Comparative Aspects of Development and Differentiation in Actinomycetes L

BACTJmOLOGICAL REVIEWS, June 1976, p. 469-524 Vol. 40, No. 2 Copyright 0 1976 American Society for Microbiology Printed in U.S.A.

Comparative Aspects of Development and Differentiation in Actinomycetes L. V. KALAKOUTSKIIP* ArN NINA S. AGRE Microbiological Ontogenesis Research Unit, Institute ofBiochemistry, & Physiology ofMicroorganisms, USSR Academy ofScience, Poustchino on Oka, Moscow Region, 142292 USSR INTRODUCTION ...... 469 Aims and Scope ...... 469 Suitability of Various Types of Cultures for Studies of Development and Differen- tiation ...... 470 Growth of Actinomycetes ...... 471 LIFE CYCLES IN THE ACTINOMYCETALES ...... 472 Generalized Scheme of Events Accompanying Reproduction ...... 472 Multiplicity of Reproductive Patterns ...... 474 Rare and Unconfirmed Modes of Reproduction ...... 475 STATIC ASPECTS OF DIFFERENTIATION ...... 475 Differentiation within Colonies ...... 475 Differentiation of the Mycelium: Primary versus Secondary Mycelium ...... 476 Cellular Differentiation ...... 477 Amycelial Structures ...... 479 Modes of Spore Formation ...... 481 Spores ...... 483 Structure ...... 483 Composition ...... 484 Physiology ...... 486 DYNAMIC ASPECTS OF DIFFERENTIATION ...... 487 Age-dependent Changes in Actinomycete Cells ...... 487 Environmental Conditions and Morphogenesis ...... 488 Metabolic Activity and Morphogenesis ...... 489 Environmental Factors in Control of Sporulation ...... 489 Nutritional limitations and sporulation: desiccation and other triggers ...... 489 Nutritional requirements for sporulation ...... 490 Supression of sporulation ...... 491 Sporulation under natural conditions ...... 491 Internal Factors in Control of Sporulation ...... 491 Variants and mutants with impaired differentiation ...... 492 Sporulation and chromosomal genes ...... 493 Sporulation and plasmids ...... 493 Proteolytic activity and sporulation and the question of RNA polymerase modification ...... 493 Involvement of specific factors in regulation of sporulation ...... 494 Spore Germination and Types of Dormancy Encountered ...... 495 Constitutive dormancy of thermoresistant endospores and an overall picture of germination ...... 496 Possibility of an alternative way for release from dormancy ...... 496 Germination of exospores ...... 498 DIFFERENTIATION AND SECONDARY METABOLISM ...... 500 Points of Similarity in Origin and Flow ...... 500 Prospects for Involvement of Antibiotics and Pigments in Regulation of Metabo- lism...... 503 Antibiotics and Their Components as Possible Building Blocks ...... 504 LITERATURE CITED ...... 506 INTRODUCTION taken as a group, would present difficulties, Aims and given the current state of our knowledge, al- Scope though they do pose intriguing and sometimes This paper is not going to suggest a new unique questions closely related to the area of model for studies in prokaryotic cell develop- developmental research. ment. It is believed that the Actinomycetales, I Present address: Microbial Ontogenesis Research Unit, nisms, USSR Academy of Science, Poustchino on Oka, Institute of Biochemistry and Physiology of Microorga- Moscow Region, 142292 USSR. 469 470 KALAKOUTSKII AND AGRE BACTERIOL. REV. The authors ofrecent reviews, dealing specif- screening for antibiotics and other products of ically or nonspecifically with particular prob- secondary metabolism. However, the very in- lems ofactinomycete development (69, 136, 235, teresting hypotheses linking secondary metab- 485), agree almost unanimously that develop- olism with differentiation (58, 449) were formu- mental studies with these forms lag behind lated using either fungi or endosporeforming those with viruses, aerobic sporeforming bacte- bacteria. These hypotheses, in our opinion, offer ria, and fungi. Whatever the reason, current very interesting perspectives for research, and trends of research with various organisms are consequently they merit extensive testing, spe- inconsistent, not only in terms of laboratory cifically with actinomycetes. Therefore, an at- techniques used, but also in the kinds of ques- tempt to examine the situation "from within tions asked, goals formulated, and ideas ex- the Actinomycetales" seemed timely (at least in ploited. Thus, periodic attempts to apply ap- helping some of our industrial colleagues to proaches becoming conventional with different realize that actinomycetes do not just grow and organisms appear merited. It is hoped that by produce everything possible, but they also so doing, the area of"lags" and potential break- somehow develop). throughs might become more precisely defined It is believed that the plan of this review will and comparative knowledge will be made more fit its aims without: (i) stating the basic prob- meaningful and thought provoking. lems, only to find supporting evidence insuffi- Now that the differences between eukaryotes cient, and (ii) describing organisms, one by one, and prokaryotes are understood, the old debate and reporting all relevant information, thus about whether the Actinomycetales are bacte- losing sight ofthe problems in the wealth ofless ria or fungi is definitely over. However, there relevant facts. are still reasons for comparing them with other Whenever there were no questions in our bacteria and fungi. minds, we used the generic names adopted in Chemical techniques, extensively employed the eighth edition ofBergey's Manual. In doubt- over the last two decades for characterization ful cases, generic names given in the original and identification purposes in microbiology and publications were retained. based mainly on determinations ofthe presence or absence of certain compounds, most often Suitability of Various Types of Cultures for confirmed the relatedness of the Actinomyce- Studies of Development and Differentiation tales to other bacteria ("true bacteria"). Using Information pertaining to development of ac- more complex features (wall structure, photo- tinomycetes currently stems mainly from stud- synthesis, gliding motility, endospore forma- ies that employ either submerged or surface tion, etc.) one can document differences be- cultures. Morphological and, very probably, tween gram-positive and gram-negative bacte- physiological and biochemical, manifestations ria. But what are the characteristic features of of differentiation in these types of cultures dif- the Actinomycetales, easily sensed by an ob- fer quite markedly. server, which support the idea of placing them To begin with, it is characteristic that most in a separate order, but which are not so easily differentiated representatives of the Actinomy- expressed in precise chemical terms? Are they cetales form hydrophobic aerial mycelia and not, to a large extent, the results of cellular spores in surface cultures. Although spore for- differentiation in these organisms? mation has repeatedly been reported to occur in Currently, Actinomycetales and fungi are be- submerged cultures, the spores and spore-bear- lieved to belong to different kingdoms of the ing structures formed under these conditions living world; it is inferred that their evolution- seem to differ morphologically and physiologi- ary separation is greater than that offishes and cally from these of surface cultures (cf. Spores, whales. Yet, striking examples ofsupposed con- below). Surface and submerged cultures might vergent evolution include not only mycelial or- differ also in the relative areas of cell contacts, ganization, but also general trends in reproduc- these tending to be more extensive in surface tive events. By comparing examples of analo- cultures. Cell crowding may well influence dif- gous differentiation, one might hope to learn ferentiation within hyphae, as seen, for in- about specific limits attainable at the prokar- stance, when submerged cultures grow in the yotic level. Relevant here also is the need to form of pellets. This influence is rather clearly understand the mechanisms allowing adapta- linked with changing gradients ofnutrient sup- tion to a different (terrestrial?) environment, in ply, accumulation of toxic substances and, which respect the Actinomycetales seem the much less clearly, with the interchange of more most capable of the prokaryotes. specific signals between cells. Physiological dif- There is one field that has involved actino- ferences between surface and submerged cul- mycetes more extensively than other microbes tures appear to be illustrated by the sometimes during the last three decades, and this is the encountered differences in their antibiotic pro- VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 471 ductivity. They are further illustrated by the Moreover, different portions of one and the effect of C-factor (see section on specific factors same hypha appeared infrequently to belong to in spore regulation, below), manifested only in different types. Different fractions of the myce- submerged cultures of strains of Streptomyces lium, obtained by centrifugation in sucrose gra- griseus. dients, appeared to be endowed with different The profound character of differences that streptomycin-synthesizing capacities in short- might be found between surface and submerged term experiments. In experiments with a re- cultures of actinomycetes are further docu- lated organism it was found (110) that the my- mented by studies of morphology during celium from a submerged shaken culture could growth ofNocardia corallina and other nocar- be separated into six fractions by density gra- diae. Here, growth on solid media takes the dient centrifugation. The fractions differed in form of a mycelium that later fragments. When stainability with methylene blue, in the rate of shaken in liquid medium of the same composi- colony formation when plated on solid medium, tion, the cell form changes to rods, which are and in ultimate streptomycin yields in trans- much shorter than the filamentous forms. fers on liquid medium (shake cultures). The Branching is rare. The rod-shaped cells divide heterogeneity of mycelia that is characteristic by binary fission. Fragmentation division fol- of submerged shaken cultures of actinomycetes lows as growth ceases (136). It also is a common probably accounts for the substantial difficul- observation of industrial microbiologists that ties encountered in attempts to correlate such fragmentation in several Streptomyces spp. is parameters of growing submerged cultures as markedly increased under conditions of sub- mycelium weight, biomass volume, and protein merged fermentation, as compared with sur- content (206), or fluctuations in deoxyribonu- face culture on solid media. Thus, different pos- cleic acid (DNA) content reported in an earlier sibilities are offered for studies in development. paper (103). Surface cultures are more suitable for studies Clearly, the continuous culture of actinomy- on differentiation within colonies, aerial myce- cetes might offer solutions to some of the above lium formation, etc. Because the growing hy- difficulties. This, however, has seldom been at- phae are attached to agar surface, their posi- tempted (38, 39, 469). Use can also be made of tion might give some information on chemotac- the fact that rather dense suspensions ofactino- tic responses and interactions (48). mycete spores, under proper conditions ofnutri- Although evidence of differentiation, both tion and aeration, can be germinated synchro- macro- and microscopic, is more clearly ex- nously and kept in synchronous growth for sev- pressed in surface cultures, most physiological eral hours. studies with actinomycetes have been con- ducted with submerged batch cultures. These Growth of Actinomycetes studies include important ones on nutritional limitations, on age-dependent biochemical How does the growth of actinomycete myce- changes, and on production of some secondary lium proceed? Some aspects of this process are metabolites. Considerable caution is in order not yet quite clear. when interpreting morphogenetic changes ob- According to cytological observations with servable in surface cultures with the aid of stained preparations (423), nuclear elements in physiological and biochemical data obtained growing Streptomyces hyphae divide not only with submerged shaken cultures. in their apical regions, but in other regions as The value of findings obtained with sub- well. These observations led the author to as- merged shaken cultures is limited, among sume that both intercalary and apical. growth other things, by the marked heterogeneity of is possible. In submerged cultures of Strepto- the mycelial mass obtained. This heterogeneity myces streptomycini, at a certain stage, a can be demonstrated simply by using centrifu- marked increase in the extension of hyphae gation in density gradients and differential was observed (111), which was not accompa- staining (495). Thus, submerged hyphae of S. nied by a corresponding increase in the rate griseus were stained with toluidine blue to re- of multiplication of nuclear bodies. This was veal nuclear elements, with Schiff stain to re- reflected in a marked decrease of the nucleo- veal polysaccharides, and with methylene blue cytoplasmic ratio along the whole length of to reveal metachromatic bodies. Each staining the hypha and tends to support the possi- procedure tended to distinguish several "types" bility of intercalary growth, at least at some of hyphae that appeared sequentially in the stages of culture development. Manometric course of actinomycete cultivation. The hyphae experiments (352) established a linear rela- thought to belong to a certain type using one tionship between the growth of several mycelial procedure appeared to belong to a different type organisms (streptomycetes and fungi) and the when another staining procedure was used. cubic root of the oxygen volume consumed. Be- 472 KALAKOUTSKII AND AGRE BACTIERIOL. REV. cause a similar relationship was found with So far, along with observations on growth of other bacterial cultures, a three-dimensional actinomycete hyphae cited above, this evidence mode of growth for the mycelial organisms stems from data illustrating the ways in which studied was suggested. The interpretation of the apices differ from the rest of the hyphae. the physical aspects of the three-dimensional These include differences in isoelectric points growth model is not quite clear, however. (183) and increased levels of activity of such Time-lapse microphotography experiments enzymes as alkaline phosphatase, catalase, and with growing N. corallina cells (54) indicated peroxidase (162). Apical regions seem also to be the mycelium grows predominantly at the api- more responsive to alterations in the tempera- ces and in regions where transverse septa are ture of the medium. In an attempt to analyze formed. In the latter case, growth is confined to the reasons for "ring" formation in giant colo- the opposing ends of the cells that are just nies of Thermoactinomyces vulgaris, it was separated by the newly formed septa. It is shown, for example, that certain kinds of rings known (203) that the new wall is formed at the were formed in response to brief drops in tem- equatorial regions, accompanying septum for- perature. According to microscopy observa- mation in certain gram-positive cocci. It is pos- tions, the apices of growing hyphae underwent sible that a similar phenomenon takes place autolysis when the cultures were subjected during septation of the mycelium in actinomy- briefly to decreased temperatures (46). Ger- cetes. minal tubes of the same organism responded According to Gottlieb (171), the growth of similarly, and a maximal effect was noted at actinomycete mycelium, like that in fungi, is 20 C, as observed by one of us (N.S.A.). This almost entirely confined to apical regions. Re- event is reminiscent of hyphal tips of fungi cent extensive light microscopy observations of bursting in response to environmental (includ- Streptomyces hygroscopicus hyphae growing in ing temperature) changes (33). The latter event surface culture (456) showed that growth is re- is thought to reflect increased sensitivity of the stricted to apical regions about 20 ,um in apical cell wall to autolytic enzymes (408). It length. Nuclear bodies were seen dividing should be emphasized, however, that subcellu- along this region, and no side branches were lar structures (vesicles and "Spitzen-K6rper"), seen to be formed here. In the next 80 to 110 whose presence is thought to be characteristic gum, there is no growth, but side branches are for fungal apices (181), have not been reported formed. Nuclear bodies divide at those sites in the hyphal tips of actinomycetes. Although where side branches emerge. Still further from an observer ofdevelopmental patterns in bacte- the apex, the "old" portion of the hypha begins, ria often meets with phenomena of "polarity" which does not grow and does not form side (207, 483), the biochemical basis for such phe- branches. Observations with submerged cul- nomena is far from clear. Perhaps the growing tures of S. streptomycini (111) also demon- hyphae of an actinomycete might eventually strated the inverse relationship between the become an interesting object for study and con- ability of individual hypha to extend longitudi- trol of polarity and dominance in procaryotes. nally and to form side branches. It seems likely that more decisive information on the relation- LIFE CYCLES IN THE ships that exist during various growth phases ACTINOMYCETALES between the ability of actinomycete hyphae to elongate and to branch might be obtained by Generalized Scheme of Events using the "growth unit" concept. As suggested Accompanying Reproduction originally for fungi (516), this unit relates total Developmental events in actinomycetes, as hyphal length to the number of apices found in other organisms, are most fully manifested along it. From the observations on the growth in the course of their reproductive cycles. Fig- of actinomycete hyphae on solid media, one ure 1 shows several examples of the latter and gets the impression that the rates of their is aimed at giving an overall picture of the growth and frequency of branching are only main modes of reproduction reported in repre- partly genetically determined and depend sentatives of this group of microorganisms. A heavily on cultivation conditions. These pre- number of organisms belonging to Actinomyce- liminary observations clearly await substantia- tales and related groups do not form specialized tion by more exact techniques. reproductive cells. Vegetative reproduction in Some evidence of indirect and preliminary these organisms is achieved by cell septation character indicate that in actinomycetes a kind (this process has several modifications) and of mechanism might be operating that results budding. in the so-called apical dominance - a well- As far as the mycelial stage is concerned, the known phenomenon in mycelial fungi (32, 573). simplest forms are represented by mycococci VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 473

FIG. 1. Reproduction cycles in selected representatives ofActinomycetales. Numerals refer to certain stages in the life cycles. Solid lines denote presence ofthe corresponding stages; dotted lines denote absence ofa stage. (1 -4) Vegetative stages; (5-7) spores. (1) Motile cells; (2) nonmotile cells; (3) mycelium; (4) fragmenting mycelium; (5) spores formed on substrate mycelium; (6) spores formed on aerial mycelium; (7) motile spores. Schematized according to information given in descriptions of: Mycococcus (112); Oerskovia (417); Intraspor- angium (241); and other genera (42). (112, 278). These organisms form neither myce- tum formation and genophore segregation in lial nor rodlike forms; their cells undergo irreg- Nocardia seems to be sufficiently well coordi- ular division (Fig. 2) that results in the forma- nated (136), so that no nonviable cells are tion of elements of uneven shape and size. Oc- formed as a result of the process. casionally, motile, flagellated cells are formed. Flagellated, motile, sometimes branching Rodlike and coccoid cells in representatives of elements are formed on fragmentation of myce- Mycobacteriaceae (474) and Actinomycetaceae lia in Oerskovia spp. (439) can develop into a transient mycelium, Septa are formed in two perpendicular planes which is quite unstable in most forms and is not during mycelial fragmentation in Dermatophi- detectable in some others. Along with septa- laceae (167; see also Fig. 2). This process is tion, Actinomyces spp. were reported to be able distinctly different from that in Nocardia be- to reproduce by budding (128; see also Fig. 2). cause of an additional plane of fragmentation Enlarged cells of unknown reproductive value and shows greater regularity than is seen in are sometimes observed ("clubs" in Actino- mycococci. Separated cells are often flagellated, myces spp.; "swollen cells" in Arachnia sp.). actively motile, and sometimes called zoopores. Motile forms are unknown in representatives of In Geodermatophilus spp., which do not form these families, except in the genus Mycoplana, specialized spores, reproduction involves cell whose suggested affiliation (87) is not quite septation and budding (221; see also Fig. 2). certain at the moment because of its gram neg- The actinomycetes that retain unfragmented ativity. (although seldom unseptate) mycelium during In Nocardia (361) and Oerskovia (417), the long periods oftheir life cycle usually reproduce mycelial stage persists considerably longer by forming asexual spores. Earlier proposals then in the above-mentioned organisms. concerning autogamy associated with sporula- Mycelial fragmentation can be regarded as a tion (23, 267) seem not to be warranted (see special form of vegetative reproduction. It is below). As one may see in Fig. 1, there are observed in many actinomycetes and is fairly many types of reproduction involving asexual characteristic for those called "nocardioform" sporulation. Spores may be formed on substrate by some authors (416). The process of fragmen- and/or the aerial mycelium as single cells or in tation in Nocardia spp. involves formation of chains of various length, or harbored in special multiple septa that divide the growing hyphae vesicles (sporangia), and may be endowed with into more or less regular elements. Contrary to flagella. some earlier suggestions (1), the process of sep- The initial steps of spore formation in several 474 KALAKOUTSKII AND AGRE BACTERIOL. REV. 1 a a, c 4z T, a0> ,- .-6 (41ff L=r~v l-= a~~~~ d'

