length, the filaments are retained on theshown by unicellular blue-green algae and myxo- filter, and the filtrate consists almost exclusivelybacteria. of gonidia, which can be concentrated to any

Provided that large numbers of gonidia are required density by centrifugation. Studies ofconcentrated over a relatively small area and that slide cultures prepared from gonidial suspensionstheir motility is unimpeded, the next stage of the of different densities show that the initial densitylife cycle is gonidial aggregation to form rosettes. of the gonidial population is of cardinal impor-Plate III, figures 8 to 13 depict the course of tance in determining whether aggregation willgonidial aggregation in a single microscopic field occur. When the gonidia in a slide culture areover a period of 55 minutes. Three individual separated from one another by an average dis-rosettes have been lettered in order to facilitate tance of 60 ,u or more, no rosettes are formed.the comparison of successive photomicrographs. Instead, each gonidium eventually settles down,

In the earliest observable stage of aggregation develops an individual holdfast, and grows out(figure 9), three gonidia form a cluster in close into a single filament (figure 16). The gonidia areapposition at one pole of the cell. Additional reproductive structures, and it is probable thatgonidia glide into the developing rosette until the period during which active movement caneventually a tightly packed structure is formed, occur is limited, just as in the case of a protistanthe component cells radiating in all directions zoospore. Consequently, a gonidium which doesfrom the central point of attachment. Up to a not manage to find its way into a rosette duringcertain point of development, aggregation is a this period is perforce condemned to isolatedreversible phenomenon, and rosettes containing development. A second factor which may wellas many as 12 individuals may redisperse com- operate to prevent rosette formation in dilutepletely. Larger rosettes appear to be incapable of gonidial preparations is the weakness of thedisaggregation, probably as a result of the fact intercellular chemotactic gradients. It seemsthat some of the component cells have already almost certain that chemotaxis plays a role inbecome sessile. Secretion of a holdfast common to aggregation since chance collisions could neverall the cells begins shortly after they have become cause the pattern of accumulation shown insessile. The holdfast is a very inconspicuous figures 8 to 13.structure, barely perceptible in living, unstained Since isolated gonidia can give rise to normalpreparations. The best method which we have filaments which in time liberate gonidia again, itdiscovered for making it readily visible is to flood a follows that rosette formation is not an obligatoryslide culture with an aqueous 10 per cent (w/v) feature of the life cycle. Furthermore, the factsolution of nigrosine. The nigrosine is adsorbed by that an isolated gonidium produces only onethe holdfasts, which appear as black bodies in the filament shows that rosettes cannot be formed bycenter of the rosettes, contrasting well with the basal cell division in several planes; evidently,unstained cells. The holdfasts at the bases of cell division is rigidly restricted to a single plane.isolated filaments are also clearly demonstrableby this technique (Plate IV, figure 14). Physilogy of LeucothrixEach gonidium in a rosette grows into a multi- Since Leucothrix so closely resembles Thiothrix

cellular filament, which continues to elongate morphologically, the possibility existed that ituntil maturation occurs and a fresh crop of might be a facultatively chemoheterotrophic


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52 RUTHHAROLD AND R. Y. STANIER [VOL. 19

member of the Thiothrix group. Accordingly, Leucothrix was previously used by Trevisanwashed filaments of Leucothrix were placed in sea (1879) as a synonym in part of Beggiatoa."water containing small amounts (approx. 1.5-150 This brief history aroused our curiosity, andmg per liter) of hydrogen sulfide and examined at

after some difficulties we succeeded in obtaining aintervals for the presence of intracellular sulfurglobules. Noo evidence of sulfur accumulation was cop of Gese' opsulm Dergon.uglobu*es. No evidence of sulfur accumulation was

marinis, elementa topographiae historiconaturalisobtained. Our observations on enrichment cul-freti Oeresund. Leucothrix is described with clas-tures also make it somewhat improbable that

Leucothrix is capable of oxidizing hydrogen sul- sceal terseness in the enumerati algarum iridium,fide. It has often been observed in algal infusions u t . S tl

