Zeitschrift für Allgemeine Mikrobiologie: Volume 24, Number 8 [Reprint 2021 ed.] 9783112565681, 9783112565674

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ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS VOLUME 24 • 1984 • NUMBER 8

AKADEMIE-VERLAG • BERLIN ISSN 0044-2208

Z. allg. Mikrobiol., Berlin 24 (1984) 8, 5 0 5 - 5 8 4

EVP 2 0 , - M

Instructions to Authors 1. The journal publishes original papers, short communications, and review articles. Submission of a paper implies that it has not been published or has not been submitted for publication elsewhere. Manuscripts should be sent in duplicate with one set of the original illustrations to the Editor-in-Chief: Prof. Dr. U. Taubeneck, DDR-6900 Jena, Beutenbergstr. 11. 2. Manuscripts should preferably been written in English but may also be submitted in German. They should be typed double-spaced and be accompanied by a title page comprising: name and address(es) of the institution(s) where the work was done, title of the paper, and the complete name(s) of the author(s). Each paper must begin with a brief summary in English. Original papers should be divided into sections headed: Introduction, Materials and Methods, Results, Discussion, Acknowlegements, and References. A short title for use as running head should be provided. The exact mailing address of the author to whom correspondence, reprint requests etc. are to be addressed must be given at the end of the paper. 3. Tables, illustrations, and descriptive legends of the illustrations must be submitted on separate sheets. Each table should have a heading. The size of illustrations should not exceed the maximum printing area of 12 cm X 19 cm or 4.7 inches X 7.5 inches, respectively. 4. Literature citations in the text should be by author and year of publication. If there are more than two authors, only the first should be named, followed by "e< a l " . References should include only publications cited in the text. They should be given in alphabetical order: a) Books:

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ZEITSCHRIFT FÜR ALLGEMEINE MIKRO- BIOLOGIE AN

INTERNATIONAL

JOURNAL

ON

MORPHOLOGY, PHYSIOLOGY, GENETICS,

H E R A U S G E G E B E N VON

W. Fritsche, Jena G. F. Gause, Moskau 0 . Hoff mann-Ostenhof, Wien A. A. Imseneckii, Moskau R. W. Kaplan, Frankfurt/M. F. Mach, Greifswald 1. Malek, Prag W. Schwartz, Braunschweig C. Weibull, Lund

UNTER D E R CHEFREDAKTION VON

U. Taubeneck, Jena

A N D ECOLOGY OF MICROORGANISMS U N T E R MITARBEIT VON

J . H. Becking, Wageningen H. Böhme, Gatersleben M. Girbardt, Jena S. I. Kusnecov, Moskau 0 . Necas, Brno C. H. Oppenheimer, Port Aransas N. Pfennig, Göttingen I. L. Rabotnova, Moskau

REDAKTION

U. May, Jena VOLUME 2 4 • 1984 • N U M B E R 8

AKADEMIE-VERLAG • BERLIN

R

-

Ma

y> J e n a

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Erscheinungstermin: September 1984. Bestellnummer dieses Heftes: 1070/24/8. © 1984 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 75218

Zeitschrift für allgemeine Mikrobiologie 24 (1984) 8, 507 — 514

(Ain-Shams University, Faculty of Agriculture, Department of Microbiology, Shobra, Cairo, Egypt, and 1 ) Institut für Mikrobiologie der Universität Göttingen, D-34 Göttingen/FRG.)

Characterization of two strains of Tobacco Mosaic Virus by biological, biochemical, immunological and electron microscopical techniques A . M. E . EL-AHDAL, F . M A Y E E 1 ) , S. I. E L A F I F I a n d E . K .

(Eingegangen

am

ALLAM

4.1.1984)

Two strains of Tobacco Mosaic Virus (TMV) isolated from tomato plants were characterized by biological, biochemical, immunological and electron microscopical techniques including immunoelectron microscopy. The biological tests indicated, on the basis of symptoms observed on differential hosts, differences between the two strains. However, detailed studies with the other techniques mentioned above did not reveal significant differences.