FIG. 2. Septation in actinomycetes. (I) Septation in vegetative cells. (a) Transverse septation common for majority ofactinomycetes; (b-d) septation in diverse planes as exemplified in: (b) Mycococcus spp. cells; (c) vesicles ofFrankia spp.; (d) chlamydospores ofM. chalcea. (II) Types of budding encountered. (a) Side bud formation in Actinomyces spp.; (b) polar bud formation in Geodermatophilus sp. (daughter cells are budded from a stalk ofthe mother cell). (III) Septation in sporulating hyphae. (a) Septation in sporulating hyphae of Streptomyces spp.; (b, c) sequential division in sporulating hyphae: (b) basipetal in Micropolyspora spp.; (c) acropetal in Pseudonocardia spp.; (d) "cascade" septation in Actinomadura dassonvillei; (e) septation in two perpendicular planes characteristic ofDermatophilaceae. oligosporic actinomycetes can be viewed as bud- The ability ofStreptomyces spp. to form some ding, since the characteristics of the process specialized cells along with aerial conidia on meet the main criteria for budding (207) in substrate mycelium was mentioned and illus- other bacteria. trated long ago (278, 326, 536, 537). They were So far, it has not been possible to grow repre- pictured as enlarged, thick-walled cells var- sentatives of Frankia spp. in laboratory cul- iously called "chlamydospores" or "arthro- tures. Their reproduction cycles were inferred spores." Their exact structure and origin have and reconstructed from observations made on not been reexamined since the advent of elec- slices of infected plant tissues. Two main forms tron microscopy. Chlamydospores and micro- are seen: branching septate mycelium (usually cysts were also sometimes observed in Nocar- associated with young nodules) and spherical dia spp. (361). Some Streptomyces spp. carry vesicular bodies (up to 5 um in diameter). Vesi- single spores or chains of spores on'substrate cles are found on the mycelial hyphae in termi- mycelium (36, 192, 465, 466), in addition to the nal, subterminal, or intercalary positions (37, characteristic chains of arthrospores on aerial 344). Vesicles are abundant in nodules that mycelium. actively fix molecular nitrogen. In dead tissues, In a study on an unusual species of Actino- irregular polygonal endophyte cells are seen planes, A. armeniacus, mention was made of (0.5 to 1.0 gum in diameter), which are believed the ability of this organism to form two types of to represent a resting stage capable of survival spores: (i) motile, peritrichously flagellated zoo- in soil after autolysis of the plant cells (437). spores in sporangia on substrate mycelium, and (ii) chains ofStreptomyces-type arthrospores on Multiplicity of Reproductive Patterns aerial mycelium (245). These varied in propor- The species of actinomycetes, like the fungi, tion to the cultivation conditions employed. A are often endowed with more than one potential similar situation is also met with in other spe- reproductive route. Mention has already been cies of Actinoplanaceae (563, 565). Species of made of the ability to reproduce by mycelial Kitasatoa (358) and Pilimelia (249) also form fragmentation and sporulation. The additional motile spores in vesicles and nonmotile spores reproductive unit is sometimes different from in chains. the mere fragment of the mycelium. Micromonospora spp. usually have single VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 475 spores but seem to form occasional spores in streptomycetes, "living crystals" were reported pairs; they also form terminal, intercalary, and to occur (381), which are able to germinate to interminate chlamydospores (335). Pseudono- give rise to the mycelium on fresh media. In the cardia were reported (193) to produce three hands of other authors (331) S. sindenensis types of spores, according to their origin: (i) yielded supposed zoogametes, along with usual terminal or lateral chains of spores arising on mycelium, fruiting bodies, and motile ele- the mycelium, formed in acropetal fashion; (ii) ments. fragmentational spores irregularly formed on Insufficiently explored is the interesting sug- the mycelium; (iii) blastospores, produced in gestion (279) that some actinomycetes form, in the basipetal fashion and seen in twos and the course of autolysis, submicroscopic bodies threes in short chains on mycelial segments. that may pass through conventional bacterio- Actinomonospora lusitanica was reported to logical filters and regenerate under certain con- form large spores (2.5 to 3.0 ,um in diameter), ditions into initial cultures. which are positioned terminally or laterally STATIC ASPECTS OF on the hyphae, but formation of chlamydo- DIFFERENTIATION spores, arthrospores, and endoconidia was mentioned by the authors who first described the Differentiation Within Colonies organism (336). The colonies formed by actinomycetes in sur- Descriptions of life cycle traditionally serve face cultures may be roughly assigned to one of as one of the milestones in the systematics of the following three types. (i) The first type actinomycetes. In view of the above, considera- consists of pasty rough or smooth colonies that ble patience is required to provide conditions can be easily detached from the solid media. that allow for expression of as many of the These are seldom covered with aerial mycelium reproductive patterns as possible. The condi- and usually are formed by actinomycetes with a tions permitting less conventional routes of dif- transient mycelial phase. (ii) The second type ferentiation in actinomycetes, as well as physi- includes colonies like those of Sporichthya, ological events accompanying them, are almost which are almost devoid of substrate mycelium untouched by experimental studies. and consist ofaerial hyphae attached to the medium through special holdfasts. (iii) The third Rare and Unconfirmed Modes type consists of compact, leathery colonies usu- of Reproduction ally bearing aerial hyphae and firmly attached There are hints that at least some actinomy- to the substrate by hyphae that penetrate the cetes possess much more complex life cycles substrate. The latter usually are found in ac- than is usually believed. A word ofcaution is in tinomycetes that have a persisting mycelial order, since some of these were inferred from stage. One may usually distinguish in the col- microscopy observations on highly heteroge- onies of the type (iii) those portions that lie nous (cf. above) cell preparations and stained beneath the level ofsubstrate and those that lie smears. This seems to be particularly applica- above it in the form ofeither "substrate" and/or ble to the formation of the much-debated "ini- "aerial" hyphae. tial cells" (267), or the complex life cycles of Since substrate and aerial hyphae seem to Streptomyces spp., which are supposedly ex- differ in some ways (see below), one may ques- hibited in submerged liquid cultures (400, 401). tion whether or not submerged cultures of acti- The scheme of the life cycle of some freshwater nomycetes are physiologically equal to surface streptomycetes (433) also seems insufficiently cultures devoid of aerial mycelium. This ques- documented. According to the latter, the change tion is largely unsolved. However, it might from haploid to diploid state ofthe mycelium is be noted that the terminology sometimes used accomplished by a sexual process, involving by those who study streptomyces, which in- fusion of zoogametes. volves "vegetative" and "generative" mycelium Nakazawa (379, 380) described a complex life (meaning substrate and aerial mycelium, re- cycle in Streptomyces sindenensis, which in- spectively), is hardly justified, because many volves formation of special amycelial "fruiting actinomycetes form reproductive structures on bodies" carrying motile isogametes, which fuse the substrate or primary mycelium. Formation and form zygotes. The latter develop into my- of spores in shaken cultures by Streptomyces celium or special vesicles, which can be culti- spp. was noted long ago (63), as was the forma- vated separately or together, the latter condition of sporangia by Streptosporangium sibiri- ton being inductive for the eventual formation cum under similar conditions (223). Consider- of fruiting bodies. To complicate matters fur- ations ofconditions and stimuli that are respon- ther, in the culture of this and some other sible for the tendencies of growing submerged 476 KALAKOUTSKII AND AGRE BACTERIOL. REV. hyphae of actinomycetes to form tight clumps studies of"thin" mycelium of this kind are lack- or to repel each other remain largely specula- ing. tive; research in this field clearly lags behind Actinomycete mycelium shows one of the fol- work done in the same area with fungi (405). lowing types of branching, which are also The real picture of differentiation in colonies known for mycelial fungi: (i) monopodial, (ii) of several Streptormyces spp. (423, 550) seems to dichotomous, and (iii) verticillate. Monopodial be much more complex than is usually depicted branching is most common. Dichotomous (e.g., the scheme of Hopwood et al. [213]). The branching is characteristic of species of Acti- difference might be explained by the fact that nobifida, whereas species of the genus Strep- actual colonies of actinomycetes usually in- toverticillium are noted for the verticillate volve the interaction of several generations of branching of their aerial sporogenous hyphae. both primary (substrate) and secondary (aerial) Although dichotomous and verticillate branch- hyphae, which sometimes overgrow each other. ing is found in fungi of the genera Thamni- Strain- and medium-dependent differences are dium and Spicaria, respectively, only the prominent in actinomycetes. It was shown, for overall analogy seems to be justified at present, example, that in contrast to the parent culture since the underlying mechanisms are virtually the surface layers of colonies of one of the var- unknown in actinomycetes. In the actinomy- iants of Streptomyces variabilis are actually cete genus Dermatophilus, the side branches of composed of primary hyphae (532). Besides lo- the mycelium have a tendency to grow perpen- cal gradients in supply of nutritional factors, dicularly to the parent hyphae. As already lytic enzymes, etc., the picture of differentia- mentioned, many, but not all, actinomycetes tion within colonies is further complicated by form two kinds of mycelium: primary (sub- the local accumulation of antibiotics and proba- strate) and secondary (aerial) mycelium. The bly other factors that limit growth. At least for latter is currently believed by most investiga- some antibiotics (heliomycin), quite distinct tors to arise directly from the primary myce- sites of accumulation within colonies of the lium. producer were revealed (531). The following points (taken from the work of Obvious age-dependent differences among Higgins and Silvey [205]) briefly summarize populations of cells constituting a colony are the differences between the aerial and sub- reflected in such well-known phenomena as the strate mycelium in the Streptomyces sp. stud- fragmentation of the mycelium. This (if it does ied by the authors mentioned above. (i) The take place at all) spreads from the center of a aerial mycelium is slightly thicker. (ii) It usu- colony. Much less obvious are the reasons for ally has a dark, insoluble pigment associated changing patterns of sporulation in colonies of with its outer envelope and looks gray ifviewed Streptomyces massasporeus (464), in which in reflected light. (iii) It shows less tendency to coiled aerial sporophores predominated in the branch. (iv) The mycelium shows almost no center of the colonies, while single spores or tendency to penetrate the medium. (v) It forms short chains of spores were seen at the periph- spores through fragmentation. (vi) In contrast ery. to the substrate mycelium, the aerial layer is An observer of actinomycete colonies is hydrophobic. These points of difference are also tempted to assume, with Pollock (409), but on evident from studies of many other authors. different grounds, that an individual cell, ex- Cytological observations on young aerial hy- emplified by a fragment ofa hypha, is not equal phae of Streptomyces antibioticus suggest that to the whole "living organism." The fungal col- they contain nuclear elements that are richer ony was likened (179) to a city, populated with in DNA as compared with substrate hyphae nuclei. By analogy, an actinomycete colony (423). Data on metabolic activities of aerial hy- might be likened to a students' dormitory, with phae are sparse. Cytochemical techniques (162) rules and regulations that are incompletely de- applied to young, emerging aerial hyphae of fined and only half-understood. Streptomyces spp. showed increased levels of sulfhydryl compounds as well as increased activity of enzymes such as alkaline phosphatase, Differentiation of the Mycelium: Primary catalase, and peroxidase. As the cultures en- versus Secondary Mycelium tered the sporulation phase, the levels of activ- The width of mycelium in actinomycetes usu- ity of these enzymes decreased. ally varies from 0.5 to 1.5 gum. Mycelium be- In the respirometric experiments of Erikson comes thicker in some nutritionally unbalanced and Webley (145), the cells of Thermoactino- cultures. Some authors have reported the occa- myces vulgaris aerial mycelium, sampled be- sional occurrence ofcells that were 0.1 gm (237, fore the onset of sporulation, were shown to 288) or 0.1 to 0.2 ,um thick, but systematic have a respiration rate higher than that of my- VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 477 celial cells sampled from the bottom of a liquid matophilus spp., is also known to occur in Acti- culture. A study of a mesophilic Streptomyces nomyces spp. (128). In vegetative hyphae of sp. (probably S. griseus) (154) concluded that Nocardia and Streptomyces spp., transverse the substrate mycelium may be regarded as fac- septa seem to start in their "older" portions, ultatively aerobic, whereas the aerial myce- and then the activity spreads toward hyphal lium is obligately aerobic. This conclusion was apices. It is worthwhile comparing this se- based on experiments employing respiratory quence of events with those observed during poisons and cultivation in changing atmos- sporulation (see below). In Streptomyces mega- pheres. sporus (121) and Mycobacterium tuberculo- The aerial mycelium of actinomycetes might sis (21), cells separate by constriction in addi- be considered as belonging to those rare or even tion to septation. unique procaryotic vegetative cells that are A similar process was described also in Ar- kept functional in the absence of water in their throbacter simplex (303). The cells that sepa- immediate surroundings. One can speculate rated by constriction were said to have a gram- that such a capability might be associated with negative cell wall, whereas those dividing by the first steps of some ancestral procaryotes septation had a gram-positive cell wall. from a predominately aquatic to a terrestrial Septa in the vegetative hyphae of the major- environment. In this connection, it is worth ity of actinomycetes studied (561) look single or noting that actinomycetes that are currently triple layered, depending on whether a line of isolated from aquatic environments (e.g., spe- electron-dense material in the middle of the cies of Micromonospora, Actinoplanes, and septum is revealed (Fig. 3). It was suggested some others) are usually represented by forms (298) that the failure of septate Streptomyces devoid of aerial mycelium. hyphae to separate into individual fragments The hydrophobic nature ofthe outer envelope might be due to local deficiencies in the activity of aerial cells might be relevant to the initial of lytic dechaining enzymes. orientation of these mycelial cells and their The growing transverse septa in fragmenting emergence through the gas-liquid interphase. Nocardia hyphae usually are split longitudi- What maintains the growth of aerial hyphae nally, starting from the ends adjacent to hyphal out of the medium is unclear, but negative walls (Fig. 3). Cytologically, the process of cell geotropism is certainly excluded. Presumably, septation in Nocardia is said (136) to be remi- some barrier exists that protects the cytoplasm niscent ofthat in Arthrobacter crystallopoietes. of aerial hyphae from rapid desiccation, and The persistence of the outer-wall component this may be related to specific structures, var- and its rupture in later stages of daughter cell iously called "surface sheath," "surface mem- elongation has been implicated in the forma- brane," or a "microcapsule." The use of term tion of characteristic V-form cells. (Compare "membrane" seems inappropriate because of this with events accompanying spore delimina- confusion with the outer membranes of gram- tion inActinomadura spp.). However, light mi- negative bacteria. croscope observations (360, 522) point to possi- Some characteristics of surface sheath struc- ble existence of variant modes of fragmenta- tures are given in Table 1. tion. Along with the indispensable and ever-pres- Cellular Differentiation ent intracellular structures, one finds in the The structure of actinomycete cells is similar hyphae of actinomycetes some variable ele- to that of other gram-positive bacteria. ments, as well as some rare and unusual struc- The formation of septa is observed more or tures. As in other procaryotes (467), these com- less frequently in the hyphae of all actinomy- ponents may be found enclosed within membra- cetes (Fig. 2). These are thought to be formed in nous sacs or lying free in the cytoplasm, form- a manner analogous to that of septa in other ing various inclusions. Localization of polysac- gram-positive bacteria (133, 204). charides, polyphosphates, and lipids was dem- Transverse septa are the type found most onstrated cytochemically (5, 107, 561), and often in mycelium of actinomycetes (Fig. 2). the presence of poly-,8-hydroxybutyric acid Occassionally, possibly during regulatory im- might be inferred from chemical determina- balance, the growing septa are split and divide tions (250). Vacuoles in the actinomycete cells the cytoplasm into several regions (Fig. 2). Ex- may have widely differing configurations. amples of this are seen during chlamydospore Some of the vacuoles, enclosed in a nonunit formation in Micromonospora chalcea (337) membrane, look very similar to gaseous vacu- and are regularly observed in vesicles ofFran- oles in other bacteria (561). kia (37, 152). Formation of septa in two perpen- Rodlike and tubular structures reminiscent dicular planes, considered to be usual for Der- of raphidosomes (440), viruses, or defective 478 KALAKOUTSKII AND AGRE BACTERIOL. R-EV. TABLE 1. Aerial mycelium and spores ofactinomycetes: some distinct characteristics of their surface structures Organism studied and mate- Refer- Methods applied Results and comments' rial examineda ence Aerial mycelium outer surface: some distinguishing features S. coelicolor, Streptomyces spp.: 138 LM of Sudan black-stained prepa- Differential staining of AH; washings SH, AH, spores rations prior to and after wash- with acetone, ethanol, ether, chloro- ings with solvents form, and some other solvents pre- vents staining; presence of lipids associated with AH surface inferred S. violaceus, Streptomyces spp.: 234, 285 LM in incident and incident polar- AH and spores distinctly differ from SH SH, AH, spores ized light; Sudan black B stain- in associated demonstration of bire- ing; exposure to solvent wash- fringence, yield interference colora- ings tion, and stainability; these distinc- tions are abolished after acetone, methanol, and ethylacetate washings and are reduced following exposure to water vapors; regular organization of AM surface layers with a likely participation of lipoidal material suggested S. griseus, S. viridochromo- 123 Electrophoresis following treat- Changes in electrophoretic mobility fol- genes, S. finlay; spores ments with enzymes, surface- lowing lysozyme but not SDS and li- active agents, and chemicals pase treatment; surface location of lip- specific for certain functional ids questioned; dominance of carboxyl groups and amino groups on surfaces inferred Surface sheath: elementary structures and their features S. coelicolor: SH, AH, spores 214 TEM of carbon replicas Surfaces of AH and spores distinctly different from SH in possessing a delicate envelope inlaid with rodlike elementary structures S. venezuelae: AH, spores 51 Elementary structures present: their lo- 135 TEM of thin sections, carbon rep- calization altered following treatments licas; exposure to solvent with ethanol, xylol and benzene; rods washings removed by acetone washings followed by dehydration with ether; sodium hy- droxide probably solubilizes elementary structures S. coelicolor: AH, spores 555 TEM of negatively stained prepa- Rodlike elementary structures (12 to 20 (smooth) rations, freeze-etching-, expo- by 40 to 250 nm) tend to be assembled S. glaucescens, S. viridochro- sure to solvent washing in pairs; those seen on preparations mogenes, S. spadius, S. viola- 551 TEM of negatively stained prepa- probably represent parts of longer ceoruber: AH, spores rations, carbon replicas structures; not removed by solvent washing S. viridochromogenes: All, 552 TEM of negatively stained prepa- Elementary structures of slightly differ- spores rations, freeze-etching ing shape and size invariably present S. glaucescens, S. arcimycini: 553 TEM of negatively stained prepa- on AH and spores; kinds of ornaments spores rations, freeze-etching formed on either AH or spore surfaces S. griseus, S. venezuelae, S. vi- 557 TEM of negatively stained prepa- tend to be similar in species with ridochromogenes, S. finlay, rations, freeze-etching, carbon smooth spores and different in species S. glaucescens: AH, spores replicas with spiny spores Position and integrity of elementary structures not changed after treatments with solvents Micropolyspora spp.: AH, 9 TEM of negatively stained prepa- spores rations S. roseoflavus var. roseofun- 71 TEM of negatively stained prepa- Elementary structures destroyed by brief gini: AH and spores rations prior to and after ace- acetone washings; closely similar tone extraction structures reassembled in vitro from acetone extracts of AH.