* l l j . . . , ~~~~~rare 1we ut h rgnldsrpinwhich also contained numerous Beggiatoa and a , e quote the original description:Chromatium, and although the cells of these Leucothrix mucor nob. in stagnis submarinisorganisms were packed with sulfur, the interior plantas filis suis radiantibus mucorum modoof the filaments of Leucothrix was at all times obducit.sulfur-free. Character genericus. Fila alba et caespites etWe have always grown Leucothrix in the com- stratum, radiis longissimis emissis, formantia,

plex medium described in the preceding section, articuli distincti, interstitiae hyalinae simpli-but Dr. S. H. Hutner has very kindly examined cissimae absque constrictionibus, sporidianumerosa pulveracea alba.its nutrient requirements for us, and he reports Hoc genus a Callothrice, cui proximum est, et(personal communication) that it requires no indole caespitis, valde radianti, et interstitiisgrowth factors, and can use a considerable variety multo distinctioribus, ab omnibus confinibusof sugars and other simple organic compounds as sporidiorum indole valde recedit. Motiunculaesources of carbon and energy. ut apud ceteras Oscillatorineae interdum cons-

Leucothrix is a strict aerobe. The temperature pici possunt.maximum lies at 30 C, and the optimum at about25 C. It grows best at a salt concentration (syn-thetic sea water) of 16 g per liter. Growth is still Leucothrix mucor nob. send out its radiatingpossible at a salt concentration of 3 g per liter, threads in marine pools much as molds do.but the morphology is abnormal. Generic characters. White threads forming

both mats and layers, and sending out very longLeucothrix, Chlamydothrix, Pontothrix: a Tangled radiating strands, distinct articulations, with

Thread very simple unconstricted hyaline interstices,When we started our studies on Leucothrix, we sporidia numerous, powdery, white..eognidiscThis genus may be separated from Calothrix,

recognized its unawareol similanprevit to which it is close, by the nature of the mats,

Thsripthions wh eerenwreofi microorgany-rvu which are clearly radiating, by its much moredescriptions of chemoheterotrophic microorgan- distinct interstices, and by the highly localizedisms possessing this peculiar structure. Hence, wse formation of sporidia. Motile sporidia are pos-believed that we had discovered a new organism; sible, as is sometimes observed in the otherand since its physiological differences from Thio- Oscillatorineae.thrix clearly warranted generic segregation, itbecame our task to propose a name. Leucothrix In this translation, articulations should be con-seemed an apt choice, but on consulting Buchan- strued as transverse walls, interstices as cells, andan's General Systematic Bacteriology (8) we found sporidia as hormogonia or gonidia.that it had been previously used. Buchanan's There can be no doubt that Oersted describedaccount of its history follows: an organism closely similar to, if not identical

"Leucothrix. A generic name adopted from with, the organism which we have isolated: a

Oersted (1844) by De Toni and Trevisan (1889) 'Our copy was obtained from the Yale Uni-to serve as a subgenus of Leptotrichia Trevisan. versity Library.The type species is Leptotrichia (Leucothrix) 2 Kindly prepared for us by Dr. R. C. Baci-mucor (Oersted) Trevisan. The subgeneric de- galupi, Curator of the Jepson Herbarium at thescription given is simply "Marinae, majores." University of California.


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marine colorless blue-green alga of filamentous motility. They are always unbranched and ofstructure, showing a radial arrangement of very striking length. Threads 0.5 cm. or more inlong threads. The identification receives further length are not uncommon. In microscopic prepa-support from Oersted's statement that Leucothrix rations they lie in tangled masses, or form ropesis similar to Calothrix since it is the blue-green composed of numerous, often hundreds, of more

I. . or less parallel or twisted single threads.algae of this general type, now placed in the The threads are not uncommonly surroundedfamily Rivulariaceae, which most closely resem- by a gelatinous sheath of variable thickness,ble in vegetative construction the microorganism which is not directly visible as a rule. When itisolated by us. The only other known microorgan- attains an appreciable thickness it can be seenism to which Oersted's description could possibly in water mounts; and by the use of India ink,be applied is Thiothrix, but the statement "hya- even the thinner sheaths appear as a lightline interstices (cells)" does not fit, since in nature border around the filaments.the filaments of Thiothrix are as a rule almost The filaments without sheath are 1-3 1A inopaque on account of their sulfur content. Hence thickness, fully formed filaments being ordi-

we accept Gersted's name for the organism which narily 2 ;L thick. The filament consists of cellsaveraging 1-5 Z in length, which can be dis-