The tomato is an economically important crop in Egypt. Poor growth and low yield and quality of tomatoes were a common complaint among the growers in the last few years. This has been attributed to virus diseases. A B O - E L N A S E R ( 1 9 7 6 ) showed that not only Tomato Mosaic Virus, but also Tobacco Mosaic Virus might infect tomato plants. In a number of cases analyzed ( A B O - E L N A S E R 1 9 7 6 ) , it was difficult to decide whether an isolated virus in fact belonged to the Tobacco Mosaic Virus group, or to the Tomato Mosaic Virus group, or even to a third group with less clearly defined biological symptoms. The purpose of the present investigation is to examine the differences between one strain of Tobacco Mosaic Virus (TMV I) and one virus strain (TMV II), isolated from a tomato plant, as TMV I, but belonging to the third of the above mentioned groups. The analysis was performed with a variety of techniques including examination of biological symptoms, electron microscopy of infected leaves by ultrathin sectioning, and electron microscopical, biochemical and serological analyses of isolated virus particles and their components. Materials and methods Source of virus strains: The two strains originally isolated from tomato plants grown in Egypt were obtained from the Virus Laboratory of the Faculty of Agriculture, Ain-Shams University, Cairo, Egypt. Both strains were biolocically purified by the local lesion technique using Nicotiana glutinosa; they were propagated on Nicotiana tabacum var. SAMSON. Differential hosts: Plants from two families represented by six species were inoculated with the two virus strains. These plants were Chenopodium amaranticolor, Nicotiana glutinosa, N. rustica, N. tabacum var. W H I T E B T J K L E Y , N. sylvestris and N. paniculata. Culture of virus: N. tabacum var. SAMSON was used as systemic host for both virus strains. Three weeks after inoculation, the infected plants were collected. Unless otherwise stated, the harvested leaves were stored at —20 °C until required. Virus purification: The infected leaves were ground in a blender with 2 ml of 0.1 M sodium phosphate buffer (pH 7.2) per g of leaves. The resulting juice was filtered through cheese cloth and clarified by centrifugation at 10000 X g for 10 min at 4 °C. Virus from the clarified sap of each strain was sedimented by ultracentrifugation at 110000 X g for 1 h at 4 °C. The pellets were resuspended in 0.1 m sodium phosphate buffer (pH 7.2) and again centrifuged at 10000 X g. The complete procedure was repeated once. The final pellets were resuspended in 0.1 M sodium phos33»

508

A . M . E . E L - A H D A L et

al.

phate buffer (pH 7.2) and stored at —20 °C. The infectivity assay for purified virus suspensions of the two strains under investigation was carried out using Nicotiana glutinosa as local-lesion host. Density gradient centrifugation: Aliquots of purified suspensions of each of the two virus strains were layered on top of sucrose density gradients (step gradients) formed from 4 ml 10%, 7 ml 20%, 7 ml 30% and 7 ml 40% succrose in tridistilled water in polycarbonate tubes. The samples were centrifuged a t 34000 x g for 2.5 h at 4 °C. The gradients were fractionated by collecting drops from a hypodermic needle inserted through the bottom of the tubes. Nucleic acid preparation: The RNA from the two virus strains was prepared by repeated phenolextraction. I t was resuspended in 0.1 M Tris-HCl, p H 8.5, containing 0.01 M EDTA, and stored at

- 2 0 °C.

Degradation of virus proteins: The virus proteins were degraded by the acetic-acid method

(FRAENKEL-CONRAT 1 9 5 7 ) .

Determination of the molecular weight of the coat proteins: The molecular weights of the coat proteins of TMV I and TMV I I were determined by sodium dodecylsulfate Polyacrylamide gel e l e c t r o p h o r e s i s ( W E B E R et al.

1972).