phage particles (298, 509) have been observed. formation of these structures is linked with Electron-transparent regions of irregular autolysis (8, 550), accumulation of antibiotics configuration containing no membranes are of- (75, 298), or phage infection (432). ten seen in the cytoplasm of hyphae from the Actinomycetes share with fungi not only my- idiophase stage of Streptomyces cultures. The celial organization per se, but also some struc- VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 479 TABLB 1-Continued Organism studied and mate- Refer- Methods applied Results and comments' rial examineda ence Spore appendages: origin, involvement of elementary structures Streptomyces spp.: AH, spores 530 TEM of intact spore chains Spores with appendages are formed in- (with differing surface archi- side AH wall tectures) S. togocaenais: AH, spores 12 TEM of spore thin sections The outer-wall component probably in- (spiny) volved in formation of spore appendages S. violaceu8: AH, spores (spiny) 425 TEM of intact spore chains and 548 thin sections Origin ofspore appendages not related to S. venezuelae AH, spores 51 TEM of thin sections, carbon rep- AH walls. Surface sheath, as a distinct (smooth) licas structure, directly involved in origin of S. griseus: AH, spores (smooth) 560 SEM of intact spore chains and appendages; kinds of appendages pro- S. finlay: AH, spores (hairy) TEM of thin sections duced do not reflect differences in mode of spore formation Streptomyces app.: AH and 551 TEM of negatively stained and Spore appendages consist of elementary spores (with different surface 552 freeze-etched cells, thin section- tubular, grooved, and similar struc- architectures) 553 ing, carbon replicas tures found also in the surface sheath; conelike bundles of elementary structures form spiny appendages; hairy ones represented by bundles of in- tertwisted elementary structures Environment and formation of spore appendages S. violaceus: AH, spores (spiny) 321 TEM of intact spore chains follow- Presence and general structural features ing cultivation on different nu- of appendages almost unaffected by trient media nutritional conditions S. hygroscopicus: spores (ru- 104 TEM of carbon replicas Presence and structure of appendages gose) 105 varies depending on relative distance Streptomyces spp.: spores (spiny) of spores formed from the agar surface (relative humidity gradient important?) Spw. e appendages: response to treatmentts Streptomyces spp.: AH and 151 TEM of spore chains (intact and Spore silhouettes not affected by wash- spores (with differing surface after washing with solvents) ings with chloroform and some other appendages) solvents Streptomyces spp. 341 TEM of carbon replicas and intact Appearance of spore appendages grossly spore chains (prior to and after changed following lysozyme treatment lysozyme treatment) I SH, Substrate hyphae; AH, aerial hyphae; LM, light microscopy, TEM, transmission electron microscopy; SEM, scanning electron microscopy; SDS, sodium dodecyl sulfate. bOur comments are given in parentheses. tural features that might result from it. Among lensis were different in that they were only 8 to these one might list the intrahyphal hyphae 9 nm thick, much less than most flagella (431). found in Streptomyces roseoflavus var. roseofungini (70), Micropolyspora spp. (8), and Mi- Amycelial Structures croellobosporia flavea (561). As in fungi (333, 517), the intrahyphal hyphae in actinomycetes Some actinomycetes form structures that are found not only in ageing cultures; they are arise from mycelial cells but differ markedly thought to assume an important role in the re- from them. The role of these structures in reproduction of over-wintering cells of Frankia production is not quite clear; in any case their spp. (345). formation does not represent the sole way of The motility of actinomycete cells, whether multiplication. specialized (zoospores) or unspecialized (myce- Sclerotia persistently formed by Chainia spp. lial fragments), is always linked with posession (242, 505) belong to this category. In the forma- offlagella, and all types offlagellation are to be tion of sclerotia, the mycelial hyphae thicken found. Peritrichous flagellation is rather rare. and become septate, and many lipid-containing The flagella of Actinoplanes spp. were said to vacuoles are formed. Intercellular cementing be reminiscent (317) of those in Pseudomonwas, material is formed, which contains L-2,3-di- whereas the flagella of Dermatophilus congo- aminopropionic acid. The lipids are mainly tri- 480 KALAKOUTSKII AND AGRE BACTERIOL. REV.

+ rI

a c

I+v+ T**0~~~~~ z0

4' 2DD 00s A~~~~~~~~~A

FIG. 3. Diagrammatic presentation ofstructural organization and formative events in septae ofactinomycetes. (a, b) Single- or triple-layered septa found in the majority ofvegetative cells studied. (c, d) "Split" septae: (c) found in fragmenting hyphae ofNocardia spp. and in sporulating hyphae of many actinomycetes; (d) revealed in S. coelicolor (554). (e, f) Multicontour sporulation septae: (e) found in S. viridochromogenes; bear several layers ofdouble unit membranes (568); (fl found in S. ribosidifuscus (568). (g, h) Sporulation septa with intersporal pad: (g) the pad persists in mature spore chain (A. dassonvillei [562]); (h) intersporal pads destroyed during later stages of spore formation in M. rectivirgula (117); at a certain stage of spore formation a plasmodesm is distinguishable within the intersporal pad. Solid areas, cell wall and septal material; dotted areas, intersporal material. glycerides of branched fatty acids of the iso/ lium and pigments. anteiso series having 15 to 16 carbon atoms. Close associations of hyphae are met with in These transformed and fattened cells are coremia, produced by some Streptomyces spp. packed in hyphal mats (322). It is thought (278, 326). No signs of special transformations (561) that the structure and functions of scle- of hyphae in coremia were reported so far. It is rotia in Chainia are reminiscent of those in interesting to note, however, the ability of such fungi as Sclerotinium rolfsii and Verti- neighboring coremia to extend hyphae that cillium alboartrum. form "bridges" through the air and fuse (180). Hyphal aggregates formed by certain Strep- The vesicles formed by Frankia spp., already tomyces spp. on some media (160) are probably mentioned, may be surrounded with capsular not related to sclerotia (242), nor are the "gran- material, with a thickness that depends on the ules" described by Baldacci et al. (26). The in- age and condition of the plant host (345). triguing feature of these granules is that they Some actinomycetes excrete slimy sub- yield, upon inoculation on fresh media, colonies stances, which might contribute to the forma- ofactinomycetes that might differ from the par- tion of cell agglomerates. Some are polysaccha- ent colonies in ability to produce aerial myce- rides (403), probably enriched in ferric hydrox- VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 481 ide (278). The specific glycolipid-"wax D"- The protoplasmic regions enveloped by the in- produced by some mycobacteria (170) contains ner layer of the hyphal wall then become con- components that are common to wall murein densed, leaving some electron-transparent and probably originate from transformations of space between the sporangial and spore walls. the latter. Coating the cells with wax D proba- Spores, shaped like sporangia, are liberated as bly offers additional protection (124). Immuno- soon as sporangial wall (bearing a surface chemical evidence was said (308) to suggest sheath that is characteristic for aerial myce- excretion ofmembrane complexes, consisting of lium of actinomycetes) is broken. mucopolysaccharides and a phospholipid, dur- Spores in most actinomycetes are formed ex- ing the growth ofmycobacteria, nocardiae, der- ogenously. Formation ofspores in Streptomyces matophili, and streptomycetes. The "sulfur spp. starts by division of nucleoids and changes granules" formed by Actinomyces spp. in vivo in the membranous structures. The latter show consist of mycelial hyphae cemented with a in thin sections as thickening of the peripheral polysaccharide-protein complex. The mineral plasma membrane and the appearance of a portion of granules includes apatite and cal- number of mesosomes. Later on, sporulation cium phosphates (155, 404). Central regions of septa accompanied by mesosomes start to form Streptomyces scabies colonies appear to be cov- (51, 163, 425, 554, 568). The wall of the develop- ered with a "blanket" ofsome extracellular ma- ing exospore seems to be formed with help from terial (126). the cytoplasmic membrane of the correspond- Occasionally, round or clublike vesicles of ing region of the sporogenous hyphae, in con- increased size (up to 3 to 5 ,um in diameter) are trast to the situation with endogenous sporula- found in submerged Streptomyces cultures tion. The subsequent processes transform the (529); swollen cells ofDactylosporangium thai- thickening and stratifying hyphal wall into landense are thought (462) to represent reac- spore wall (121, 164). However, it seems more tions to unfavorable growth conditions. Sporan- probable that the sporal walls, or at least their gia ofIntrasporangium calvum (241) have been inner layers, are synthesized de novo on the placed (318) in the same category. cytoplasmic membrane (337, 554). Intracellular transformations accompanying Modes of Spore Formation sporulation are manifested also by the nucleoid The spores formed by actinomycetes fall and membranous bodies assuming a more com- within two groups: endogenous and exogenous, pact form. Spore nuclear areas remind one of according to the mode of their formation. those in Streptococcus faecalis cells in which Endogenous formation of thermoresistant protein synthesis has been blocked (91). The spores inside the cytoplasm of parent hyphae consequences of all these transformations be- was studied in the thermophilic actinomycetes come evident on examining fixed preparations Thermoactinomyces and Actinobifida (86, 89, of sporogenous cultures of actinomycetes by 116, 118, 310). Structurally, it follows essentially electron microscopy: spores are usually much the same sequence depicted for spore formation less damaged by fixation and sectioning than in other gram-positive bacteria (150). Some dif- vegetative hyphae. Also, the internal struc- ferences in the regulation of spore formation tures of spores are often less resolved, which probably exist, asjudged from the final location probably reflects incomplete penetration of the of spores on the mycelium. In T. vulgaris, fixatives. spores often reside in the hyphae and are par- In some actinomycetes, like Micropolyspora tially extruded in the course of formation and flavea (424) and Micropolyspora viridnigra (7), maturation; in Thermoactinomyces sacchari, an unusual central and granular mass, absent most spores are found terminally, and in Acti- in vegetative cells, is found in spores and is nobifida dichotomica only terminally. termed the "central body." Spores ofthese orga- Spore formation in Planomonospora and nisms also contain large vacuoles enclosed in a Dactylosporangium was also said to be endoge- unit membrane. nous (463, 561), but the process seems to be very The variations in exogenous spore formation different from what is believed to represent among actinomycetes mainly involve the origin "true" endogenous sporulation in bacteria. The and fate of sporulation septa, which, contrary difference is especially pronounced in the fate of to the situation found in vegetative mycelium, presporal and sporangial membranes and their seems to be under more stringent control. participation in the formation ofspore coats fol- These temporary and spatial controls bear upon lowing "engulfment." According to these au- the frequency and the character ofthe transfor- thors, the initial steps of spore formation in the mations that newly formed septa undergo. Nas- above-mentioned actinomycetes include split- cent spores in chains (Fig. 2) are delimited by ting ofthe parental hyphal wall into two layers. septa appearing in sequential order, which may 482 KALAKOUTSKII AND AGRE BACTERIOL. REV. be different in various actinomycetes (compare, side of the septa. At which stage (if at all) the for instance Micropolyspora spp. [283, 320] and spores formed become efficiently separated Pseudonocardia [194]). The sequence reported from the mycelium is not clear. The presence of for Pseudonocardia seems especially interest- plasmodesms has been reported only for spores ing, since the nascent spores seem to retain ofMicropolyspora rectivirgula (117) and Strep- vegetative functions (transport of metabolites tomyces viridochromogenes (568). from hyphae to apical buds) for rather extended Actinomycetes that form exogenous spores periods of time. fall within two groups according to whether the In Streptomyces spp. (51, 164, 425, 554, 560) surface sheath is present on the sporulating and some sporangium-forming actinomycetes hyphae (561). Surface sheath is absent on spor- (317, 319), septa seem to be formed in two ulating hyphae of Micromonospora, on sporu- stages. First, the sporophore is separated by a lating substrate hyphae of actinomycetes, and single septum from the corresponding hyphae. on fragmentation spores of Nocardia spp. The Then the sporogenous hyphae is itself divided ornamentation on spores of Micromonospora by a number of septa almost simultaneously chalcea results from projections, present on the into approximately equal-sized cells that even- outside layer of the parental hyphal wall (337). tually are transformed into spores. The simul- When present, the surface sheath envelops the taneous character of multiple septation in sporulating hyphae as a kind of "stocking." In Streptomyces sporophores reported by a major- the course of late stages of sporulation, the ity of investigators probably needs further ex- surface sheath behaves as a separate entity, perimental proof. This might be not easy to seemingly independent of the transformations obtain, since the process is rapid enough to affecting the sporophore wall. The sheath be- obscure time-lapse observations. If the septa- havior, however, seems to be intimately con- tion actually proceeds in a basipetal manner, nected with the resulting spore surface archi- such a sequence would be interesting to com- tectures (Table 1). pare with an opposite one, found in fragment- The coat of vesicles in actinomycetes is ing vegetative hyphae. thought to be homologous to the surface sheath Occasionally, in M. viridinigra (7) and Acti- of aerial mycelium. The use of the term "spo- nonadura dassonvillei (562), septa are formed rangium," as suggested (77) for the former not quite regularly, dividing the sporogenous structures, seems not to be quite justified ifone hyphae into unequal compartments. In A. das- considers the fine details of spore formation sonvillei, the extended cells resulting from the within vesicles. It has been suggested (82, 563) first round of septation may be divided by sec- that the term sporangium be retained exclu- ondary septa. sively for actinomycetes with endogenously Once formed, sporulation septa in actinomy- formed spores and thus establish closer links cetes may develop further and undergo rather with the common bacteriological nomenclature. complex transformations that are usually not It seems worthwhile to name a few anomalies observed in vegetative cell division. The main in spore formation, since they may help to find types of sporulation septa are diagrammati- eventually the "weak points" in the regulation cally presented in Fig. 3. They differ in shape of sporulation. The number of spores in chains and as to whether special wall thickenings and seems usually not to be under a stringent con- intersporal material are present (these struc- trol. Probable exceptions are represented by tures also show various patterns in develop- Microbispora (390) and Microtetraspora spp. ment). (504). Formation ofpairs ofspores inM. chalcea Functional implications of the differences was interpreted (337) as an indication of impre- mentioned above are not clear. The structure of cise regulation of septum formation. the sporulation septa seems not to affect the Anomalous and badly germinating spores final form ofthe sporophore (i.e., straight, flex- were found in some Micropolyspora spp. (7), uous, spiral, etc.). The latter is thought to be and these showed much-thickened walls, ab- acquired before extensive development of sence of the central body and vacuoles, and septa, during the first sporulation stage (215). accumulation of electron-dense (presumably Scanning microscope observations (25, 27) are polyphosphate) granules. Three types of spores in agreement with this view, so it is believed differing in ultrastructure were revealed in that the sporophore architecture is governed by thin sections ofStreptomyces megasporus (121). events that occur in the mycelium before the The defective nature of sporulation is here sug- onset of sporulation. The zig-zag form acquired gested by low germinability, unusual spore by the A. dassonvillei spore chains during one size, and location of the nucleoid, etc. of the stages of sporogenesis is thought (562) to Summarizing this section, we would like to result from spore disjunction starting from one point out that the wide spectrum of structural VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 483 modifications during the basic process of cell about 5 nm. In this respect, these spores resem- septation for exospore formation in actinomy- ble those ofBacillus coagulans (176). The inter- cetes fits well with the hypothesis (209) that nal structure seems not to differ in any signifi- sporulation is a modified procaryotic cell divi- cant respect from that of spores in Bacillus and sion. The hypothesis fits even closer than it Clostridium spp. They have a cortex and an does for sporulation in bacilli, for which it was elaborate system of outer coats. Details of spo- originally suggested. One reason for this is the roplast are not easily seen in thin sections of possibility of observing some of the likely "in- dormant spores. In contrast to Thermoactino- termediate steps" that are preserved and func- myces spores, those of Actinobifida dichotom- tioning. On the other hand, cellular differentia- ica possess an exosporium. tion in actinomycetes is so rich in terms of re- In their fine structure, spores ofPlanomono- sulting forms, it is no wonder that one finds spora and Dactylosporangium resemble exoge- difficulties in describing them in bacteriological nous spores of actinomycetes, which show a terms and seeks refuge in the more developed rich diversity of structure. For the purpose of mycological classification of spore types (82). rather superficial and pragmatic classification, one might distinguish here the following main Spores types. We now turn to the characteristics of spores (i) Spores that do not contain novel internal produced by actinomycetes and the structural structures that are completely absent in vege- diversity that they exhibit. They fall within the tative cells. One would place into this category broad definition of a spore (488) and the fonna- the spores of Streptomyces spp. (most often tion of these cells may represent the main studied) and several other actinomycetes, means of reproduction in actinomycetes; there which form chains of spores and vesicles with is no doubt that they are important. spores (43, 164, 317, 319, 463). No substantial More information is available on spores pro- differences were recorded in the structure of duced on the aerial mycelium than on spores spore nucleoid, ribosomes, membrane struc- produced in submerged cultures and within tures, and vacuoles compared with analogous agar. It is known that Streptomyces spp. do structures ofvegetative cells. However, the cor- produce sporelike structures in submerged cul- responding spore components often look more tures (63), which probably differ slightly from densely packed, the number of mesosomes mycelial cells in thermoresistance (131). They being reduced. The spore wall is usually 1.5 to also differ from the aerial spores of the same 2.0 times thicker than the vegetative wall, and species in hydrophobicity and requirements for two to three layers differing in electron density germination (240). So far, studies on submerged can be revealed within it. spores have been hindered by technical difficul- Similar internal structure but thicker walls ties in obtaining uniform material. Their fur- and more elaborate multilayered external coats ther investigation would be interesting in con- characterize the spores ofMicropolyspora spp., nection with possible attempts to observe the Saccharomonospora spp., Thermrnomonospora "microcycle sporulation" in actinomycetes. curvata, Actinobifida chromogena, and Der- Since the properties of spores of actinomy- matophilus congolensis (119, 195, 431). The cetes were the subject of some of the recent spores of M. chalcea (337) are similar in that reviews (81, 83, 235, 248, 485), what follows is a they have much-thickened walls, which proba- condensed summary of some of the relevant bly account for rather high refractility char- earlier works, with the emphasis on less-devel- acteristic of spores of Micromonospora spp. oped and newer areas. The middle wall layer in spores of M. chalcea Structure. The structural differences be- and A. chromogena (342) was termed "cortex." tween endo- and exospores are pronounced. The use of the term in the above cases seems Endospores produced by thermophilic actino- inadequate because of the distinctly different mycetes are prominent among actinomycete origin of the cortex of true endospores and in- spores because ofpolygonal outlines on the out- ternal-wall layer of the above spores. ermost surfaces (3, 79). On the surface of spores The question of how many nuclear elements the pentagonal and hexagonal areas are may reside in a Streptomyces spore is still unre- slightly curved inwards. Similar surface archi- solved. There are indications that most spores tecture was earlier revealed in some Bacillus of this type contain one nucleoid (50, 111, 114, spores (211). Scanning electron microscopy and 257, 258, 423), although claims of finding two application of the freeze-etch techniques ena- nuleoids are also known (147, 445, 446). bled investigators (329, 365, 556) to reveal that (ii) Spores that do contain structures absent the outer coat ofT. vulgaris spores actually con- in vegetative cells. These structures may be sists of parallel rows of fibrils each measuring represented by large vacuoles and the forma- 484 KALAKOUTSKII AND AGRE BACTERIOL. REV. tion of a structure of unknown origin and com- The spores ofS. streptomycini and some ther- position termed "central body." Spores of this mophilic Streptomyces spp. contain increased category may differ in the structure of their amounts of Ca and Mg (236), and these spores envelopes, and the presence of marked inter- also seem to accumulate Mn and Ni (411). Ac- sporal pads is characteristic for spores of M. cording to electron spin resonance signals, flavea (424). An extensive fine, granular coat there is a possibility that Mn is present in both delimited by a triple-layered sheath distin- the free and the bound state (412), the last form guishes spores of M. viridinigra (7, 120). The giving a signal similar to that in bacterial surface sheath, which covers aerial mycelium spores which is ascribed to the Mn-dipicolinic in many actinomycetes, usually persists on acid (DPA) complex (567). According to conven- spores. The exceptions are represented by tional analysis, however, DPA has not been spores formed on submerged mycelium and found in any Streptomyces spores examined so spores ofMicromonospora spp. even in surface far (227). culture (561). In contrast, the endospores of thermophilic Some characteristics ofsubunit morphologies actinomycetes were shown to contain amounts encountered in surface sheath ofvarious actino- ofDPA and Ca (89, 236, 309) equivalent to those mycetes are listed in Table 1. Spore surface in some of the bacterial spores (375). Traces of architecture (smooth or bearing various ap- DPA were found in spores of A. chromogena pendages) varies among the species of Strepto- and M. rectivirgula (236). myces and is extensively used for identification The DNA content of Streptomyces spores purposes (552, 553). seems to be slightly higher, whereas the con- Elementary structures which are contained tent of ribonucleic acid (RNA), according to in the surface sheath form a kind of ornament different authors (see section on germination), that might differ among organisms. Poten- is lower or higher than in vegetative cells. Cy- tially, this provides an additional opportunity tological data (461) show figures of 94 and 50 to for identification purposes (551, 172). 69% DNA and 19 and 51 to 75% RNA for nuclear Similarity in ornament, however, does not bodies of spores and vegetative mycelium, re- prove the similarity oforganisms, since similar spectively. ornaments were found in spores of S. griseus There are indications that Streptomyces (557), A. dassonvillei (562), Streptoverticillium spore DNA differs from that of mycelium. The (84), and sporangia of Planomonospora (463). differences, revealed in studies ofS. venezuelae, Moreover, morphologically similar ornaments include (i) thermal denaturation temperature; were also revealed on spores in several aerobic (ii) buoyant density (1.722 and 1.730 g/cm3 in sporeforming bacteria (211) and conidia ofAs- cesium chloride for spores and mycelium, re- pergillus and Penicillium spp. (196, 197). spectively), and (iii) homology with respective Possible functions of both spore appendages vegetative DNA (134, 135). Interestingly and elementary structures are rather obscure. enough, the buoyant density of DNA was Suggested hypotheses range from enhancement shown to fall gradually as spores matured, of hydrophobicity and floating abilities for the this process being accompanied by lowering the spores (555) and enhancement of dissemination degree of annealing with denatured vegetative by insects, to a more extravagant one implying DNA from 100 to just 30%. It has been sug- genetic exchange between the spores through gested that altered properties of spore DNA microtubules (357). An alternative hypothesis might result from complexing with some will be formulated in the section on secondary unique spore product (135), possibly a pigment metabolism. (458). Composition. Information on the chemical The relative amount of protein in mature composition of actinomycete spores is fragmen- spores ofS. venezuelae reaches 10 to 15% (Folin tary compared with that on bacterial and some phenol assay method). Total-protein hydroly- fungal spores. Again, spores of Streptomyces sates seem to contain increased relative spp. have been a little more thoroughly investi- amounts of arginine and leucine (51, 503). gated. Spore ribosomes ofS. griseus were shown to The amount of water in these spores varies be more stable in solutions of the same ionic widely according to environmental conditions, strength than vegetative ribosomes (527). This 2 to 4% being represented by what may be was also shown to be the case with spore and called "firmly bound" water (314). Although vegetative ribosomes of Streptomyces granati- "total" infrared spectra of mycelium and spores color (367). On dialysis against 10-2 M tris(hy- seem not to differ appreciably (419), some sig- droxymethyl)aminomethane buffer containing nificant differences in the contents of individ- Mg2+, the majority of spore ribosomes formed ual components were found. aggregates, whereas vegetative ribosomes per- VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 485 sisted as 7MS monomers. Both types of ribo- mentation in the course of spore germination somes were shown to be able to dissociate into (see below). A distinct correlation was reported 505 and 30S subunits. Qualitative differences between spore color en masse and the surface were said to be found in nucleotide composi- ornamentation of spores (see Table 2). In spore tion of spore and vegetative ribosomal ribonu- homogenates, some of the pigments were re- cleic acids. ported (367, 527) as being firmly associated with The murein components of spore and vegeta- ribosomes. It is not clear, however, whether tive cells in Streptomyces seem not to differ these observations reveal the chemical poten- significantly in the relative amounts of consti- tialities of the pigments or their true localiza- tuting monomers (90), although an increased tion in situ. amount of aspartic acid residues was recorded The electron spin resonance technique indi- in spore murein hydrolysates (96). cated the presence of melanine in spores of S. Some possible differences in structure and streptomycini (413). The brownish pigment also perhaps arrangement of simple monomeric present in these spores, according to ultraviolet molecules in spores are reflected in their de- spectra and paper chromatography, was not creased staining ability with methylene blue, identifiable with vegetative actinomycete pig- altered ability to stain with a fluorescent ments of the same color (18). The brownish brightener (559), and ability to stain selectively pigment of Streptomyces venezuelae, which ac- (according to Corti [76]). In some Streptomyces cumulated in the course of sporulation, was spp. the ability to stain gram positively was shown to possess indicator properties with ab- reported to increase during spore delimitation sorption maxima at 420 and 520 nm in butanol and maturation (472), whereas in others it was at pH 3 and 12, respectively (458). Traces of reported to decrease drastically (448). Distinct streptomycin were detectable in the spores of a differences were also found in susceptibility to strain of S. streptomycini of low antibiotic ac- phage attack, although specific phages are tivity (18). absorbed on resting spores (243, 430). The intact The antigens of actinomycete spores differ spore wall of several Streptomyces spp. is not from those of vegetative cells and are recog- sensitive to lysozyme digestion and is not de- nized as a separate group of antigens (307). The stroyed by treatment with a 15% KOH solution relative amount of spores in the actinomycete (96, 476), in contrast to the vegetative cell wall. biomass might thus influence its antigenic and The spores of Streptomyces spp. are marked chemical composition (306). The antigens asso- by their coloration. The color of "sporulating ciated with spores of thermophilic actinomy- aerial mycelium" was indeed employed for sev- cetes and responsible for the allergic condition eral decades in descriptions and classification of called "farmer's lung disease" are among those cultures. Surprisingly, we know very little most intensively studied (541). It is not clear, about the pigments responsible for this colora- however, whether these antigens are associated tion-practically nothing in comparison with only with spores or with the aerial mycelium as the abundant information on pigments that dif- well. fuse into the medium or are associated with The available information on chemical com- mycelium (44). position of the spore surface sheath was men- Some of the spore pigments are probably as- tioned in Table 1. sociated with spore coat(s), as evidenced by During sporulation, some actinomycetes ex- changes in coloration of aerial mycelium in the crete and accumulate in their cultures slimy course of sporulation (215, 494) and loss of pig- substances that might hold sporulating hyphae TABLE 2. Aerial mycelium color, spore, and sporophore morphologies in Streptomyces spp.a