we have isolated and with all the more alacrity tinguished r nalynwell from oneanteI tinguished reasonably well from one anothersince it was our own choice before we knew of even in living material.his work. The threads adhere without a distinct hold-Warned by this experience, we then conducted fast to the water surface or to small solid ob-

a more rigorous search of the literature and found jects, and form bunches which hang down-that Leucothrix had been reported on two later wards.occasions. It was rediscovered in 1912 by Molisch The young colonies show a radiating arrange-(2) who, ignorant of Oersted's work, described it ment of the threads, similar to that which onebriefly under the name of Chlamydothrix labs- observes in Thiothrix. Reproduction occurs by

detachment of portions of the filament or singlepima. 1932, Ndeson and Ka sst cells. Occasionally a not very distinct holdfastamplfiedMolsch'decripion at he ame ime can be seen on young germinating filaments.

changing the name to Pontothrix longissima.Molisch described Chlamydothrix longissima in In figure 17 we reproduce Molisch's drawing of

a paper principally concerned with marine sulfur Chlamydothrix longissima. This illustration of abacteria. The relevant passage is translated young colony, which should be compared to ourbelow. photograph of rosettes in the early stages of elon-

gation (figure 15), shows that he was dealing withIn connection with these sulfur bacteria a tesm raim h nyfaueo oic'

filamentous bacterium will be described which istesm raim h nyfaueo oic'

filamentouesulfurbacteriumwillbeudesc which is account which does not tally with our findings isnot a true sulfur bacterium, but which com-monly occurs with marine sulfur bacteria and is the description of a sheath. In pure culture, Leu-a very characteristic component of the marine cothrix shows no sign of sheath formation. Thesulfur flora. It is surprising that this organism, discrepancy might be explained in two ways. It iswith its striking size, massive development and possible that under certain conditions in a naturalfrequent occurrence, has not caught the eye of environment, Leucothrix is capable of sheath for-bacteriologists long ago. I name this filamentous mation; or alternatively, that in algal infusions itbacterium Chlamydothrix longissima Molisch. often grows embedded in zoogleal masses of other

In rotting algal infusions in Trieste seawater organisms, which simulate a gelatinous sheaththere frequently occurred on the surface of the around the Leucothrix threads. The variableliquid a filamentous bacterium composed of thickness of the sheath as described by Molischshort cylindrical cells. Sulfur is never depositedin the cells, and only deposited on their surface would ben accord with either of these nterpre-when the threads lie right at the air-water inter- tations.face in contact with large amounts of oxygen. The description of the same organism given inThe threads are commonly free of sulfur, both 1932 by Nadson and Krassillnikov (3) is consider-internally and externally. ably more detailed than that of Molisch, but con-The threads recall a colorless Oscillaria, but tains a number of features which cannot be recon-

are immediately distinguishable by their im- ciled at all with our observations. Nadson and


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Krassilnikov maintained the organism in crude filaments. Hence mature filaments are character-culture for several months by successive transfer istically arranged in the form of radial colonies.in algal infusions, but did not succeed in isolating Gonidia can also develop singly, forming isolatedpure cultures. Judging from their description and filaments. Chemoheterotrophic.illustrations (reproduced in figure 18) it seems Synonym: Pontothrix Nadson and Krassil-very likely that they confused other filamentous nikov, 1932.microorganisms, also present in the infusions, One species, Leucothrix mucor Oersted 1844with developmental stages of Leucothrix. Their emend., with description as for genus.illustrations of mature sessile filaments and de- Synonyms: Chlamydothrix longissima Miolischveloping rosettes (figure 18: 3, 5, 10, 11), albeit 1912; Pontothrix longissima Nadson and Krassil-crude, fit the picture for Leucothrix; but the nikov 1932.peculiar thick cells occurring locally in normal Occurrence: Widespread on decaying plantfilaments (figure 18: 1, 2) and the emergence of material in marine littoral regions.multicellular hormogonia of an oscillatorian typefrom a sheath (figure 18: 3) do not. They failed to The Morphology and Development of Thiothrix:observe mobility of the gonidia and hence also a Reviewmissed the aggregation process. The genus Thiothrix was established by Wino-