Analysis of amino acid composition of the coat proteins: The analysis of the amino acid composition of the coat proteins of TMV I and TMV I I was performed by vising an automatic amino acid analyzer ( B I O T R O N I C , Munich/FRG) according to MOORE and S T E I N ( 1 9 6 3 ) . The samples were prepared by hydrolysis of the proteins in 6 N HCl. The hydrolysis was conducted a t 110 °C for 20 h in vacuum sealed tubes. The evaluation of the resulting amino acid patterns was done by using reference chromatograms. Rabbit immunization and isolation of IgG antibodies: One rabbit was used for each virus strain. Pre-immune serum was taken as control. Two weeks after the first injection of virus material, a second injection was made. Bleeding was carried out two weeks after the second injection. The specific antibodies were precipitated from the serum according to the procedure described by Bo W I E N and M A Y E R (1978). Extraction of the specific IgG antibodies was done using a DEAE Sephadex A50 column. The fractions were tested regarding their immunological interaction with the virus strains TMV I and TMV I I by using the O U C H T E R L O N Y double immunodiffusion technique ( O U C H T E R L O N Y 1949). After concentration, the specific IgG antibodies were stored at —20 °C. Immunoelectron microscopy: For the formation of virus-IgG antibody complexes, solutions of TMV I or TMV I I were mixed with IgG antibody solutions. The resulting solutions of complexes were made free from unreacted components (virus, antibodies) and large aggregates by sucrose density gradient centrifugation. The resulting fractions were controlled by the electron microscopic negative-staining technique. Electron microscopic techniques: Negative staining of isolated virus particles and of virus-lgG antibody complexes was performed as described by V A L E N T I N E et al. (1968) and B O W I E N and M A Y E R (1978) with 4 % (w/v) uranylacetate (pH 4.9) dissolved in bidistilled water. Preparation of isolated RNA molecules for electron microscopy was done according to the procedure described by L A N G and M I T A N I (1970), but modified by using formamide as additional component in the spreading solution. MS-2 RNA was used as standard RNA for calibration. Metal shadowing of spread RNA was done with the rotation technique with a platinum-iridium (80/20 w/w) alloy. Length measurements of isolated RNA molecules for molecular weight determination were performed with electron micrographs taken a t calibrated magnifications, with a P R O Längenmesser (BRÜHL, Nürnberg/FRG). Fixation and ultrathin sectioning of plant material were performed according to the procedure described by B E H N and A R N O L D (1974), with S P U R R low viscosity medium ( S P U R R 1969) for embedding. Post-staining of ultrathin sections was done with lead citrate (VENABLE and COGGESHALL 1965).

Electron micrographs were taken with a P H I L I P S EM hoven, The Netherlands) a t calibrated magnifications.

301

electron microscope

(PHILIPS,

Eind-

Results and discussion Biological

studies

Differential hosts as mentioned in Section "Materials and Methods" were used. Their reactions are presented in Table 1. From these data it can be deduced that there are biological differences between TMV I and TMV II both in type and in severity of symptoms, and that TMV I and TMV II, according to the grouping of TMV strains based on the differential-host method, are different strains of TMV.

509

Characterization of Tobacco Mosaic Virus

Table 1 The reactions of the differential hosts to the inoculation with two strains (TMV1, TMVII) of Tobacco Mosaic Virus Differential hosts

External symptoms with TMV I

TMV II

Chenopodium amaranticolor

local lesions

local lesions

Nicotiana glutinosa

local lesions

local lesions

Nicotiana rustica

yellow spots, necrotic spots, mosaic, top necrosis

local lesions

Nicotiana tabacum var. WHITE BUBLEY

necrotic local lesions, stem necrosis

mosaic

Nicotiana sylvestris

no symptoms

small local lesions

Nicotiana paniculata

yellow spots, mosaic, crinkling

yellow spots, necrotic local lesions, crinkling, necrosis

Fine structure analysis of isolated TMV I and TMV II

particles

From electron micrographs similar to Figs. 1 and 2, length and diameter measurements of the two virus particle types were performed. Full length virus particles showed a length of 300 nm. For both virus particle types, a diameter of 18 nm was determined. Figs. 1 and 2 show examples of micrographs typical for TMV I and TMV I I . Both particle types exhibited the characteristic dark central line and a fine striation indicating the helical symmetry of the TMV capsid. A preliminary evaluation by lightoptical diffraction (data not shown) did not reveal any significant differences between T M V I and T M V I I ; both showed a pitch of 2 . 3 nm (MANDELKOW et al. 1 9 8 1 ) . These data indicate that significant structural differences between TMV I and TMV I I could not be observed. Molecular weight of the coat proteins of TM V I and TM V II SDS polyacrylamide gel electrophoresis revealed a molecular weight for the coat proteins of TMV I and TMV I I of 17500. This value is in accordance with known data (TSUQITA 1 9 6 2 ) . Differences between T M V I and T M V I I could not be observed. Amino acid composition of the coat proteins of TMV I and TMV