Sections Color of myceliuinsporulating aerial Sporophores % Strains studiedSpore surface Azureus Bluish, bluish-green Spiral (93) Appendages (90) Cinereus Gray Spiral (80) Appendages (70); spores with hairs found almost exclu- sively in this section Roseus Rosy Straight (50) Smooth (90) Appendages (10) Helvolo-flavus Yellow, buff Straight (76) Smooth (100) Albus White Straight (77) Smooth (100) a Schematized according to data of reference 418. 486 KALAKOUTSKII AND AGRE BACTERIOL. REV. together and otherwise modify the appearance (423) suggested that many of the spores found of sporophores (217, 287, 438, 464). Staining of in colonies of Streptomyces spp. were not via- slimy substances produced by S. hygroscopicus ble. The duration of viability in actinomycete (438) seems to indicate the presence of polysac- exospores is thus comparable to that of asexual charides. conidia in fungi (435). Physiology. Metabolic activity of intact, dry Resistance to deleterious agents. Most of the Streptomyces spores is low, as shown by respi- actinomycete spores studied share a high resist- rometric experiments. Respiration of ungermi- ance to dessication (41, 407, 478) which proba- nated spores is rather resistant to cyanide inhi- bly has important ecological implications. It bition (245). These observations correlate with might be responsible, at least in part, for the diminished amounts of cytochromes b, c, and a relative abundance of actinomycetes in arid (500). However, a number of active dehydro- soils. The biochemical basis for this enhanced genases, as well as the enzymes of phosphorus resistance is especially intriguing in Strepto- and nitrogen metabolism, were detected in myces spores because their structure and chem- Streptomyces spores, the level of the former ical composition is not different from vegetative sometimes exceeding that in 2-day-old vegeta- cells. Vegetative mycelium of most actinomy- tive mycelium (239, 459). An increased level of cetes seems to be less resistant to desiccation catalase was also reported in spores ofS. strep- and lyophilization (198, 299, 506, 515). When tomycini; a portion of this activity could be dried spores are stored in air at different rela- removed by washing with water (477). Photo- tive humidities, the most deleterious for preser- reactivation ofStreptomyces spores is well doc- vation of viability appeared to be the range of umented in Kelner's (253) pioneering work. relative humidity from 60 to 90% (314). Spectrometry revealed electron spin reso- Compared to vegetative cells, Streptomyces nance signals in dry S. streptomycini spores spores appear to be more resistant to mechani- (411) characteristic of metabolically active cal abrasion (473), diluted acids, Formalin, hy- yeast, bacterial, and animal cells (106, 219, drogen peroxide, and some alcohols (17), proba- 328), but absent in the innately dormant bac- bly also phenol (315) and chloroform (60). In- terial spores (567). When kept under suitable creased resistance to radiation was found to temperature and aeration conditions, the spores correlate with hydration level, number of nu- of S. streptomycini, whose germination is pre- cleoids per cell, and the stage of gemination vented by elevated salt concentration, are able (383, 423). Nucleic acids were shown to be the to incorporate exogenous 32P mainly into their radiation-sensitive target in both dry and wet RNA fraction (238). Calorimetric measures of spores (224, 225, 377). Actinomycete (Strepto- their activities (247) bring to mind certain myces ?) populations in soils were reported (66, plant seeds during the so-called dead phase of 549) to be more resistant to soil treatment with their germination (61). Therefore the senescent methylbromide, 1,3-dichlorpropane, and some spores of Streptomyces seem to be attractive other chemicals, as compared with fungi and objects for studies of this reversible state and some groups of bacteria. the maintenance of viability in procaryotic According to most authors, who have been cells. discussing the subject for six decades, exospores The spores of T. vulgaris were shown to be ofStreptomyces spp. arejust slightly more ther- devoid of measurable respirometric activity moresistant than vegetative cells. As with (145) and deficient in cytochrome a (500). vegetative cells of other bacteria, resistance of Duration of viability. The maximal duration these spores to dry heat is much higher than of viability for spores of T. vulgaris surviving that of wet spores (16, 149, 226). In a set of in lakes at 5 to 6 C has been reported to be 100, typical experiments, it was shown (355) that even 1,500, years (88), although earlier workers viable fragments of vegetative mycelium were (326, 389, 455) mentioned rather rapid loss of still recoverable after exposure of suspensions viability in cultures of the thermophilic actino- in water at 45 C for 15 min. Heating spore mycetes studied by them. Duration of viability suspensions of various Streptomyces spp. at 60 of actinomycete endospores is thus comparable C for 15 to 45 min left no viable cells. Rod- to that of other bacterial endospores (475). and coccus-like vegetative fragments of no- The maximum value for maintenance of via- cardiaform Streptomyces variants (378) were bility by air-dried Streptomyces spores so far shown to possess slightly enhanced heat re- reported is 14 years (248). No viable spores were sistance compared with ordinary fragments found in dry cultures stored for 42 years (422). of vegetative mycelium in parent cultures, Successful storage of Streptomyces and No- whereas similar fragments of A. viscosus (57) eardia spores in distilled water for 4 years has showed no such difference. been reported (364). Cytological observations Studies of heat resistance of -Streptomyces VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 487 spores usually rely on conventional techniques amino acids and glucose. Presence ofinhibitors involving suspensions of heated cells plated on of nucleic acids biosynthesis induced formation solid nutrient media. Deviation from such tech- of nonmotile flagella. niques appeared to be instrumental in reveal- ing spores to regain their the ability of such DYNAMIC ASPECTS OF viability after heating. If the spores were kept DIFFERENTIATION in a humid atmosphere or in distilled water after heating at 99 C for 10 min, an appreciable Age-Dependent Changes in proportion of them recovered viability (313), Actinomycete Cells thus opening prospects for the investigation of mechanisms involved in repair ofheat damage. Age-dependent changes were most often Studies of such mechanisms employing bacte- noted in submerged cultures of actinomycetes. rial cells or fungal spores (29) are usually done To summarize observations with several Strep- with suspensions subjected to less drastic heat- tomyces spp., one may conclude that during the ing. trophophase the majority ofcells in populations The remarkably increased resistance of dor- are basophilic (high RNA content), with in- mant actinomycete endospores toward moist creased rates of protein synthesis and DNA heat can be correlated with their structure and synthesis as well as replication. During the composition. As with bacterial endospores, idiophase, these rates and the stainability of however, it is still uncertain whether increased the cytoplasm decrease (62, 63, 423, 457). Dur- Ca and DPA content is primarily responsible ing the periods of a maximal rate of biomass for increased thermoresistance, and whether accumulation, enlarged nucleoids rich in DNA the cortex is likely to exert pressure on the were observed in Streptomyces hyphae and sporoplast through contraction (325) or expan- termed "polyenergid nucleoids" (423). Their sion (174). functional significance and possible relation to The spores of other Actinomycetales studies the concept ofchanging gene dosage (316) await occupy an intermediate position between the further studies. Anyway, the fact remains that extremes exemplified by Streptomyces and the relative amount of DNA (per unit of dry Thermoactinomyces. This was shown to be the weight) fluctuates significantly in developing case with Nocardia, Micromonospora, Pseu- submerged cultures of actinomycetes. donocardia, Micropolyspora, and s6me others Age-dependent changes were revealed in the (144, 149, 236, 538). Since many of these spores plasma membrane of the mycelium of Strepto- contain no DPA or only traces, have no cortex, myces albus (372), the difference in membrane- and possess walls of different degrees of thick- bound nicotinamide adenine dinucleotide, re- ness, it has been suggested (83; Cross, personal duced form, oxidase levels being especially communication) that these spores might be ar- prominent in young and older cells. In the ranged in a row in which each succeeding mem- course of S. antibioticus development in sub- ber of the series would be characterized by in- merged culture, a redistribution of lysine and creased heat resistance and wall thickness. DAP isomers in the cell walls was reported They would thus resemble a row of cortex-less (427). Marked changes in age-dependent cell re- mutant Bacillus spores in which at least a por- sponse to Gram stain were noted in a study of tion of the functions supposedly carried by the Streptomyces erythreus (426), as well as in vari- cortex would be transferred to spore wall lay- ous representatives ofActinomycetales (165). ers. Accumulation of volutin granules in actino- Motility. Active motility of zoospores ofActin- mycetes cells was shown to be associated not oplanes spp. was stimulated in the presence of only with ageing, but also with nutritional im- several amino acids (iarginine being espe- balances (182). As in other organisms, volutin cially efficient) and urea (78). Disruption of the granules in actinomycetes contain high-molec- sporangium and release of motile zoospores in ular-weight complexes of orthophosphate with Actinoplanes sp. was stimulated by a wetting various ions and they are believed to play an solution, Tween-80 (202), and additives that af- important role in regulation of phosphate and fect mobility. Spores, although possessing fla- cation levels within the cell (395). gella, were not motile when released from old In N. rubra cultures (366), mycelioid, bacil- sporangia if suspended in water or solutions lary, and coccus-like cells predominated during containing phosphate and amino acids. Addi- logarithmic, stationary and autolytic stages, tion of a suitable carbon and energy source to respectively. Accordingly, the relative amount the solution restored the motility of zoospores. of DNA (per biomass unit) varied in the range Deflagellated zoospores (after ultrasound treat- 2.2 to 3.0 to 1.5%; RNA, 20.1 to 10.0 to 4.0%. ment at 4 C) showed resynthesis if the spores Protein content fell from 50 to 45% during the were placed in nutrient solution containing early logarithmic stage and then was constant 488 KALAKOUTSKII AND AGRE BACTERIOL. REV. until autolysis, when it fell to 31%. The total several carbohydrates to the growth medium of lipid content rose from 9.9 to 20.0% during loga- N. corallina tended to reduce mycelial growth; rithmic phase, remained constant, and then and on a medium with fructose the organism reached 30.85% during the autolytic phase. grew predominately in the coccoid form (542). The growth of Arthrobacter globiformis cul- Some Streptomyces spp. formed septate nocar- tures freshly inoculated into a complex medium dioforms on synthetic media with fructose; was preceded by a 4-h lag period (482). RNA with certain cultures the effect was reversible synthesis began immediately after inoculation; (384, 386). synthesis of protein and DNA began an hour A. crystallopoietes grew in coccoid form on a later (compare with data on arthrospore germi- mineral synthetic medium with glucose, and nation, see below). The differential rate of syn- the transition to rodlike forms was controllable thesis of the above macromolecules appeared to by addition ofpeptone or by one ofthe following be the same before the onset ofcell division and individual compounds: succinate, butyrate, ly- during a brief period of growth as rods. It was sine, asparagine, arginine, phenylalanine. The concluded from similar experiments with A. rodlike forms persisted in cultures until these crystallopoietes that morphogenesis in these or- compounds were completely exhausted (137). ganisms might involve transient incoordina- Two important differences were found in the tion of macromolecular syntheses and cell divi- sphere and rod peptidoglycans (290, 291): (i) the sion (136). polysaccharide backbone averaged 40 hexosa- Environmental Conditions and mines in the spheres and 114 to 135 hexosa- Morphogenesis mines in rods; (ii) the peptide cross bridges of the rod peptidoglycans were formed by single L- Environmental variables seem to exert a pro- alanine residues; the spheres contained some nounced effect on morphogenetic events in acti- alanine cross bridges, but more than half ofthe nomycetes. So far, most of the conclusive evi- bridges were comprised of iLalanyl-glycyl-gly- dence, reveiwed by Ensign (136), stems from cine. The significance of these differences to work done with forms producing transient or morphogenesis is not quite clear at present, but multiseptate mycelium. These are suitable for they are discussed (136) in connection with the such studies, as pointed out by Heinzen and possible changes in wall flexibility, accompany- Ensign (190), because the predominantly myce- ing morphogenetic transitions. lioid growth and regular septation appear to be Cell septlation early in the mycelial growth of separated in time sequence. N. turbata appeared to be stimulated by de- An example of the effect of complex nutrient creased temperature of incubation, decreased additives is given in studies with Nocardia sp. relative humidity (surface cultures), and by ad- 721-A, which formed clublike cells on a medium dition of Tween 80. On synthetic media with with meat extract. These cells underwent irreg- nitrates, the vegetative growth was rather ular septation; they did not appear on nutrient scanty and was accompanied by frequent septa- agar and mineral synthetic media with glucose tion. With ammonium salts, growth was pro- or saccharose (35). moted, the frequency of septation being re- Among the effects of more specific variables duced. Some individual amino acids stimulated one might cite (i) the limitation of manganese, growth ofrodlike cells, whereas casein hydroly- which causes inhibition of septation in N. sate caused the appearance of various irregular opaca (545); (ii) the promotion of mycelium de- dividing cells. Among the carbon sources velopment and inhibition of motile-cell forma- tested, starch, glycogen, and, to a certain de- tion in Dermatophilus by sodium and potas- gree, glucose inhibited fragmentation and pro- sium ions (436); and (iii) induction of R (motile moted extension of the mycelium (142). Sphere- rods) to C (irregular cocci) transition in Geo- rod transition in N. corallina growing on syn- dermatophilus sp. by mono- and divalent cat- thetic medium was markedly promoted by the ions and amines (222). Gas and temperature addition of acetate, propionate, 3-hydroxybu- variables were also shown to be important in tyrate, butyrate, succinate, Krebs cycle inter- determining predominantly mycelioid or diph- mediates, tyrosine, and peptone (190). theroid growth, as shown by work with Actino- The processes of crystallization are likely to myces sp. (232) and Arthrobacter sp. (468), re- be involved in formation of some surface struc- spectively. tures of actinomycetes, and such processes also The utilization of particular carbon and ni- depend heavily on environmental variables. trogen sources by growing actinomycete cul- Thus spiny quasi-crystalline projections on tures frequently appears to be correlated with sporangia ofMicroechinospora grisea appeared predominating cell morphologies. Addition of when the organism was cultivated at 37 C (but VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 489 not 20 and 26 C), probably under conditions of murein structure accompanying morphogene- enhanced desiccation (273). sis might be causally related. It is not clear, As one may see from Table 1, the only envi- however, which factor(s) might control the en- ronmental variable that looks likely to affect zyme activity itself (136). the formation of the surface sheath is the rela- As exemplified by the use ofmetabolic inhibi- tive humidity of the atmosphere, to which the tor, morphogenetic events in actinomycetes nascent aerial hyphae are exposed. might be controlled at several levels. Thus, Some unspecified variables believed to be re- vacuolization in A. viscosus was shown (57) to lated to the host defence mechanisms induce be markedly sensitive to inhibitors of protein pathogenic anaerobic and possibly aerobic acti- synthesis, while being rather unsusceptible to nomycetes to form "clubs" and some other inhibitors of nucleic acid (except of rifampin), structures while in the host body (351, 433). It is cell wall, and membrane biosynthesis. unclear whether vesicles ofFrankia spp. belong to this category. Formation of clublike bodies Environmental Factors in Control of was sometimes mentioned to occur in vitro (34, Sporulation 233) but on highly complex media. Nutritional limitations and sporulation. Des- iccation and other triggers. There are many Metabolic Activity and Morphogenesis reports documenting enhancement of sporulation in Streptomyces on media containing min- The possible relationship of the growth rate eral rather than organic sources of nitrogen, on and morphogenesis was noted in N. corallina "diluted" or "starvation" media (133, 168, 169, and A. crystallopoietes because many (but not 277, 278, 282, 376, 454, 507). Variants of S. all) nutritional compounds implicated in the griseus, either ultraviolet induced or obtained sphere-rod morphogenesis in these organisms in the course of spontaneous variability and (see above) enhanced the growth rate (190, 338, devoid of aerial mycelium and spores (11), re- 339). The connection between the growth rate tained their phenotypes through many pas- and cell differentiation might be of a more gen- sages if they were transferred on complex me- eral nature and was implicated in the sporula- dia with peptone or glycerol, but rapidly re- tion ofbacilli (94). According to the hypothesis, versed to parental phenotypes if transferred on the primary effect of starvation is to reduce the media with mineral sources of nitrogen. It was growth rate, this reduction being involved in also shown with submerged cultures of several triggering of morphogenesis. Streptomyces spp. that exhaustion of nutrients The cells of N. corallina during the transi- (in particular, phosphorus) might be among tion state from spheres to rods lost the ability to conditions favoring sporulation (122, 572). The oxidize glucose. Interestingly enough, the role of specific limitations, however, was very likely explanation was found in altered cell seldom studied systematically. One would note permeability toward this sugar (55), and the in this context some studies, showing retarda- utilization of fructose and caproate was not al- tion of sporulation in Streptomyces spp. by ad- tered. Ability to oxidize glucose was restored on dition of a carbon source to a medium, in which fragmentation of rods into coccoid cells. Later this source was exhausted but the nitrogen on, using labeled glucose, it was shown that source was still present (171). Future studies of utilization of glucose by rodlike cells is in fact this kind are likely to meet with some technical inhibited incompletely (190). Individual sub- difficulties because the majority of Strepto- stances that induce and support the growth of myces spp. freshly isolated from soil might be A. crystallopoietes in the form of rods also re- considered oligotrophic in that they can be pressed glucose assimilation and catabolism. successfully transferred without losing their ca- Inhibition of glucose permease was thought to pacity to sporulate for a rather long time on occur. Those substances (e.g., glutamate) that solid media containing just agar and its impuri- promoted increased growth rate without affect- ties. ing morphogenesis did not affect glucose metab- Unlike the majority ofprocaryotes, but much olism (283). The level of wall-bound N-acetyl- like fungi, actinomycetes may form aerial my- muramidase activity correlated positively with celium, which in many instances, at least in the change in the length of the polysaccharide surface cultures, serves as a prerequisite for backbone in the murein of A. crystallopoietes: sporulation. Whereas nutritional limitation it remained low when the cells grew as rods might be regarded as a kind of a general stimu- and then increased sharply with depletion of lus directly or indirectly inducing sporulation peptone when the rods were fragmenting into both in bacteria and fungi, there are some other spheres. The changes in enzyme activity and environmental stimuli that induce aerial my- 490 KALAKOUTSKII AND AGRE BACTERIOL. REV. celium formation and hence sporulation in peptone, urea, glycerol, and sodium chloride mycelial fungi but are not on the list ofcommon (189). Different batches of peptone varied sig- inducers of spore formation in bacilli. The case nificantly in their effect on sporulation; and in point is desiccation, which, according to meat extract was found to be useless. Addition Sussman (487), is likely to act under natural of Mg, Fe, and Mn had a slight positive effect. conditions on a rather extended time scale. This In synthetic media, glycerol and urea appeared characteristic delay might be incompatible to be the best among C and N sources tested, with the growth rate of most bacteria but quite respectively, and the addition of CaCo3 pro- compatible with the growth rates of fungi and moted sporulation. many actinomycetes. Several observations sug- Specific examples of mineral requirements gest, indeed, that aerial mycelium formation in include the stimulation of sporulation in Strep- actinomycetes is responsive to this stimulus tomyces spp. by cobalt (201) and sporulation in (237). As with fungi (14, 153), sporulation in thermophilic actinomycetes by mixtures of submerged cultures of Streptomyces was found trace elements (501). Extracts of soils differing (460) to be enhanced by an increased concentra- in their relative trace-element content stimu- tion of ions. It is possible that this variable late sporulation ofStreptomyces spp. at various exerts its effect by lowering water activity in rates (302). The requirement for cobalt was said the media and thus mimicking desiccation. to be specific, and this ion could not be replaced Lower agar concentrations in solidified me- by iron. There is an obvious possibility that dia tend to favor vegetative growth, whereas orthophosphate-metal complexes (mentioned higher agar concentrations tend to enhance the above), accumulating in the volutin granules of onset of sporulation in Streptomyces spp. (189). actinomycetes, might serve as depositories, There is some indirect and chance evidence masking ionic requirements for sporulation that aerial mycelium formation in actinomy- (should specific ones really exist). cetes is enhanced in heteroplasic systems. Re- Sporulation of Actinoplanes spp. in labora- sumption of aerial mycelium formation and tory culture was stimulated by several amino sporulation by actinomycetes subjected to soil acids, as well as by preparations of humic and culture might perhaps be relevant here (139, fulvic acids (564, 566). Aerial mycelium forma- 140, 230, 293, 302; see next section for alterna- tion by T. vulgaris on a complete synthetic tive explanation). A likely explanation for simi- medium including 18 amino acids and several lar phenomena in fungi involves increased P02 vitamins was blocked by excluding methionine in surface films matched to the obligatory aero- from the component amino acids (544). In a bic metabolism associated with conidiogenesis variant of S. coelicolor, production of aerial (520). Anaerobiosis and a sufficiently increased mycelium and associated activities were not C02 tension inhibit sporulation in Strepto- observed on synthetic Czapek agar but reap- myces spp. (189). peared after the addition of biotin to the me- Nutritional requirements for sporulation. dium. Culture filtrates of several microorga- Belief in a requirement for additional nutri- nisms had the same effect (284), probably be- tional factors for aerial mycelium formation cause of the presence of biotin. and sporulation in some actinomycetes stems Sporulation in Streptomyces spp. seems to from not infrequent observations of stimula- cease to depend, at a certain stage, on exoge- tion of these events in impure cultures of acti- nous nutrients and proceeds endogenously nomycetes mixed with bacteria (191) and the (e.g., in water) both in submerged (382) and in effect ofunspecified metabolites ofother actino- surface cultures (47). In the former case, spore mycetes (115). Systematic efforts to dissociate formation was supposedly observed in the first the effect of nutrients on growth and sporula- generation of vegetative cells formed following tion have not been consistent. As a result, spore germination. In the latter case, sporula- according to Freeze (156), it is often difficult to tion in aerial hyphae of a definite age was not say whether the condition studied is necessary, supressed by addition to the medium of glucose pleiotropic, or irrelevant for sporulation. and growth-prohibiting concentrations of peni- The "maintenance" media for streptomycetes cillin. are usually those that ensure abundant sporu- pH values might be critical for sporulation in lation, and these media often include complex actinomycetes (470), because slightly alkaline plant materials such as tomato paste, yeast conditions often favor sporulation, as shown, extract, oatmeal extract, etc. (294, 421). Six for instance, in a study with A. alba (330). species of Streptomyces (including strains like An intriguing but insufficiently documented S. griseus and S. lavendulae), deliberately cho- effect on aerial mycelium formation in Strepto- sen as "bad spore producers," produced their myces spp. 63 to 67 might derive from the nutri- best sporulation on solid medium containing tional status of the culture used as inoculum VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 491 (231). An inoculum from soil agar ensures a might function as more thanjust a substrate for more abundant formation of aerial mycelium protease action. A case in point is histone, on synthetic Czapek agar. Whatever the real which was shown to penetrate the cells ofEsch- reasons, it is interesting to recall the findings of erichia coli and B. cereus and affect protein Davies et al. (93) with B. subtilis which indi- synthesis (possibly at the level oftranscription) cate that chromosome packing and position are and morphology ofthe respective bacterial cells dependent on the nutritional status of the cell. (356, 398, 399, 481). In its turn, the chromosome state is likely to Prolonged cultivation of actinomycetes on affect cell behavior at some stages of the devel- laboratory media leads to a poorly understood opmental cycle. condition termed "degeneration," which is most It would also be interesting to correlate the frequently associated with loss of the ability to above observation with the common practice in form aerial mycelium, spores, antibiotics, and the antibiotic industry of using empirically de- pigments (299, 429). The proportion of dormant veloped media to grow the material for subse- spores in the population of degenerated T. vul- quent use as inoculum for fermentation. garis cultures has been shown to increase (6). Suppression of sporulation. Aerial mycelium Sporulation under natural conditions. Di- formation in the mesophil S. albus (151) and rect optical microscope observations in situ by some thermophilic actinomycetes (81, 141) ap- earlier workers (139, 292, 479) suggested that pears to have narrower temperature optima actinomycetes (probably Streptomyces spp.) compared with vegetative growth. Replace- form aerial mycelium and sporulate in soil. ment of air with pure oxygen in the gas phase Krassilnikov (280) noted a reduced formation inhibited aerial mycelium formation by Micro- of the extensive mycelial network of actinomy- monospora (Thermactinomyces) vulgaris grow- cetes in soil. More recent observations with the ing as a surface film on liquid medium, the ef- help of pedoscopes (13), direct electron micros- fect being attributed to inactivation of some copy of soil suspensions (388), and fluorescent thiol enzymes (543). In