Like Molisch, Nadson and Krassilnikov gradsky in 1888 (6) but is still very imperfectlystressed the fact that even when growing in algal known. During the course of time, seven speciesinfusions richly populated by sulfur-filled fila- have been proposed; they are differentiated bvments of Beggiatoa and Thiothrix, the threads of the width of the filaments and by habitat. VanLeucothrix never contain sulfur. Niel (9) states: "the validity of these distin-Nadson and Krassilnikov's observations on the guishing characters is, however, doubtful because

natural distribution of Leucothrixaree of consid- their constancy has not been sufficiently estab-erable interest. It was first seen by Nadson of lished; so far, the morphology of the Thiothrix1898, growing in decaying algae on the shore of species has not been studied in pure cultures."Helgoland. In 1914, he found it in a similar habi- The morphology and development of the varioustat at Naples, and in 1915 at Sevastopol. The forms which have been described show a sub-study reported in 1932 was made on material stantial uniformity, and accordingly in this re-again obtained from the Black Sea, and it was view we shall attempt a general description andalso observed at that time on algae collected at elucidation of the life cycle, without analyzingAlexandrovsk on the PolarSea. Coupled with our separately the data on each of the allegedobservations and those of AMolisch, this shows "species"chat Leucothrix is a widespread and common The study of Thiothrix is made peculiarly diffi-marine saprophyte in littoral regions, a fact which cult by its growth requirements. So far as islends plausibility to the existence of a very early known, it is an obligate chemoautotroph whichdescription in the algological literature, viz, that obtains its energy by the oxidation of H2S. Henceby Oersted. its development is dependent on the presence of

This survey of the somewhat devious history of C02, 02, and H2S; furthermore, as Keil (10) hasLeucothrix can be terminated most appropriately shown, the organism can tolerate only rather lowby an amended diagnosis. concentrations of H2S. This constellation of re-

Leucothrix Oersted 1844 emend. Colorless quirements adequately explains the fact that,immotile filaments up to 5 mm in length, tapering with one dubious exception (10), pure culturesfrom base to apex, and commonly attached bas- have never been obtained. The best availableally to solid substrates by an inconspicuous method for studying the development of Thio-holdfast. Reproduction by means of gonidia, thrix is microculture on slides, devised by-Wino-which are detached either singly or in small chains gradsky (6).from the apical portion of the filament. The Winogradsky's description of the morphologygonidia show gliding motility, and under favor- and life cycle of Thiothrix, still by far the mostable conditions aggregate to form rosettes con- complete one, was based on the study of suchtaining up to 50 cells. The cells in rosettes become microcultures. His conclusions are well summar-

immotile, develop holdfasts, and elongate to form ized in his description of the new genus:


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19551 GENERA LEUCOTHRIX AND THIOTHRIX 55

Non-motile filaments with transverse septa, nor Pringsheim denied the possibility that suchsurrounded by a delicate sheath, differentiated movement does exist, and indeed a denial couldinto an apical and a basal region, attached to scarcely be entertained in the face of Winograd-solid objects by means of a sticky holdfast, sky's highly circumstantial account. As Prings-filled with sulfur globules under normal con- heim (4) says: "the capacity for gliding move-ditions of existence. Reproduction by bacilli mondons,form gonidia, endowed with slow gliding movement is easily disturbed by changed conditions,ment, which fix themselves to solid objects and and the settlement of young filaments is scarcelyelongate into filaments. explicable without motile reproductive stages."

In our opinion, the failure of later observers toFigure 19 reproduces Winogradsky's original confirm this feature of Winogradsky's observa-

drawing of Thiothrix. tions simply reflects the extreme difficulty ofThe formation and behavior of the gonidia were maintaining slide cultures of Thiothrix continu-

described with great precision by Winogradsky, ously in a favorable physiological state.and since several later workers have failed to In one crucial respect, Winogradsky's descrip-confirm his account, we will translate the relevant tion of the morphology of Thiothrix in microcul-passage in its entirety. tures deviates markedly from descriptions based

on the examination of natural material. In crudeWhen the filament attains a certain length- cultures, as he himself states, Thiothrix forms