II

Table 2 presents the values measured for the amino acid composition of the coat proteins of TMV I and TMV II. Significant differences are not obvious. When compared to published numbers of amino acids for the TMV coat protein, the number of 156 amino acids both for TMV I and TMV I I is lower than that commonly found (158, found by GIBBS and MCINTYKE 1 9 7 0 ) . However, as also shown by these investigators, this number may differ from strain to strain, ranging from 155 to 158. Lower numbers than 158 are usually attributed to defective mutant strains of TMV (TSUQITA and FRAENKEL-CONRAT 1 9 6 2 ) . The TMV strains have been classified into four groups grossly differing in the contents of methionine residues; i.e. the group members contain 0, 1, 2 or 3 methionine residues per coat protein molecule. According to this classification, TMV I and TMV I I belong to the first of these groups. Histidine was also not detected. Again, no significant differences between TMV I and TMV I I could be found.

510

Al*«:.

f

2

A. M. E. El-Ahdal et al.

511

Characterization of Tobacco Mosaic Virus

•4 Pigs. 1 and 2 Isolated virus particles, negatively stained with uranyl acetate. Fig. 1, TMV I ; Fig. 2, TMV I I . Full length rods and shorter, probably broken particles are visible. Fine striations indicating the presence of helically ordered protein subunits, and a central dark line representing a central channel can be seen Fig. 3 Serological cross-reactions (OUCHTERLON Y tests) between TMV antigens and TMV antisera. The central wells contained anti-TMV I (a) or anti-TMV I I (b) serum. 1, TMV I antigen; 2 TMV I I antigen. The patterns of the major precipitation lines indicate that TMV I and TMV I I have identical serological properties within the limit of the resolution of this technique Fig. 4 Results of immunoelectron microscopy applied to the analysis of serological relationships between TMV I and TMV I I . Negative staining with uranylacetate. Inset: Isolated IgG antibody, shown at high magnification (bar indicates 25 ptm). a) Reaction between IgG I and TMV I b) Reaction between IgG I I and TMV I I c) Cross-reaction between IgG I I and TMV I d) Cross-reaction between IgG I and TMV I I Arrows indicate complexes formed by virus particles and IgG antibodies Fig. 5 Isolated denatured RNA molecules prepared for electron microscopy by the spreading technique, a) RNA from TMV I ; b) RNA from TMV I I Table 2 Amino acid composition of the two Tabacco Mosaic Virus strains TMV I and TMV I I (Moles amino acids per Mol protein subunit) Amino acid

TMV I

TMV I I

alanine arginine aspartic acid/asparagine cysteine glutamic acid/glutamine glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine valine

14.4 10.3 18.2 0.6 19.1 6.5

14.0 9.9 17.8 0.7 18.6 6.1

14 10 18 1 19 6

—

—

—

7.2 11.7 1.9

6.6 12.0 1.9

7 12 2

—

—

11.5 6.9 14.7 14.7 4.5 3.8 14.1

9.7 6.9 13.6 13.7 3.8 3.7 13.5

Immunodiffusion

probable composition of TMV I and TMV I I

—

10 7 14 14 4 •4 14 156

experiments

Fig. 3 demonstrates serological relationships between TMV I and TMV I I (for details, see figure legends). From the patterns of the major precipitation lines it can be seen

512

A . M . E,!. EL-AHDAL et

al.

Characterization of Tobacco Mosaic Virus

513

•4 Figs. 6 to 9

Ultrathin sections through cells of infected Nicotiana tabacum leaves.