Streptomyces spp., aer- microscopy (359, 575) suggest that the type of ial mycelium formation was severely inhibited sporulation characteristic of actinomycetes in by adding to the medium 0.01% anionic deter- laboratory cultures is indeed observed in soil. gent (143) or lecithin (139). The frequency of sporulation is probably lower illumination with visible light appeared to than would be deduced from the widespread suppress aerial mycelium formation in several occurrence of these organisms. Some earlier Nocardia, the effect being dependent on the data (268) might be interpreted to suggest that incubation temperature (362). spores and probably other cells formed by acti- It is a common laboratory observation, men- nomycetes in soil are more resistant to heating. tioned by several investigators, that the inclu- This property is probably utilized in some ofthe sion of meat extract in nutrient media (as con- techniques for preferential isolation of actino- trasted with peptone in low concentration) re- mycetes from soil, employing heating at ele- tards aerial mycelium formation and sporula- vated temperatures (90 to 100 C) for rather ex- tion in many different actinomycetes (59, 276, tended periods of time (up to 2 h) (2, 80, 397, 569; to cite only a few). In fact, severe difficul- 518). ties encountered in the systematics of actino- Some patterns of sporulation in actinomy- mycetes encountered before the introduction of cetes were observed occurring in situ in cotton synthetic mineral media are at least partly ex- fibers (453) and soil (4) which were not seen in plainable by almost exclusive use of media con- laboratory cultures. Whether these can be at- taining meat extracts, sera, blood, and similar tributed to some still uncultivable forms (like materials commonly used in medical diagnostic Frankia spp.) or represent alternative modes of media. reproduction in some already known forms is The general suppressive effect ofthese media unclear. on aerial mycelium formation and sporulation is still unexplained. One might speculate on the Internal Factors in Control of Sporulation possible inhibitory effect of the polyamines and The developmental program of all the living some special kinds of proteins present in the organisms is thought to be determined by the extracts. Polyamines are thought to assume sequential expression of genetic activity, the some histone-like functions in bacterial cells latter being regulated by specific activation and (444). Their regulatory functions in vivo were repression of the corresponding genes. The shown infrequently (220), whereas their regula- repression hypothesis (348) as applied to micro- tory activity in vitro extends to the level of organisms has been less thoroughly investi- DNA-dependent RNA polymerase (22). Some of gated experimentally, which does not make it the basic proteins, present in the medium, any less attractive or disprove it. 492 KALAKOUTSKII AND AGRE BACTERIOL. REV. From the biochemical viewpoint, the distinc- the structures in question at all). These were tion of control mechanisms operating on tran- subdivided as follows: (i) "bald" (no aerial my- scriptional, translational, and post-transla- celium formed); (ii) aerial mycelium appears tional levels is important. The scarce informa- but the spiralization of sporophores does not tion on transcriptional and translational con- take place; (iii) spiralization is initiated but not trols available with actinomycetes will be pre- completed; (iv) spiralization is completed, but sented in the following section, as well as in the sporal septa are not formed; (v) sporal septa are one dealing with secondary metabolism. formed, but spore walls do not thicken and Mounting evidence stemming from the work round off; (vi) spore rounding-offinitiates but is with sporulating bacteria (275, 441, 480) sug- not completed; (vii) final stages of sporogenesis gests that at least some spore proteins might be are blocked, with accumulation of the grey products ofvegetative genes. Besides emphasiz- spore-associated pigment released from sporo- ing the importance of post-transcriptional con- phores. trols, these facts might also reflect the economy The above categorization is also a good sum- of information stored in chromosomal genes. mary of the sequence of events that occur dur- The last principle seems to be especially rele- ing sporogenesis in S. coelicolor. In other spe- vant when differentiation and morphogenesis cies of the genus, some modifications are likely in procaryotes is considered. Possibilities for to be encountered, taking into consideration the involvement of epigenetically regulated morphological differences in the last steps of processes in morphogenesis are further exem- sporogenesis (Fig. 3). Less clear on the scale plified by the ability of several large molecules is the position of the so-called nocardioform to associate into supramolecular complexes us- mutants, whose origin is frequently mentioned ing the conformational specificity stored in in the literature. Usually, the complete loss of these same molecules. Well-studied examples aerial mycelium production and sporulation is include the assembly of flagella, pili, poly-,3- accompanied in these mutants by the tendency hydroxybutyrate granules (308), and possibly of the colony mycelium to develop septa and also some surface structures in gram-negative fragment, much as in Nocardia spp. (see, for bacteria (508), spore coats, and more complex instance, 70, 378, 386). These mutants have not cell organoids (308). The importance of eluci- been subjected to genetic analysis, which might dating the possible matrix activity of the pre- present additional difficulties, because some of formed structures also emerges from these these mutants are reminiscent of the "abnor- types of studies. mal" sporulation mutants of bacilli (449) i.e., Finally, we must identify and discover the they present morphological features not found nature ofchemical signals generated within the in the process of normal sporulation. In this developing cells which are probably involved in case, the cascade of events leading to sporula- triggering some phases of developmental tion could possibly be blocked here at some events and play a role in coordinating meta- early step that is still associated more closely bolic activities within the differentiating cell. with cell septation. It is interesting to recall in These offer interesting and little-explored pros- this context the hypothesis of Schaeffer et al. pects for research with actinomycetes. (450), which states that the majority of Spo Variants and mutants with impaired differ- mutants in bacilli are in fact membrane mu- entiation. Reports on the occurrence of caco- tants. Apparently, a back mutation in some genic (e.g., blocked in the differentiation pro- "early" genes might release a normal sporula- gram itself or mechanisms allowing it to be ful- tion process. Mutation from sporeless state to filled [156]) variants in actinomycetes ap abundant sporulation was said to be possible peared very early. Papers on selection are with Streptomyces mediterranei (146). flooded with references to variants, depicted in An example of conditional mutants is seen in such terms as "morphological" with changed a S. coelicolor strain requiring biotin for sporu- color of aerial mycelium and even as "bald." lation (284). Mutants requiring C and A factors A strict distinction was not always made be- for sporulation (which will be dealt with in tween stable and unstable, including condi- more detail below) in a certain sense also be- tional, mutants. long to this category, and they certainly fit into A systematic study of a group of stable devel- the second category of cacogenic mutants opmental mutants of Streptomyces coelicolor those in which the genetic program for differen- was made by Hopwood and his colleagues (213). tiation is not missing, although the possibility These seem to belong to the category ofmutants for its fulfillment is impaired. in which the activity of genes absolutely neces- Less interesting is the group of mutants that sary for the synthesis ofcorresponding enzymes exhibit impairment of both differentiation and structures is blocked (e.g., with the excep- mechanisms and those involved in vegetative tion of protogenic revertants, they fail to make growth. These are probably represented by the VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 493 "dwarf" colonies not infrequently noted during has already revealed several interesting facts selection of mutants. The physiology of these and clarified the formulation of certain prob- mutant strains is almost unstudied. lems, a word of caution as to the probability of Besides being a result of the application of success in achieving ultimate goals using only physical and chemical mutagens, mutants and this approach might be in order. As pointed out variants with impaired differentiation among by Dworkin (130), the main hindrance to pursu- the Actinomycetales frequently arise in the ing genetic analysis beyond the present level is, course of spontaneous variability (537). Some even with Bacillus, a poor understanding of complex media are reported to provoke this what the products of sporulation genes might variability (and are used in studies of its lim- be. The situation with actinomycetes is cer- its), whereas others reduce it (and are recom- tainly even more difficult. mended for storing of the strains) (300, 301). Indeed, similar ignorance ofStreptomyces fur- Streptomyces variants with possible blocks in ther hampers the tentative assignment of spe- differentiation, most often encountered in se- cific functions to individual sporulation genes lection work, were categorized (301) into the ofS. coelicolor A3 (2) suggested by Chater (68). following groups; (i) nocardioform; (ii) asporo- Based on experience with double sporulation genous, or "white"; (iii) oligosporic; and (iv) mutants, the author discusses the possibility "dwarf-colony" type. Others were affected in that whiG and whiH gene products might be pigment production. Among the effective envi- involved in the cell wall changes required for ronmental influences that were reported to in- the initiation and localization of sporulation duce developmental variants, one should note septation. The coiling is thought to be a second- increased CO2 tension (378), undefined compo- ary consequence of these changes. The whiA nents in certain batches of meat extract me- and whiB gene products are supposed to be dium (Oxoid) (26), and growth on fructose- directly involved as structural or catalytic ele- containing media (385). ments in the development of sporulation septa. Streptomyces variants with impaired sporu- Sporulation and plasmids. Plasmids were im- lation so far studied were not found to possess plicated in the control of some phenotypic char- alterations in cell wall components which are acters in Streptomyces spp. including tyrosin- used in taxonomy (304, 486). ase inheritance in S. scabies (177, 178) and Sporulation and chromosomal genes. Since fertility types in S. coelicolor A3(2) (213). Since the genetic aspects of differentiation in actino- plasmid-associated genes are believed to be mycetes were extensively dealt with in several more sensitive to certain treatments than chro- recent competent reviews (69, 215) we shall mosomal genes, an attempt was made to elimi- briefly mention some points that seem to us nate plasmids from several Streptomyces spp. important for the general picture. by treating the cultures with heat and acridine It was shown (63, 213) that such S. coelicolor orange (394). The treated cultures lost the abil- mutant phenotypes as bid and whi are blocked ity to form aerial mycelium and spores. The in the activity of chromosomal genes. The posi- results were interpreted as indicative in terms tion of some of these genes on the circular S. of plasmid involvement in determination of coelicolor chromosome has been mapped. The sporulation. It would be pertinent to say that a total number of genes participating in the de- similar hyphothesis regarding sporeforming velopmental program in this organism was es- bacteria was widely discussed about a decade timated to be about one dozen, which is signifi- ago and abandoned on several grounds. One of cantly less than the number thought to partici- these was a demonstration that the results of pate in Bacillus sporulation according to some acridine treatment (not to mention heat treat- estimates (24), but not significantly less accord- ment) are probably not confined to elimina- ing to others (216). As shown, however, by stud- tion of plasmids (49). Interestingly enough, use ies on genetic aspects of sporulation in Asper- of acridines in experiments with Nocardia sp. gillus nidulans, the number of genes impli- (56) did not lead to results expected from the cated in sporulation is very likely to increase as earlier experience with E. coli. Results from the subtleties ofbehavior ofa particular system experiments on mapping sporulation genes on become better understood (see 73, 354). As in the chromosome of S. coelicolor, mentioned Bacillus spp., sporulation genes in S. coelicolor above, also make positive control of sporulation do not seem to come from a single region, but in actinomycetes by plasmids rather unlikely. rather are found to be scattered throughout the To our knowledge, the possibility of negative circular chromosome with occasional cluster- control has not been tested. ing. Proteolytic activity and sporulation and the Although the application of genetic analysis question of RNA polymerase modification. to studies on differentiation in actinomycetes Several observations suggest that total (108, 494 KALAKOUTSKII AND AGRE BACTERIOL. REV. 286, 295) or a limited (e.g., that of aerial myce- ture (50, 510) of RNA polymerase modification lium; 10, 509) autolysis accompanies or coin- in vivo seem not to be understood in full detail, cides (at least in time) with sporulation in acti- the potential significance ofthe event prompted nomycetes. Electron microscope pictures sug- some initial studies with actinomycetes. The gest that controlled autolysis plays a part in approach taken by Chater (67) made use of a later stages of spore formation in some Strepto- correlation that exists (546) between rifampin myces spp. (Fig. 3) and especially in M. rectivir- resistance and changes in the RNA polymerase gula (117). For modification of the developing of bacteria. It was also reported that rifampin- spore wall (rounding: stage III of Wildermuth resistant mutants of B. subtilis show an ab- and Hopwood [554]), a number of autolytic en- normal sporulation process (113). However, zymes are probably responsible, including pro- work with S. coelicolor A3(2) seems to suggest teases, peptidases, amidases, and murami- that the RNA polymerase of the actinomycete dases. is less sensitive to rifampin than is the corre- Although actinomycetes are used for indus- sponding enzyme of other bacteria tested. Not a trial production of some proteolytic complexes single rifA mutant among the approximately (e.g., Pronase and others), specific conditions 100 tested was without morphological abnor- leading to "oversynthesis" or derepression of malities that might have been associated with extracellular and, especially, intracellular pro- the mutation. A substantial number of "bald" teases have been studied insufficiently. Some colonies were obtained; in all cases, however, observations (65, 324) suggest that increased this latter marker appeared to segregate in production of extracellular proteases is stimu- crosses from rifampin resistance. It was con- lated by nutritional limitations. It is difficult to cluded that, ifsome changes in the RNA polym- say at the moment whether there is a specific erase of the actinomycete that affect its matrix kind of limitation that favors protease produc- specificity do occur during sporulation, they are tion or derepression. Recent results with A. confined to that portion ofthe enzyme molecule nidulans suggest that limitation in either car- that is not essential for the antibiotic to exert bon, nitrogen, or sulfur source might stimulate its effect. synthesis and the secretion of extracellular Involvement of specific factors in regula- neutral and alkaline proteases (74). In most tion of sporulation. Some observations suggest instances so far reported (with the probable that developing cultures of actinomycetes may exception of Clostridium sp. [343]), one ob- produce and sometimes excrete substances that serves a general correlation between such probably participate in regulation of differen- events as onset of nutritional limitation, lower- tiation. Examples from earlier works include ing of the growth rate, and sporulation. The diffusible substances that cause aerial myce- causal type of relationship between the above hum production in old surface colonies ofStrep- events, although shown in Bacillus, awaits tomyces spp. (115) and some substances in spent experimental determination in actinomycetes. broth filtrates reported to enhance septation in Actinomycetes also produce several sub- N. corallina (54). Occurrence of the latter was stances called protease inhibitors (373, 524). recently questioned (190). Three types of fertil- These compounds are mainly represented by ity variants revealed in S. coelicolor were rather low-molecular-weight peptides (158, 524, found to differ in their possession ofthe plasmid 525), and their spectrum ofinhibitory activity is called SCPI, which is found only in IF ("initial rather wide (514). Although aimed at regula- fertility") and NF ("normal fertility") but not tion of protease activities in other organisms, in UF ("ultrafertile") strains (69, 112). When these inhibitors have not been looked upon as cultivated on agar medium, IF and NF strains potential regulators ofprotease activities in the inhibited production of the aerial mycelium by actinomycete cultures producing them. UF strains. The inhibition was ascribed to a Many Streptomyces spp. produce serine pro- chemical factor diffusing through agar; in cer- teases, which differ in some properties from tain concentrations, it inhibited only aerial my- bacterial and fungal proteases and are related celium production. to some animal proteases (40% homology with Experiments on the so-called cosynthesis of bovine trypsin was reported by Hartley [188]). some antibiotics firmly established the ability It was the serine protease of B. subtilis which of several nonproductive strains of actinomy- was thought (323) to be responsible for modifi- cetes to complement each other, probably in the cation of DNA-dependent polymerase; this early stages of antibiotic precursor synthesis, event constitutes an important transcriptional thus allowing the complex biosynthetic path- control mechanism of sporogenesis in bacilli way to be successfully finished (363). Particular (332). mutants might bear deficiencies not only in Although the exact cause (374, 396) and na- their ability to synthesize secondary metabo- VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 495 lites, as exemplified by several antibiotics, but passed from 52-I to 45-Il by bringing the former also in their ability to synthesize secondary into contact with DNA from the latter during structures, exemplified by spores. These lesions several passages (526). are also repairable in some instances, and stud- Significant differences were found between ies on metabolic complementation have helped the strains in submerged cultures on complex to reveal some interesting examples. media. Whereas the 52-I strain was character- Factor A. The existence of this factor was ized by an "attenuated" life cycle (ending in revealed in the course of studies on streptomy- autolysis without sporulation), the develop- cin biosynthesis by parent and mutant cultures ment of 45-II under similar conditions was ac- of S. streptomycini (255). Among the mutants, companied by apparently normal sporula- a number ofcultures were detected that lost not tion. In the cultural broth of 45-11, an active only the ability to synthesize streptomycin, but complex was found to accumulate, called "fac- also lost the ability to form aerial mycelium tor C" ("cytodifferentiation factor"; 491, 492, and spores ("bald" colonies in surface cultures). 493). On addition to the developing culture of Both abilities were simultaneously restored on the strain 52-I (defective in sporulation) of the addition ofculture filtrates from normally spor- partially concentrated factor C, a marked stim- ulating and antibiotic-producing cultures. The ulation of septum formation, dividing hyphae effect was traced to a rather low-molecular in sporelike segments, occurred (533). mass (242 daltons) compound, called "factor A"' The factor C has not yet been purified suffi- and having the tentative formula of C13H2204 cently to regard it as an individual substance. (406). The molecule contains hydroxy-, car- Rather, it is a complex, which is thernolabile bonyl-, and isopropyl groups. Its diacetate and (inactivated by heating for 5 min at 45 C), 3,5-dinitrobenzoate derivatives retain the bio- and non-dialyzable. It is inactivated by trypsin logical activity. treatment, but not by deoxyribonuclease, ribo- The substance was produced by parent cul- nuclease, papain, pepsin, lysozyme, and dias- tures and was effective with sporulation mu- tase treatments. Some information points to tants, in very low concentrations. The effect of the possibly important connection between fac- factor A on sporulation and streptomycin bio- tor C activity and RNA synthesis in the devel- synthesis was observable both in surface and oping hyphae. It was thus shown (496) that C submerged cultures. A possible clue to the reac- factor interfered with the endogenous biosyn- tions that might be affected in the presence of thetic activity in vitro of DNA-protein com- factor A is given by studies showing the inter- plexes from S. griseus, as well as their ability ference of factor A with glucose 6-phosphate to bind purified RNA polymerase from E. coli. dehydrogenase activity (535). It is interesting The activity was not observed, however, with to correlate this finding with the changing pat- DNA-protein complexes fromE. coli K-12. Fac- tern of glucose catabolism in developing Nocar- tor C interfered also with the activity ofE. coli dia and Arthrobacter cultures mentioned ear- RNA polymerase if calf thymus DNA was used lier in this review. as a matrix. Prior and separate incubation of In another study on metabolic complemen- both DNA and E. coli RNA polymerase with tation in streptomycin biosynthesis and sporo- factor C resulted in similar interference with genesis of S. streptomycini, it was shown that the enzyme activity. Heating of factor C in some of the mutants blocked in sporulation solution destroyed every bit of its activity. might produce a diffusible substance(s) that The studies of various chemical factors sup- restores the normal differentiation and anti- posedly playing important roles in the coordi- biotic production by the others. The chemical nation of differentiation in Streptomyces spp. nature of the active principle was not eluci- are thus at different levels of advancement as dated (109). far as their chemical nature and mechanisms of Factor C. Comparative studies on two strains action are concerned. Comparison of data on of S. griseus, of which one was a streptomycin factors A and C seems to emphasize, as an producer (N52-I) whereas the other (N45-II) additional prospect for study, an attempt to was not, revealed only some minor differences correlate more precisely the events occurring in in the characteristics of their surface cultures the differentiation of surface and submerged (490, 534). Among the biochemical features cultures of actinomycetes. studied later, one would mention differences in hexose and pentose content in walls of hyphae, Spore Germination and Types of Dormancy the strain 52-I being also less resistant to heat- Encountered ing (30). Variants were identical in the relative Two extreme types of spores in actinomy- guanine plus cytosine content of their DNA. cetes, exemplified by the endospores of thermo- The ability to synthesize streptomycin could be philic actinomycetes and the arthrospores of 496 KALAKOUTSKII AND AGRE BACTERIOL. RiEV. Streptomyces spp., respectively, are character- and divalent ions (260). It is the initiation stage ized by distinctly different levels of dormancy, of these spores that shows the most exacting According to terminology accepted by Sussman requirement for high temperature and is proba- and Halvorson (488), these may be called "con- bly critical for the cultures developing at tem- stitutive" and "exogenous" dormancy. peratures close to the minimal cardinal point Constitutive dormancy of thermoresistant (261; see Fig. 4). Outgrowing spores of T. vul- endospores and an overall picture of germina- garis, in contrast to spores of most Bacillus tion. Like the majority of bacterial endospores, spp., appeared to be insensitive to rather high fresh, intact endospores of Thermoactinomyces novobiocin concentrations (20). spp. and A. dichotomica would fail to germi- Possibility of an alternative way for release nate if placed under environmental conditions from dormancy. Temperatures that were that are otherwise favorable for development. found to be optimal for releasing spores of T. The main steps involved in the overall germi- vulgaris and A. dichotomica from dormancy nation process (e.g., activation, initiation, and appeared to be lower than those limiting vege- outgrowth) are similar to those of spores in tative growth and seemed to be close to 20 C in Bacillus and Clostridium spp. (19, 235). Species either species (262). Ifthe temperature at which differences in the fate of cortical material re- spores were produced was lowered by 20 C, this vealed by structural studies (116, 310) corre- change in its turn lowered by about 20 C the spond to those encountered earlier with differ- maximal temperature to which these spores ent species ofBacillus (187, 311). were resistant, as well as the temperature opti- As with most bacterial endospores (325), the mally suited for heat shocking them in the dormant condition of actinomycete spores routine procedure for activation. The tempera- might be enhanced by suitable choice of tem- ture range optimal for low-temperature activa- perature and nutritional conditions for spore tion, however, was not affected by changes in formation and the relative humidity of the at- temperatures at which spores were produced, mosphere in which spores are stored. Thus, nor by the composition of the medium on which populations ofT. vulgaris spores formed at 55 C spores were produced (265). were found to be more heat resistant and con- The critical environmental variables that al- tain a higher proportion of dormant cells than low low-temperature activation to proceed in- populations formed at 37 C (266). Populations of clude certain levels of humidity (.10%) and spores formed on synthetic medium were rich- presence of molecular oxygen in the gas est in dormant spores, the degree of dormancy phase-this requirement is not usual for ac- being markedly influenced by amino acids and tivation or initiation of most bacterial endo- Ca2+ and Mg2+ levels. An inverse correlation spores so far studied (263). Cooling appeared to was found in dormancy and thermoresistance of be indispensable in conditioning the T. vul- spores formed on synthetic medium. In contrast garis endospores for germination; it was irre- to the situation with many bacterial spores, placeable by such routine activators as heat thermoresistance of T. vulgaris spores was shock and mild mechanical abrasion. found to be most critically influenced by Mg2" Only those spores subjected to limited cooling but not Ca2+ ion levels (264, 265). for rather short periods (<60 min) were found Some special features characterized the acti- responsive to the heat shock. If the cooling was vation and initiation stages in germinating en- extended, the spores underwent activation dospores of T. vulgaris. These spores could be without heat shock. In this case, heat shock conditioned for germination not only by heat was found to kill the spores, which lost their shock but also by exposure to decreased temper- thermoresistance (259; see also Fig. 5). These ature (6); they are markedly sensitive to physi- findings probably explain the controversial re- cal activating agents (259), whereas the list of ports on the possibilities of heat activation of possible initiators is remarkably wide, includ- Thermoactinomyces endospores (19, 20, 89, 152, ing sugars, amino acids, ribosides, and mono- 309) to a certain extent.