which is quite variable-one sees the initiation radial colonies, the filaments pointing out in allof the formation of gonidia. In a filament which directions from a central zone of attachment.is still young and not more than 100 lt in length, However there is no evidence that he observedthe process occurs in the simplest fashion: theterminal portion, 8-10 A long, disarticulates well formed radial colonies in microcultures. Thefrom the parent filament, remaining attached characteristic natural mature growth habit ofonly by the common sheath. At the same time, Thiothrix is beautifully portrayed in Miyoshi'sit starts to show motility, which is no more than (12) drawing (figure 20), and in a recent photo-a slight trembling; but soon the movements be- graph, kindly lent to us by Dr. E. J. Ordalcome swinging, the terminal portion making an (Plate VI, figure 21). The only illustrations of theangle with the filament, or even coming to rest early development of these radial colonies whichalongside it. All movements are quite slow, and we have discovered are those of Molisch (2);interrupted by long pauses. At a certain moment their resemblance to young radial colonies ofthe motile terminal portion attaches itself to Leucothrix is striking. Molisch makes a statementthe coverslip, as shown by th.fact that it no about these colonies which suggests that he maylonger responds to slight jarring movementswhich make the parent filament tremble. It have suspected that an aggregation process pre-then moves away in gliding fashion, drawing ceded their formation: "die jungen Keimlingeafter it the floating portion of the parent fila- pflegen nesterweise beisammen zu sitzen undment until tension breaks the connection, and wachsen strahlenartig aus."the filament retracts like a taut spring. Move- In effect, however, none of the students ofment lasts only 1-3 hours, during which time the Thiothrix has attempted to explain the character-motile gonidium covers a distance of only istic radial growth habit, and the curious devel-50-100 A. The movements are slow and irregular; opmental problem which it poses does not seem tosometimes it glides with its full length on the have been clearly appreciated. Since Winograd-substrate, sometimes it stands on end, but , . . .always remaining fixed to the glass. It finally sk' obevain sho tha th oiicivcomes to a halt and starts to elongate. a certain dispersion, only two mechanisms can be

entertained to account for the ultimately observedThe motility of the gonidia was confirmed by radial growth habit. One is the occurrence of a

one later observer, Wille (11), but since he misin- mode of cell division (e.g., basal budding or fissionterpreted the intracellular sulfur globules as gas in several planes) during the early stages of devel-vacuoles, he can scarcely be considered a reliable opment that is radically different from the regularwitness. Keil (10), the only worker after Wino- divisions in a single plane occurring later duringgradsky to explore in detail the cultivation and elongation of the filaments. The other is gonidialphysiology of Thiothrix, failed to observe active aggregation. In view of our observations on Leu-movement despite a careful search, and Prings- cothrix, it may be asserted with confidence thatheim (4) was equally unsuccessful. Neither Keil the latter mechanism is the one operative in


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Thiothrix. It remains, however, to explain why It is well known that the filamentous, glidinggonidial aggregation in Thiothrix has never been bacteria show close morphological affinities withobserved. Since most students of the group after filamentous blue-green algae, and that many ofW\inogradsky could not even detect gonidial them probably represent, phylogenetically speak-movement, their failure is readily understandable. ing,papochlorotic descendants of the Myxophyta.Winogradsky, however, examined preparations in The evidence on which this conclusion rests haswhich the gonidia were motile. It should be re- been analyzed recently in great detail by Prings-called that Leucothrix does not form rosettes heim (4), and we will not recapitulate it here.when the gonidia are widely separated from one The representatives of the Beggiatoaceae andanother; under these conditions they settle dowvn Vitreoscillaceae find their photosynthetic coun-singly and grow into isolated filaments which terparts in blue-green algae of the family Oscil-closely resemble the filaments of Thiothrix figured latoriaceae; Beggiatoa itself is structurally indis-by Winogradsky. Hence his failure to see aggrega- tinguishable from an Oscillatoria, apart from thetion was probably a consequence of the fact that presence of sulfur inclusions. On the assumptionthe gonidia in his preparations never occurred that an analogous derivation may have occurredwith the requisite abundance. in the case of Leucothrix and Thiothrix, we shallTo summarize the conclusions reached from now consider to what existing groups of blue-

this review of the literature on Thiothrix, we are green algae, if any, they show affinities. A closeof the opinion that Thiothrix is characterized by a relationship to members of the Oscillatoriaceaecycle of development in all respects identical with appears most improbable since these blue-greenthat of Leucothrix but that the existing descrip- algae do not show a marked apical-basal dif-tions (even that of Winogradsky) are incomplete, ferentiation of the filament. Furthermore, thegonidial aggregation having been completely reproductive structures of Leucothrix and Thio-overlooked as a result of the difficulties inherent thrix are quite different from the hormogonia ofin the study of an obligate chemoautotroph with the Oscillatoriaceae. A hormogonium is a shortthe particular requirements shown by Thiothrix. filament of cells which, as stressed by Geitler