Fig. 6 Large virus inclusion (V) (TMV I) surrounded by the tonoplast (T), in close neighbourhood to chloroplasts (CH) with well preserved fine structure. The virus particles are partially ordered. Fig. 7 As Fig. 6, but TMV I I . Fig. 8 Virus inclusion (V) (TMV I) in close neighbourhood to a mitochondrium (M) and to a chloroplast (CH). Ribosomes (R) and the surrounding tonoplast (T) with some adhering virus particles (VP) in the vacuole are visible. Fig. 9 a) and b), Virus inclusions (V) (TMV I). Bundles of angle-layered virus particles located in the cytoplasm, close to the cell wall (W) and to chloroplasts (CH), are visible

that both virus strains have identical serological properties within the limit of the resolution of this technique. Monoclonal antibodies were not used. Immunoelectron

microscopy

As expected from the results of the immunodiffusion experiments, TMV I and TMV I I also showed cross-reactions when negatively stained samples of various combinations of TMV I and TMV I I with anti-TMV I I or anti-TMV I-IgG. antibody preparations were analyzed (Fig. 4; for details see figure legends). These observations also support the view that TMV I and TMV I I are closely related if not identical virus strains. Molecular weight determinations

of virus RNA

Isolated denatured RNA molecules from TMV I and TMV I I were measured by electron microscopy (Fig. 5). Full length RNA molecules, when compared with the length of MS-2 RNA prepared under identical conditions, allowed a calculation of their molecular weight on the basis of the known molecular weight of MS-2 RNA (1.2 X 10® d ; F I E R S et al. 1 9 7 6 ) . T M V I a n d T M V I I R N A m o l e c u l e s b o t h h a d

a

molecular weight of 2.1 X 106 d. This value agrees well with known data (LEHNINGER 1975). Again, differences between TMV I and TMV I I could not be observed. Cytology of virus-infected

host cells

Figs. 6 to 9 show examples of ultrathin sectioned infected leaves of host plants (for details, see figure legends). Careful examination of a variety of samples from infected plants taken at different times after inoculation did not reveal any reproducible differences regarding changes in the cytology of the infected cells or the location of virus particles and their aggregates, when plants infected by TMV I or TMV I I were compared. However, a comparison of the biological differences mentioned above on the ultrastructural level demonstrated differences in the severity of infection of cells in the respective leaf areas. In conclusion, it can be stated that, on the basis of definite biological differences between TMV I and TMV I I on differential hosts, these two virus strains can be distinguished as two different TMV strains. A number of other investigators (HEROLD a n d MUNJE 1 9 6 7 , CHESSIN et al. 1 9 6 7 , RAA a n d R E D D Y 1 9 7 1 , FELDMAN a n d ORE-

MIANER 1972, VELA 1972) had used the same differential hosts in order to identify new virus strains isolated from tomato plants or other infected hosts. However, all other characteristic aspects analyzed in the present investigation did not reveal any significant differences between TMV I and TMV I I . I t has to be concluded that the techniques used, though established experimental methods, were not sufficient to detect structural, biochemical or serological differences. This situation is not unique (GRANETT and SHALLA 1969). Besides sequencing techniques, improved serological proce-

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al.

dures, especially the application of monoclonal antibodies, might give further informations for a final understanding of the correlations of infectivity and virus structure. Acknowledgements W e a r e g r a t e f u l to M. B A H N W E G for technical assistance. This investigation was m a d e possible b y t h e Channel p r o g r a m m e established b e t w e e n E g y p t a n d t h e F e d e r a l R e p u b l i c of Germany.

References * A., 1 9 7 6 . Biological a n d chemical d i f f e r e n t i a t i o n of d i f f e r e n t strains of tobacco mosaic virus. P h D Thesis, F a c u l t y of Agricultire, Ain-Shams University, Cairo, E g y p t . BEHN, W. u n d ARNOLD, C. G., 1974. Die W i r k u n g von S t r e p t o m y c i n u n d N e a m i n auf die Chloroplasten- u n d M i t o c h o n d r i e n s t r u k t u r e n von Chlamydomonas reinhardii. P r o t o p l a s m a , 82, 77—89. B O W I E N , B. a n d M A Y E R , P . , 1 9 7 8 . F u r t h e r studies on t h e q u a t e r n a r y s t r u c t u r e of D-ribulose 1,5-bisphosphate carboxylase f r o m Alcaligenes eutrophus. E u r . J . Biochem., 88, 97 — 107. C H E S S I N , M . , Z A I T L I N , M . a n d SOLBERG, R . A . , 1 9 6 7 . A n e w s t r a i n of tobacco mosaic v i r u s f r o m Lychnis alba. P h y t o p a t h o l . , 57, 452—453. F E L D M A N , J . M. a n d O R E M I A N E R , S . , 1972. An u n u s u a l s t r a i n of tobacco mosaic v i r u s f r o m p e p p e r . P h y t o p a t h o l . Zeitschrift, 75, 2 5 0 - 2 6 7 (Rev. P h y t . P a t h o l . 52, 3499).