ActimOfl 6piti~tiefn i );~; (eon. 2J ) (.J.;. 21- sQ,) (opt % 4 lw)

Bt-"t~ - P-~vtIt-J° borm...t Acfvvdej Initrate OtVw9int *"M 3Sm Slope 5for& FIG. 4. Minimal temperatures allowing germinal events in T. vulgaris spores. VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 497 Although cooling appeared to activate intact the current concept (254) ofbacterial spore acti- actinomycete endospores in situ, even in cham- vation. Besides the rather specific conditions bers with controlled humidity, the suspension necessary for it to proceed, the results of the of spores in water significantly facilitated the treatment seem to be appreciably "broader," process, as did all treatments that enhanced since the spores so activated lose thermoresis- wetting ofspores. In suspensions, activation by tance and resistance to many toxic chemicals. limited cooling was markedly enhanced in the The effect of limited cooling was found to be presence of Mg2+, followed by Ca2+ ions, irreversible (263). It thus seems possible that whereas K+ and Na+ showed no activity. The the cooling triggers and then helps to maintain effect of ions during cooling could not be as- a series of events that bring the dormant spore cribed to their activity as potential initiators, into conditions that would more correctly be de- since the initiation phase required elevated scribed as the early stages of initiation. It is temperatures to proceed. therefore suggested (262; see also Fig. 5) that Low-temperature activation seems not to fit low-temperature activation might function as a