(13), behaves both morphologically and physi-The Systematic Position of Leucothrix ologically as a unit; unicellular, motile hormo-

and Thiothrix gonia have not been described with certainty.

Leucothrix and Thiothrix clearly belong among It is for this reason that we chose to employ for

colorless, filamentous, gliding organisms, the reproductive cells of Leucothrix and Thio-thec incudealsth 'a se'u thrix the regrettably ill defined term gonidia, inwshich include also the Beggiatoacene sensuPringsheim (4) and the Vitreoscillaceae (7). In his preference to the well defined but inapplicablemasterly analysis of the relationships between term hormogonia. In order to make perfectlybacteriaandblue-green alg, P m () hd. clear the distinction between these two kinds ofbacteria an bleglealae lrnse4 a

reproductive structures, and also to confer aalready recognized the important morphological repodutiv sructures an als to onfer a.. .~~Tithi and. Beg.iaoa.an much-needed precision on the term gonidium, wedifferences between Thitothrix and Bseggiatoa and .mu. .dif ce ebtwn o fla s shall redefine it as follows. A gonidium is a uni-Thioploca the two ote grup of fiamntu cellular reproductive structure capable of gliding

sulfur bacteria with which it had been associatedin the family Beggiatoaceae, and he placed it in movement which is produced by abscission from the

aseparatefamilythe Thiotrichace* We f 1 terminal portion of a multicellularfilament.aseparatefamly, the lhotri a. We fully Sessile filaments with a pronounced apical-concur ^vith the systematic judgment impliedbyppconcur with the systematic judgment*pi by7*I1 basal differentiation are found most characteris-this proposal. Thiothrix and Leucothrix stand a-

part from all other filamentous, gliding bacteria as tically among blue-green algae of the familymuch more highly differentiated organisms. This Rivulariaceae. Furthermore, many members ofis shown by the functional and structural polari- the Rivulariaceae share to a greater or lesserzation of the filament; by the restriction of motil- extent the characteristic growth habit of Leuco-ity to specialized reproductive cells; and above thrix and Thiothrix, producing tufts or radially-all, by the occurrence of an aggregation stage. In arranged groups of filaments. Indeed Oerstedfact, Leucothrix and Thiothrix rank with the fruit- already drew attention in his original descriptioning myxobacteria and the higher actinomycetes of Leucothrix to its structural similarities toas the most complex of bacteria. Calothrix, a blue-green alga now placed in the


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Rivulariaceae. There are, however, several fea- Leucothrix (Chlamydothrix) was included in thetures of Leucothrix and Thiothrix which do not fit Chlamydobacteriaceae by Molisch (2), and avery well the postulate that they are apochlorotic similar position for Thiothrix was recently mootedrelatives of the Rivulariaceae. The first is the by Bisset and Grace (16), who incorrectly as-absence of a sheath. Most (all?) members of the sumed that the gonidia are immotile. The bestRivulariaceae have a definite sheath. Secondly, studied members of the Chlamydobacteriaceaethe filaments in the Rivulariaceae are usually are filamentous organisms of the Leptothrix-terminated basally by a specialized cell, known as Sphaerotilus type (17) which reproduce by meansa heterocyst; this feature is lacking, however, in of flagellated swarmers (see Stokes (18) for superbHomeothrix and Ammatoidea. Homeothrix further illustrations of their structure). They are thusresembles Leucothrix and Thiothrix in that it does eubacterial forms, albeit quite different in con-not show false branching, which is otherwise struction and development from filamentouswidespread in the Rivulariaceae. But despite eubacteria of the Caryophanon type. The groundsthese resemblances, there is one major objection for assuming a relationship of Leucothrix andto regarding Leucothrix and Thiothrix as apo- Thiothrix to these bacteria are no stronger thanchlorotic relatives of rivularian forms like Homeo- the grounds for assuming a relationship tothrix; namely, the apparent absence in the Rivu- Caryophanon.lariaceae (and indeed in all other filamentousblue-green algae) of reproductive structures ACKNOWLEDGMENTShomologous with gonidia, which can undergo We are deeply grateful to Dr. N. Palleroni foraggregation. It remains possible that such fea- his willing and expert assistance with the photog-tures may be found in blue-green algae of a riv- raphy of Leucothrix; and also to Drs. R. C.ularian type since these organisms have been Bacigalupi and M. Doudoroff for putting theirdescribed for the most part in superficial fashion knowledge of Latin and Russian, respectively, aton the basis of observations on natural material, our disposal.and an aggregation stage, if it existed, could beoverlooked very easily under these conditions, as REFERENCESthe early history of Leucothrix shows. The habit 1. OERSTED, A. S. 1844 De regionibus marinis,of the colony, particularly in unbranched forms elementa topographiae historiconaturalislike Homeothrix, is at least suggestive of the possi- freti Oeresund. J. C. Scharling, Copen-bility that an overlooked aggregation stage may hagen.exist. Were such to be found, the systematic 2. MOLISCH, H. 1912 Neue farblose Schwefel-