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0. Vn

FiG. 5. Temperature regimes and early germinal events in T. vulgaris spores (D. Sp., dormant spores). Activated spores: (1) ASP-I, spores subjected to brief (up to 60 min) cooling; (2) AS.-HI-as AS,-I, but additionally activated by heat shock; (3) ASp-III, spores activated by prolonged cooling. These do not need heat shock for full activation. This stage is irreversible. IS,, Initiated spores. Schemes of internal organization of AS are not presented since no reliable data are yet available. Conditions (temperature/time) oftreatments are shown beside arrows pointing to final results of treatment. 498 KALAKOUTSKII AND AGRE BACTERIOL. REV. partial alternative to the usual endospore acti- might serve the purposes of analysis better vation pathway. The irreversibility of the pro- than premature adoption of a common set. cess resembles the results of ageing ofbacterial It should be noted that assisted wetting of spores; its results and temperature characteris- Streptomyces spores (an operation unnecessary tics also resemble the so-called "after-ripening" and hence unstudied with the majority ofbacte- encountered with some fungal spores (488). rial spores) leads to some activation, causing The question of why spores of thermophilic additional difficulties in attempts to operation- actinomycetes appeared to be so sensitive to ally define "intact" spores. Dried spores from limited cooling, and whether they are unique freshly grown cultures are sometimes pre- in this respect, offers interesting prospects for sumed to be intact. But drying inself might future research. Preliminary speculations might inflict some dormancy, as shown recently with take note of the possible structural rearrange- vegetative cells of certain gram-positive bacte- ments of water molecule aggregates at temper- ria (327), rendering them dependent for subse- atures of 15 to 20 C (92, 125), to which the spore quent "activation" on mild heat shock. It is also membrane next to a water-rich and "expanded" clear that extensive washing of spores might cortex (174) in a thermophilic organism would potentially alter their nutritional and ionic re- be especially sensitive. The possibility of an quirements for germination. At least some ofthe enzymatic reaction(s) being derepressed and changes inflicted on the surface sheath of proceeding below the growth temperature spores by washing seem to be reversible (Table minimum seems also feasible and is supported 1, Fig. 6). by the oxygen requirement for completion of Since germ tube formation in several Strepto- low-temperature activation. myces spores is significantly affected by the Finally, one probably should not be content nutritional environment, whereas the wetted with a silent acceptance of the notion that the spores are endowed with several enzymatic ac- development of a thermophilic sporeforming tivities, it was thought that their limited meta- organism such as T. vulgaris would depend, bolic dormancy is not constitutive but largely under natural conditions, on the presence of exogenous. However, a recent and important temperatures close to optimal. The drawing in study employing S. viridochromogenes arthro- Fig. 4 presents a scheme that emphasizes the spores (208) suggests that the situation, at least minimal temperatures allowing development with some species, might be different, and the to proceed. There is a high probability that process of swelling (Fig. 6) actually may repre- neither in soil nor in the infected tissues of sent a sum total of at least two processes. animals do the spores of T. vulgaris remain The authors devised a simple technique, dormant, even under conditions that do not which enabled them to obtain S. viridochro- allow vegetative growth. mogenes spores in a less altered state prior to the Germination of exospores. Unfortunately, start of germination experiments (although the several studies made on germination of brief sonication of suspensions was still em- Streptomyces spores employed different species ployed). Using these spores, convincing proof of the genus. A look at the diagram (Fig. 3) that both activation and initiation are involved illustrating structural aspects of sporulation in in the overall germination process was pre- several Streptomyces spp. would strengthen the sented. Activation was accomplished by a mild expectation of differences in germination. Spe- heat shock (55 C for 10 min) and was reversible. cies differences might be pronounced regarding Initiation was accomplished in solutions con- the duration of certain germinal stages, nutri- tainiing L-alanine, adenosine, glutamic acid, p- tional requirements, the fate ofouter-wall com- aminobenzoic acid, Mg2+ and Ca2+ ions in the ponent(s) during the emergence stage, etc. presence of CO.,. The process resulted in phase- Nevertheless, an attempt to correlate struc- darkening and excretion of some substances tural and physiological findings of various au- from '4C-labeled spores. Although the above thors seems to be justified and is presented in events are quite reminiscent of those accompa- Fig. 6. nying germination of bacterial endospores, As one can see, it was decided that the termi- some marked differences in the behavior of the nology for the earlier stages of Streptomyces Bacillus and Streptomyces spore systems were spore germination be retained for the present, also revealed. Activation was found to be ac- despite variance with that commonly adopted companied by pronounced rise in adenosine 5'- (173) in studies ofBacillus spores. Besides the triphosphate level and respiration. Character- reasons for doing so already discussed by istics ofthe initiation process included a further Atwell and Cross (19), we think that even tem- increase in the rate of endogenous metabolism, porary retention of different terminologies rise in adenosine 5'-triphosphate content, rapid VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 499 Summary ofcharacteristics attained at stages ofgermination Nonwettable Wettable (surface re- Volume increase Heat resistance lost versibly modified) Respiration manomet- Respiration detectable Phase darkening Membrane rearrange- rically undetectable (partially KCN- ments likely insensitive) Organic free radical A number of C-, N-, Rearrangement of pa- Maximal aerobic respi- detectable and P-metabolism en- rarnagnetic centers (Mn`, ration and heat produc- zymes revealable; endo- Fe+++) tion genous reserves start to be utilized Release of ions starts Full sensitivity to lyso- (Ca+', Mg++, Mn++) zyme and exogenous phage Release of organic compounds likely; pigment(s) lost RNA and protein DNA s:ynthesis Sensitivity to svntiuhe& nohsahlv probablly starts ionizing radia- ar here near h(ere tion decrease I ,r-likely.. 1. Sensitivity to ionizing Nuclear division starts radiation increases InaV