bakterien. Centr. Bakteriol. Parasitenk.,position of Leucothrix and Thiothrix could be con- Abt. II, 33, 55-63.sidered as settled; but for the time being, it is far 3. NADSON, G. A., AND KRASSILNIKOV, N. 1932from clear. La structure et l'6volution de Pontothrix

In conclusion, two other systematic proposals longissima Nads. et Krassiln. (Chlamydo-concerning Leucothrix and Thiothrix should be thrix longissima Molisch) une algue incolorementioned, if only to be dismissed. Peshkoff (14) du groupe de Schizophyceae. Dokladyincluded Nadson and Krassilnikov's Pontothrix Akad. Nauk SSSR, 1932, A, 243-247. Rus-longissima (which is identical with Leucothrix sian with German summary.mucor) in the order Caryophanales, whose best 4 PRINGSHEIM, E. G. 1949 The relationshipstudied representative is Caryophanon ltaum. between bacteria and myxophyceae. Bac-Caryophanon is a multicellular, filamentous bacteriol. Revs., 13, 47-98.

5. LYMAN, J., AND FLEMING, R. 1940 Compo-terlum whaic iS motile by means of peritrichous sition of sea water. J. Marine Research, 3,flagella and reproduces by binary fission of the 134-146.filament. Peshkoff (15) regarded it as intermedi- 6. WINOGRADSKY, S. 1888 Beitrage zur Mor-ate between bacteria and blue-green algae, but as phologie und Physiologie der Bacterien. I.Pringsheim (4) pointed out, there is no real basis Zur Morphologie und Physiologie derforthiscontentionand .isinfSchwefelbacterien. A. Felix, Leipzig. Re-forthiscontentionandiisinpublished as: Contribution A la morphologiecomplex eubacterial form. Hence Leucothrix et physiologie des sulfobacteries. In Wino-

(Pontothrix) has no place in an order of which gradsky, S. 1949 Microbiologie du sol,Caryophanon is the representative type. pp 83-126. Masson et Cie, Paris.


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7. PRINGSHEIM, E. G. 1951 The Vitreoscil- 14. PESHKOFF, M. A. 1948 Order Caryopha-laceae: a family of colourless, gliding, nales. In Bergey's manual of determinativefilamentous organisms. J. Gen. Microbiol., bacteriology, pp. 1002. 6th ed. The Wil-5, 124-149. liams and Wilkins Co., Baltimore, Md.

8. BUCHANAN, R. E. 1925 General systematic 15. PESHKOFF, M. A. 1940 Phylogenesis of newbacteriology. The Williams and Wilkins microbes, Caryophanon latum and Caryo-Co., Baltimore, Md. phanon tenue, organisms which are inter-

9. VAN NIEL, C. B. 1948 Family Beggiatoa- mediate between blue-green algae and theceae. In Bergey's manual of determinative bacteria. J. Gen. Biol. (U.S.S.R.), 1, 598.bacteriology, pp. 988-989. 6th ed. The Russian with English summary.Williams and Wilkins Co., Baltimore, Md. 16. BISSET, K. A., AND GRACE, JOYCE B. 1954

10. KEIL, F. 1912 Beitrage zur Physiologie der The nature and relationships of autotrophicfarblosen Schwefelbakterien. Beitr. Biol. bacteria. In Autotrophic microorganisms,Pflanz., 11, 335-372. fourth symposium of the Society for General

11. WILLE, N. 1902 Ueber Gasvakuolen bei Microbiology, p 35. University Press,einer Bakterie. Biol. Zentr., 22, 257-262. Cambridge, England.

12. MIYOSHI, M. 1898 Studien tiber die Schwe- 17. PRINGSHEIM, E. G. 1949 The filamentousfelrasenbildung und die Schwefelbacterien bacteria Sphaerotilus, Leptothrix, Clado-der Thermen von Yumoto bei Nikko. J. thrix, and their relation to iron and manga-Coll. Sci. Imp. Univ. Tokyo, 10, 141-174. nese. Trans. Roy. Soc. (London), B, 233,

13. GEITLER, L. 1942 Schizophyta: Klasse Schi- 453-482.zophyceae. In Engler and Prantl's Natuir- 18. STOKES, J. L. 1954 Studies on the filamen-lichen Pflanzenfamilien. Vol. 1 b. 2nd ed. tous sheathed iron bacterium SphaerotilusW. Engelmann, Leipzig. natans. J. Bacteriol., 67, 278-291.


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PLATE I

---~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

PLATEIfigs.1-4.Leucothrixmucor. Successive stages in the development of colonies on agar plates.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

X400.(Enlargement,2.67X).~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~


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PLATE II

PLATE II, figs. 5-7. Leucothrix mucor. Separation of gonidia from the tip of a mature filament. Con-secutive photographs of a single filament taken over a period of 7 minutes. Dark phase contrast, X 750.(Enlargement, 1.9 X).


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PLATE III

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i_ L |__7_. ,F. _, . C _... ..... _. i,,S.-¢y- .. ....... , _,,:,#f ........ I_ . _--a..5.-| til . _ ......... , 7: N,r, !. f _r

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PLATE III, figs. 8-13. Leucothrix mucor. The aggregation of gonidia to form rosettes. Consecutivephotographs of a single microscopic field, taken over a period of 55 minutes. In figs. 9-13, three de-veloping rosettes have been lettered in order to facilitate comparison. Dark phase contrast, X 500.(Enlargement, 1.28 X).


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PLATE IV

010

PLATE IV, figs. 14-16. Leucothrix mucor. Fig. 14. Nigrosine mount from a liquid culture 12 hours old,showing the holdfast (dark area) at the base of an isolated filament, X 1,500. (Enlargement, 2.22 X). Fig.15. A field of young developing rosettes, wet mount in 0.1% methylene blue. X 1,500. (Enlargement, 3 X).Fig. 16. A field of isolated filaments, developing in a slide culture from gonida too widely separated fromone another to permit aggregation. Wet mount in 0.1% methylene blue. X 400. (Enlargement, 1.68 X).


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PLATE V

PLATE V, figs. 17-20. Illustrations of Leucothrix and Thiothrix taken from earlier publications. Fig. 17.Habit sketch of young rosettes of Chlamydothrix longissima (= Leucothrix mucor), reproduced fromMolisch (2). Fig. 18. Drawings of various stages of the life cycle of Pontothrix longissima (= Leucothrixmucor), reproduced from Nadson and Krassilnikov (3). Fig. 19. Filaments of Thiothrix growing in aslide culture, reproduced from Winogradsky (6). Fig. 20. Habit sketch of Thiothrix growing in nature,reproduced from Miyoshi (12).


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PLATE VI

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byPLATE~~~~~Vfffig 21A~iraia coonofEThiohri growingfin nature. Phoomirogap of liv~in ma^teri>al


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THE GENERA LEUCOTHRIX AND THIOTHRIX · THE GENERALEUCOTHRIXANDTHIOTHRIX RUTHHAROLDAND R. Y. STANIER...

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Transcript of THE GENERA LEUCOTHRIX AND THIOTHRIX · THE GENERALEUCOTHRIXANDTHIOTHRIX RUTHHAROLDAND R. Y. STANIER...