- ~

40-120 min120m-300 min Intact Wetted Swollen Emerging f 0 10 0, C- s a n iti H%0 essential HGO, 02, C- and N-sources Additional nutritional essential; chloramphenicol in- requirements likely; sev- terference likely eral antibiotics interfere Conditions affecting the process FIG. 6. Diagrammatic sketch ofevents accompanying germination ofStreptomyces arthrospores (schematized according to data presented in the reviews 19, 83, 248). The drawings emphasize the likely behavior of two spore wall components and the surface sheath. incorporation ofadenosine or uridine into RNA, by a considerable lag (Fig. 6). However, con- and protein synthesis, which started toward the flicting evidence was reported from studies end of the process. The initiation thus does not with S. olivaceus employing Auramine 00 for seem to be confined to a series of primarily measuring DNA levels in germinating spores. degradative processes; if these processes do oc- This was said (371) to steadily increase immedi- cur, they seem to be coincident with some early ately after placement of spores in a complex starting events involving energy-yielding and medium favoring germination. It might be in- RNA-synthesizing activities. Also, it is not teresting to learn whether this finding reflects quite clear whether or not the heat activation is actual changes in DNA content of spores or an obligatory prerequisite for initiation. Com- alterations in spore permeability and the abil- menting upon early inhibition by rifampin of ity of nucleoids to bind the dye. protein synthesis in S. viridochromogenes Discrimination between some later events, spores undergoing initiation (followed by chlor- probably associated with outgrowth, becomes amphenicol inhibition at some later stages of feasible with the observation (391) on the influ- the process), Hirsch and Ensign (208) point to ence of mild heat shock on prophage behavior the possible absence of stable messenger RNA in germinating spores of S. coelicolor A3(2). molecules in the spores, an inference which Limited heating of freshly prepared spores nei- their results make likely. ther affected their viability nor induced phage It is usually thought that DNA synthesis in multiplication. Germinated spores (3 to 4 h on a germinating Streptomyces spores is preceded complex medium) were killed as a result of 500 KALAKOUTSKII AND AGRE BACTERIOL. REV. similar heating without liberating phage prog- mycetes are more specific and exact than those eny. In spores bearing germ tubes (6 to 7 h), the for vegetative growth (aeration, pH, ionic envi- shock induced phage-lysis accompanied by lib- ronment, etc.). Manipulations ofthe amino acid eration of phage progeny. or ionic composition of the sporulation medium The nature of endogenous carbon and energy will bring no less appreciable changes in, say, reserves probably utilized by germinating heat resistance or pigmentation of their spores Streptomyces spores is likely to become clearer than similar manipulations would cause in the after exploiting the finding that trehalose ca- composition of actinomycins (252) or bleomy- tabolism is markedly increased in germinating cins (523). S. hygroscopicus spores (200). Interestingly Obligate connections between the onset of enough, it is this same sugar that constitutes secondary metabolism and the idiophase have endogenous reserves in many fungal conidia. been questioned in the case of chloramphenicol and tetracyclines. It was shown (346) that, in a batch culture, production of chloramphenicol DIFFERENTIATION AND SECONDARY on a complex medium is observed during idio- METABOLISM phase. However, on a mineral synthetic me- Points of Similarity in Origin and Flow dium the synthesis of antibiotic was also observable during trophophase. It was therefore The features common to the majority of sec- concluded that a regulatory stress triggering ondary metabolism products are currently cate- antibiotic formation ensues on a complex me- gorized (58, 98, 547) as those that: (i) are pro- dium with the onset of idiophase, whereas a duced during idiophase; (ii) do not possess a similar stress is experienced by the organism common function in the life of the producing already during trophophase on a minimal me- organisms (although they might be endowed dium. No attempt was made in this work to with specific functions in the life of particular see whether there were some signs ofdifference organisms); (iii) are produced by taxonomically in cell differentiation of the actinomycete on restricted groups of organisms; (iv) are usually complex and minimal medium, which seems to produced as mixtures of related compounds be likely. It would be pertinent to note that it that can be grouped in families or series. seems logical to regard the onset of secondary These definitions resemble very closely the metabolism as a probability process, in a man- overall view of the situation encountered with ner suggested for sporogenesis (451). Some "secondary structures" or the products of cell sporulation in batch cultures of bacilli was differentiation in actinomycetes. Some perti- not infiequently observed during logarithmic nent points should be mentioned. phase (156) and is probably associated with (i) The onset of differentiation (e.g., aerial changes in the growth rate of individual mycelium production, sporulation) in actino- cells inflicted by nutritional limitations (94). mycetes is very often associated with the estab- With tetracyclines (529), maximal accumula- lishment of limiting conditions for vegetative tion of antibiotic was found to occur during growth (some are cited above in this paper). "production phase." At that time, as shown by Supposed switches for secondary metabolism these authors, the nutritional environment was (99) are indeed very similar to the supposed changed drastically, because the phosphorus switches for sporogenesis (184, 449). The bio- source was completely exhausted. Cytologi- synthesis of siomycin was shown to be re- cally, the productive-stage cells differed mark- pressed by addition of glucose to the nutrient edly from those of trophophase stage cells. It medium, even though the sugar was quite suit- was argued, however, that the final yield of the able for supporting vegetative growth (256). antibiotic heavily depended on some critical Some of the enzyme systems participating in regulatory events, which took place signifi- secondary metabolism, like phenoxazinone syn- cantly earlier than the production itself. It was thetase, were shown to be catabolite repressed the relative concentration of the likely precur- (353). sors within the cell that was held responsible The term "idiophase" stemmed mainly from for inducing the cell to produce tetracycline. It studies with submerged cultures. Not all physi- was also suggested that the term "secondary ological features of these cultures seem to coin- metabolites" be abandoned and substituted by cide with those ofsurface cultures, at least with the term "excessive metabolites." In our opin- mycelial organisms. Several examples were ion, the strong argument for use of the later given elsewhere in this paper to demonstrate form is in the intimate connections that it im- how environmental requirements for aerial plies between primary and secondary metabo- mycelium formation and sporulation in actino- lism. However, its use broadens the category of VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 501 substances to be considered almost indefinitely. ter are meant the specific structures of sporo- Under some conditions, regulatory mutants of phores, spore-bearing vesicles, spores, and E. coli can probably be induced to overproduce their ornamentation, etc; the last characteristic certain amino acids and ribosides. However, serves as a kind of "fingerprint" for identifica- the question (97) that asks why the majority of tion. At least some of these characteristics, like antibiotics are produced not by E. coli but, spore wall ornamentation, were shown (172) to rather, by highly differentiated Streptomyces be stable and thus reliable for the purposes of spp. may be more specific and relevant to the systematics. situation. The number ofantibiotics known to be formed Concerning the importance of very early by Streptomyces spp. exceeds appreciably that events for tetracycline production, one would known to be formed by representatives of any say that an example of the effect of the nutri- other genus. This might reflect the effort de- tional state ofthe inoculum spores on the differ- voted to screening programs, and the number entiation of resultant Streptomyces colonies of species within the genus, as well as their (mentioned earlier) would suggest the impor- comparable nutritional versatility, which sim- tance of even earlier events for differentiation plifies laboratory cultivation. However, the in actinomycetes. biosynthetic abilities of species of this genus (ii) Some common function(s) for spores in are matched by the almost unprecedented bacteria and fungi is occasionally discussed, structural variables involved in differentiation. but certainly not agreed upon, and the situation The species specificity of antibiotic produc- with actinomycetes is certainly no better. The tion in streptomycetes was long debated. Some suggested hypotheses range from the role of workers believed, however, that elaboration of spores in dissemination and gene transfer to particular antibiotics might be helpful for iden- survival during periods of drought and nutrient tification at either the species (281) or subspe- limitation or brief exposures to elevated tem- cies (420) levels. This line of thought is appar- peratures. All of them might well have a ra- ently contradicted by frequent reports of the tional basis, but all of them fail to take into production of similar antibiotics by represent- consideration the almost unmatched multiplic- atives of different genera of actinomycetes. The ity of secondary structures (e.g., spores) pro- contradiction seems not to be very deep if one duced by actinomycetes, as a result of natural recalls what was said on the possibilities of selection. alternative ways of reproduction in actinomy- Although industrial strains of actinomycetes cetes. Besides, actinomycetes usually produce a most certainly belong to categories of regula- mixture of antibiotics, and it is the ability to tory mutants, some minor quantities of anti- produce a certain mixture which might be spe- biotics are usually produced by cultures freshly cific. There are numerous reports of strains isolated from nature. On the other hand, it is a losing the ability to produce a particular anti- practice to subject actinomycete cultures to soil biotic and almost no reliable information on the transfer when they lose ability to differentiate production of one type of antibiotic instead of as a result oflaboratory manipulations. A num- another (say, tetracycline instead ofa polyene). ber of genes, the activity of which is inferred to (iv) The "serial" character of secondary struc- be required for tetracycline (528) and strepto- tures in actinomycetes is documented with no mycin (101) biosynthesis, lie close to those less certainty than the production offamilies of shown to be required for sporogenesis in Strep- antibiotics by them. One would likely point to tomyces. Both sets of data seem to emphasize certain types of spores produced, "sections" that neither sporulation nor antibiotic biosyn- recognized within the genus Streptomyces ac- thesis is insignificant in terms of the genetic cording to sporophore morphology (199), and burden for the producing organism. The fre- "series," recognized (28, 161) according to the quent occurrence in actinomycetes of enzymes color of the sporulating aerial mycelium. that might transform antibiotics, including Although information on sporal pigments is those produced by the same cultures (101, 402), meager, one would be inclined to consider them serves to strengthen this line of reasoning. as allied to secondary metabolites, since their Possible specific functions of antibiotics pro- formation was usually not observed during the duced by actinomycetes will be dealt with later. vegetative growth of corresponding cultures (iii) The recognition of production by taxo- under submerged or surface conditions. Inter- nomically restricted groups of organisms re- estingly enough, a kind of correlation seems to quires the identification and systematization, exist between the coloration of sporulating aer- which is principally based on characteristic fea- ial mycelia ofStreptomyces spp. and the result- tures of their secondary structures. By the lat- ant morphologies of sporophores and spores 502 KALAKOUTSKII AND AGRE BACTERIOL. REV. (Table 2). Less correlation is observed between Besides reflecting definite chemical and possi- the above characteristics and the colors of col- ble physiological individuality ofantibiotics for ony pigments and those diffusing into the me- the producing organisms, these apparently con- dia. troversial examples ofcorrelations suggest that Some of the morphologically peculiar actino- different antibiotics might be linked in their mycetes are noted for their ability to produce biosynthetic routes to quite different ("early" special types ofantibiotics (e.g., Streptoverticil- versus "late") developmental stages. Interrup- hum spp. very often produce polyenes (271, tion of development at some medium stage 519]). As with bacilli, the possibilities of links would severely affect the biosynthesis of late- between differentiation and antibiotic biosyn- stage-associated secondary products but would thesis in actinomycetes are further emphasized not affect, or even stimulate, accumulation of by drastic alterations in the ability to produce products associated with the early stage. "Ab- antibiotics by variants and mutants with im- normal" mutants, as shown with bacilli, are paired differentiation. specifically interesting for their inabilities to Some earlier studies in the area were re- sporulate normally while overproducing some viewed by Schaeffer (449). However, possible of the structural entities presumably needed on correlations were not sought consistently, and later stages of sporulation. some of the best papers on selection often con- However, the present situation is still most tained some rather indecisive formulas of the closely described by a formula, suggested by sort: "... Our superior antibiotics producers Weinberg (547): "changes in abilities to differ- were morphologically identical to the parent entiate are usually accompanied by significant cultures, except that some of them produced changes in levels of secondary metabolites pro- spores of lighter coloration" (129). Since later duced." correlations were sometimes encountered in the There are some reasons to think that meta- antibiotic potency and spore surface ornamen- bolic machinery of actinomycetes involved in tation (114), many of the doubtful cases earlier biosynthesis of antibiotics might deviate from reported merit reexamination. that involved in primary metabolism. Peptide Positive correlations between the ability to moieties of actinomycins (252) and echinomy- form aerial mycelium and spores, on the one cins (523) are thought to be synthesized without hand, and some of the secondary metabolite(s), the participation ofribosomes, as earlier shown on the other, include the following examples (to to be the case with peptide antibiotics of bacilli mention only a few): (i) pigment and character- (340). There are, however, some exceptions istic earthy smell (geosmine?) in a Strepto- (293). In six species of Streptomyces studied myces sp.; (ii) pigment, smell, and antibiotic in (229), the first enzyme in the biosynthetic chain Streptomyces sp. (378); (iii) antibiotics in S. leading to aromatic products appeared to be venezuelae and S. kasugaensis (394); (iv) pig- resistant to feedback inhibition by trypto- ment in S. longispororuber (148); and (v) pig- phane, tyrosine, and phenylalanine. Some pe- ment and antibiotic in S. coelicolor (284). Par- culiarities of biosynthetic control mechanisms ticularly relevant to those examples is the re- are probably responsible for the appearance, in sumption of streptomycin formation in aerial antibiotic molecules of actinomycetes, of such myceliumless mutants following factor A addi- rare or even unique compounds and moieties as tion in parallel with the resumption of appar- D-amino acids in peptides, the azide group of ently normal differentiation (225). Also, accord- azaserine, the vinyl group of sarcomycin and ing to observations of one of us (N.S.A.), anti- primocarcine, the mitosane of mitomycins and biotic and extracellular proteases appeared to porphyromycin, etc. (275). be synthesized by aerial mycelium-producing There is also an impression that genetic reg- variants of T. vulgaris var. calvum. Workers ulation, especially ofsome final steps in biosyn- engaged in the selection and storing ofStrepto- thesis ofsecondary products in actinomycetes, is myces strains tend to regard the aerial myce- not subject to very tight control. This specula- liumless cultures as poor or zero producers of tion is supported both by the fact that mixture antibiotics (299, 300, 429). However, as shown, of very similar molecules are often produced in the studies with factor C and its effect on and by the frequent occurrence of products that sporulation in submerged cultures and on reach their final form after some modifications, streptomycin biosynthesis (see above), there depending on environmental conditions. Possi- are some instances of negative correlations. ble examples of the latter situation would in- Some examples of such type correlations with clude "protoactinorodine" (53) and, especially, surface cultures were given by Demain (100) "proviridomycin" (45, 349, 350), a protopigment, and several by Kalakoutskii and Nikitina the final color ofwhich would depend on availa- (246). bility in excess of a particular (among several VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 503 possible) ions in the medium at a certain criti- The possible regulatory functions of at least cal period of time. Even streptomycin seems to some antibiotics are further exemplified by ex- be "adjusted" to its final and more active form treme sensitivity to an antibiotic at certain as a result of wD-mannosidase action, the activ- developmental stages. Thus, spore germination ity of enzyme being dependent on glucose ex- in S. griseocarneus was inhibited by 5 ug of haustion in the fermentation medium (102). streptomycin, whereas mycelial growth oc- Some analogy can probably be drawn between curred in the presence of 200 to 300 ,ug of the such behavior of secondary metabolites and antibiotic (40). such events in differentiation as a postulated Reports on the growth-inhibiting activity of modification ofvegetative into sporal ribosomes antibiotics were soon followed by studies indi- as a result of desiccation (269), chemical and cating possibilities for inhibition of particular physical changes in B. subtilis spores undergo- enzymatic reactions, either in vivo or in vitro. ing "maturation" in spent medium (95), and Examples include inhibition (393) or stimula- self-assembly of sporal coat proteins in vitro tion of endogenous respiration in S. griseus by into differing structural units depending on pH streptomycin, differential inhibitory activity of (15, 176). oxytetracycline on active and inactive strains of Although there are several common points in S. rimosus (392), effect of novobiocin on carbon, the pathways of secondary metabolism in such phosphorus (511), and nucleic acid (132) metab- diverse organisms as plants, fungi, and actino- olism in S. spheroides, as well as in vitro inhi- mycetes, the latter seem to utilize some of the bition by the antibiotic of succinate (512) and reactions preferentially or more frequently. pyruvatedehydrogenases. The polyene nystatin Among these, Turner (521) distinguishes the was reported to inhibit in vivo the activity of following: (i) incorporation of the whole intact glucose 6-phosphate dehydrogenase in the my- carbon skeleton of glucose (streptomycin) or a celium of the producer (513). One might infer portion of it (erythromycin) into a secondary from reports on interference of antibiotics with metabolite; (ii) Shikimate pathway for biosyn- particular enzymatic activities involved in the thesis of aromatic moieties of antibiotic mole- primary metabolism of the producing cells that cules (chloramphenicol, novobiocin); (iii) ex- at least part of the activities might be con- tensive employment of the pentose phosphate cerned with events associated with blocking cycle reactions for biosynthesis of nucleoside- vegetative growth. Inhibition per se, however, containing secondary metabolites; (iv) rather is not the equivalent of regulation. It can be extensive conversion (cycloserine) or incorpora- noted that, in one instance at least, the inhibition (gougerotin) of amino acids into secondary tion of respiration and glucose dehydrogenase metabolities; (v) extensive employment of poly- activity caused by accumulation of a polyene propionate and less extensive employment of antibiotic in the cell of S. roseoflavus var. ro- polyketide routes; and (vi) the rather frequent seofungini mutant appeared to be reversibly occurrence, among secondary metabolites, of abolished by illumination at a certain stage of terpene- and sterol-derived compounds. the actinomycete (499). Extreme cases of antibiotic interference with Prospects for Involvement of Antibiotics and the metabolism of producing actinomycetes are Pigments in Regulation of Metabolism probably exemplified by reports on mutations Earlier investigators noted, first of all, that caused by streptomycin (127) and some other accumulation of a certain antibiotic above a antibiotics (218, 519). critical level would inhibit or completely sup- The studies on the effects of antibiotics on the press vegetative growth of the producing orga- producing actinomycetes were, perhaps quite nism (540). Extensive use of this finding was naturally, dominated by the assumptions de- made in selection for strains with elevated ac- rived from results of pioneering investigations tivities. In might be also inferred from these employing "test" microbial systems for the elu- data that, in a physiologically heterogeneous cidation of target reactions for antibiotic action. population of actinomycete cells synthesizing On rather rare occasions, however, it has been an antibiotic, there would be a continuously possible to demonstrate that appropriate target increasing selective pressure favoring further structures, molecules, or reactions are absent activities of those cells that are already adapted or not employed by antibiotic-producing actino- to certain levels of antibiotic concentration. As mycetes; e.g., S. antibioticus (antimycin A pro- shown recently with S. griseus producing strep- ducer) has been shown (428) to be deficient in tomycin (64), however, the drug caused no the target for antimycin A in the respiratory change in the proportion of resistant variants, chain. The ribosomes of S. erythreus appeared which could be scored on streptomycin-contain- not to bind erythromycin to the extent of even ing media. the ribosomes of an erythromycin-resistant 504 KALAKOUTSKII AND AGRE BACTERIOL. REV. strain of E. coli (502). With the S. griseus regulation of genetic activity (135). In another strain producing macrotetralide, however, it study, the pigment associated with sporogene- appeared to be very difficult (251) to prove the sis in S. venezuelae was shown to induce sporu- hypothesis (571) involving the ionophore-type lation in vegetative hyphae of the same orga- antibiotics in ion transport in the producing nism several hours before the sporogenesis organism. started without addition of the pigment (458). There is some evidence that the kind of bio- The glucoside antibiotic aureovocin was shown logical activity expressed by antibiotics in the to be able to bind to the ribosomes of the pro- producing organism might differ from that ulti- ducing cell (370). A similar possibility was dem- mately expressed in the cells of "test" microor- onstrated for a melanoid-like pigment and ganisms (see also next section). For instance, spore ribosomes of S. granaticolor. The com- the protein biosynthesis of a chloramphenicol plexing of the pigment with sporal ribosomes producer appeared to be completely inhibited in appeared to be sufficiently stable: complexes vitro by the antibiotic. The accumulation of were not dissociated by treatment with ammo- antibiotic, however, seemed to induce profound nium chloride solutions or other routine purifi- changes in the cell permeability, thus blocking cation procedures (367). There is not much cer- the access of the drug to a sensitive target area tainty, however, in the answer to the question (342). of whether the binding of pigments actually As shown with S. aureofaciens and chlortet- occurs in vivo. racycline (166), a minor modification (removal Ribosomes of S. aureofaciens mycelium ap- of a water molecule in the "C" ring) of the peared to bind tetracycline molecules, the max- antibiotic molecule renders it five times more imal level of binding reaching 320 molecules toxic for the producer and less toxic for routine per ribosome. Most of the drug molecules were test microbes. Casual observations on profound bound reversibly with both ribosomal protein biological side effects of molecules of polyenes and RNA, whereas 1 molecule per ribosome (452) or of penicillin (270), and the suggested was bound irreversibly. Although the protein- enzymatic activities of polypeptides (523), synthesizing system of S. aureofaciens ap- might reveal further facets of their activities peared to be more resistant to tetracycline inhi- irrelevant to the suppression ofE. coli but rele- bition compared with that of other sensitive vant to their interaction with other molecules bacteria and even nonproductive strains of S. in the differentiating cell of the producing orga- aureofaciens, the antibiotic was inferred to nisms. play some regulatory role in metabolic changes The interesting hypothesis on the involve- during development (368, 369). Electron mi- ment of polypeptide antibiotics in regulatory croscopy of ribosomal preparations, obtained events during endospore formation is being during subsequent development stages of S. widely discussed and developed as applied to aureofaciens in submerged cultures, demon- Bacillus spp. (101, 210, 288, 442, 443, 447). Di- strated the increasing tendency of ribosomes rect involvement of antibiotics produced by ac- to form aggregates; this property correlated tinomycetes in the regulation of developmental with accumulation of tetracycline (334). Other events in these organisms has not been docu- studies point to the accumulation of nucleic mented. acid degradation products in the culture media Indirect evidence suggests that actinomycin as a likely consequence of tetracycline accumu- D, produced by S. antibioticus, might affect in lation by the producing strain (471, 574). a regulatory manner the RNA synthesis in the producing cell (489, 570). Some ofthe secondary Antibiotics and Their Components as Possible metabolites might probably enter (or localize Building Blocks in?) such sites within the producing cells that Industrial microbiologists usually categorize are directly concerned with transcription or the antibiotics into those (i) retained in the translation. mycelium; (ii) liberated into the medium; and Differences in buoyant density and melting (iii) accumulating both in the broth and myce- points of DNAs from spores and vegetative cells lium. Until recently, relatively little attention in S. venezuelae were already mentioned. It has been paid to locating the exact places was shown, however, that washing of spores within the actinomycete cell where antibiotics with ethanol or acetone prior to DNA extrac- are being synthesized and accumulated and tion resulted in DNA preparations that were how those that are poorly soluble (or almost almost identical as judged by above parame- insoluble) in water leave the cell and accumu- ters. It was suggested that the spore DNA is late in an aqueous milieu. Considerations ofthe complexed with a spore-specific antibiotic, the "late" consequences of antibiotic production phenomenon being directly concerned with the (e.g., killing or not killing potential enemies, VOL. 40, 1976 DEVELOPMENT IN ACTINOMYCETES 505 etc.) were, perhaps involuntarily, given prior- at the cell surface or in the medium (298, 558) or ity in speculations on the biological impacts of in granules within the hyphae or in the me- antibiotics. dium (flavofungin-type polyene; 296, 410). The Yet there is some information pointing to the crystals and granules accumulated in shaken suggestion that at least some antibiotics (more cultures apparently have no relation to the correctly, components ofthe' molecules) might normal morphogenetic process in streptomy- assume a structural role within the producing cetes. A different picture, however, was ob- cell, being incorporated into cell organelles. The tained from a surface culture of a mutant of streptidine moiety ofthe streptomycin molecule S. roseoflavus var. roseofungini with impaired was supposed to be incorporated into cell wall of differentiation. This mutant was blocked in S. griseus (489, 539). The presence of strepti- aerial mycelium formation and sporulation, dine in acidic hydrolysates of the mycelial wall but its ability to produce a pentaene-type anti- of S. griseus mutant strain 45-H was directly biotic, roseofungin, was simultaneously en- demonstrated (489). Streptomycin could be re- hanced severalfold (387). Grown in a solid leased on lysozyme treatment of this culture synthetic medium, the mutant cells were (31). These findings correlate with results from shown by electron microscopy to be surrounded Szabo's group on.the developmental character- by numerous thin structures having a tubular istics of active and inactive strains, as well as appearance, external diameter of about 20.0 on effect of C-factor on the former. N6N6-deme- nm, and an inner channel of about 8.0 nm in thyladenine, a component of the antibiotic diameter (70). These were isolated in pure puromycin, was shown to accumulate in aerial fraction (72) and later shown to be composed spores, but not in the vegetative mycelium ofS. mainly of the polyene antibiotic with the ad- alboniger, which produced puromycin during mixture of C0,1-0C8 fatty acids and several ions, sporulation (448). including Ca2+ and Mg2+ (415). The tubular Observations on the localization of antibiot- structures appeared to be completely soluble in ics in developing surface and submerged cul- acetone; when the relative proportion of water tures of active producers often contradict the in solutions was increased, they underwent a oversimplified expectations based on diffusion self-assembly or crystallization-like process against concentration gradients. and reappeared in the solutions (72). A struc- In the colonies of S. olivocinereus, an anti- tural similarity could be seen in both naturally biotic of the heliomycin type accumulates on produced and reassembled tubular structures the periphery of the advancing substrate myce- containing the polyene and those elementary hum and in minor quantity in the aerial myce- structures that can be revealed in the surface lium (531). Special "vesicles" were revealed in sheath ofseveral actinomycetes, including that the submerged culture ofS. aureofaciens which of the normally differentiated parent strain of produce tetracycline at the onset of active bio- S. roseoflavus var. roseofuingini. Tubular struc- synthesis. No such bodies were seen in cultures tures in the surface sheath of aerial mycelium of a low-producing strain. A very significant of the latter strain appeared to be remarkably portion of antibiotic activity was shown to be sensitive to brief acetone washings; they were associated with the vesicles, thought to be a almost completely dissolved. Acetone extracts mechanism for liberating antibiotic from the of the aerial mycelium, scraped from surface producing cell which is analogous with reverse cultures of this strain, yielded, on subjecting pinocytosis (297). them to dilution with water, a spectrum of dif- The cisternae and other cytoplasmic mem- ferent structures, some of which were quite brane-associated bodies filled with electron- reminiscent of those seen on the aerial myce- dense material and found in the periplasm ofS. lium surface (71). Later on, initial "tubular" vinaceus were supposed to represent main sites structures were also recovered from such ex- of viomycin biosynthesis (298). tracts (498). The conditions that led to self- aAn interesting picture presented by cultures assembly of differing structures were defined ofactinomycetes producing the water-insoluble (497) and the similarity of their chemical com- antibiotics of the polyene family. Submerged position to the original tubules was demon- cultures of these actinomycetes accumulate an- strated (414). The above facts, although still tibiotics in the form ofcystals located at the cell fragmentary, may be taken to support the hy- surface or in the medium (298, 558) or in gran- pothesis that a pentaene macrolide antibiotic, An interesting picture is presented by cul- superproduced by a developmental mutant and tures of actinomycetes producing the water- excreted into the solid medium, undergoes insoluble antibiotics ofthe polyene family. Sub- there a crystallization process, which mimics merged cultures of these actinomycetes accum- the events involved in the formation of surface ulate antibiotics in the form of crystals located sheath in a normally differentiating parent 506 KALAKOUTSKII AND AGRE BACTERIOL. REV.

strain. In the latter case, however, one pre- surface sheath structure at the onset of autoly- sumes that limited amounts of antibiotic are S's. transported to the cell surface at a certain It is tempting to speculate also that incorpo- stage (emergence of aerial hyphae) and uti- ration of a polyene antibiotic into a protective lized there for construction of sheath compo- film on the generative cells of actinomycetes nents, with a likely participation of self-as- might be favored by natural selection, since it sembly phenomena in the formation of sheath is the mycelial fungi that are likely to compete subunits. with actinomycetes in a heterophasic environ- An independent study (313) demonstrated re- ment such as the soil. The structurally incorpo- versible reconstruction of surface unit struc- rated surface-exposed antibiotic would thus be tures on spores ofS. streptomycini, the process looked upon not as a weapon for unlimited being independent of the viability of spores but chemical warfare, but more as a shield for dependent upon temperature and relative hu- limited protection against potential aggressors. midity of the milieu. Some properties of polyene macrolide anti- ACKNOWLEDGMENTS biotics would seem to be compatible with the We wish to thank David Gottlied, Department of hypothetical role discussed for them above. (i) Plant Pathology, University of Illinois, for encour- Members of the polyene macrolide family of aging us to write the review. Thanks are due to J. C. antibiotics are produced by an unprecedented Ensign, Department of Bacteriology, University of variety of species within the genera Strepto- Wisconsin, and T. Cross, Bradford University, myces, Streptoverticillium, and Chainia (185). U.K., for letting us see some of their results prior to publication. We were supported by the USSR Acad- (ii) Some anomalies in the behavior of these emy of Sciences. antibiotics in solution (185), as well as their "all or none" effect on susceptible organisms (186), LITERATURE CITED would suggest a definite tendency to form supramolecular structures of a micellar nature. 1. Adams, J. N. 1966. Studies on the fragmentation of Nocardia erythropolis in liquid me- The surface-active nature of polyene molecules dium. Can. J. Microbiol. 12:433-441. is directly related to their structure; conforma- 2. Agate, D. D. 1966. Suitability of the high tem- tional and hydration-level changes in these perature pre-incubation method for isolation molecules would be reasonably expected to ofpectinolytic actinomycetes. J. Indian Inst. have a significant effect on their behavior at Sci. 48:98-101. interface. Interaction with ions (those that ac- 3. Agre, N. S. 1962. 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