ABO-EL NASEE, M .

FIERS, W . , CONTERERAS, R . , DUERINCK, F . , HAGEMAN, G . , ISERENTAT, O . , MERREQAERT, J . , M I N J A N , W . , M O L E M A N S , F . , R A E Y M A E X E R S , A . , VAN D E N B E R G H E , A . , V O L E C K A E R T , G . a n d Y S E B A E R T , M . , 1 9 7 6 . Complete nucleotide sequence of bacteriophage M S 2 - R N A : P r i m a r y a n d secondary s t r u c t u r e of t h e replicase gene. N a t u r e , 2 6 0 , 5 0 0 — 5 0 6 .

FRAENKEL-CONRAT, H., 1957. D e g r a d a t i o n of tobacco mosaic v i r u s w i t h acetic acid. Virol., 4, 1-4. FRAENKEL-CONRAT, H., 1962. I n f e c t i v i t y of tobacco mosaic v i r u s f r o m tobacco leaves t r e a t e d w i t h 2-thiouracil. Virol., 17, 9—21. GIBBS, A. J . andMclNTYRE, G. A., 1970. A method for assessing the size of a protein particle from its composition: Its use in evaluating data on the size of the protein subunit of plant virus particles. J . gen. Virol., 9, 51 — 69. GRANETT, A. L. a n d SHALLA, T. A., 1969. Cytological d i s p a r i t y b e t w e e n t h e serologically indistinguishable isolates of tobacco mosaic virus. P h y t o p a t h o l . , 55), 1028. H E R O L D , F . a n d M I T N J E , K . , 1 9 6 7 . An u n u s u a l k i n d of crystalline inclusion of tobacco mosaic virus. J . gen. Virol., 1 , 3 7 5 — 3 7 8 . L A N G , D. a n d M I T A N I , M . , 1 9 7 0 . Simplified q u a n t i t a t i v e electron microscopy of biopolymers. Biop o l y m e r s , 9, 3 7 3 - 3 7 9 . L E H N I N G E R , A . L . , 1 9 7 5 . Biochemistry. W o r t h P u b l i s h e r s Inc., New M A N D E L K O W , E . , S T U B B S , G . a n d W A B R E N , S., 1 9 8 1 . S t r u c t u r e of t h e

York. helical aggregates of tobacco

mosaic v i r u s protein. J . Mol. Biol., 152, 375 — 386. MOORE, S. a n d STEIN, W. H., 1963. Chromatographic d e t e r m i n a t i o n of amino acids b y t h e use of a u t o m a t i c recording e q u i p m e n t . I n : COLOWICK, S. P . a n d KAPLAN, N. 0 . (Ed.), Methods in E n z y m o l o g y VI. Academic Press, New Y o r k — L o n d o n —San Francisco, p p . 819 — 831. O U C H T E R L O N Y , Ö . , 1 9 4 9 . A n t i g e n - a n t i b o d y reactions in gels. Acta p a t h o l . microbiol. Scand., 2 6 , 507-515. RAA, M. H . R .

a n d R E D D Y , D . V . R . , 1 9 7 1 . A n e w s t r a i n of tobacco mosaic virus on Lycopersicon esculantum. I n d i a n P h y t o p a t h o l . , 24, 672 — 678. SPTJRR, A. R., 1969. A low-viscosity epoxy resin embedding m e d i u m for electron microscopy. J . U l t r a s t r u c t . Res., 26, 31—43. TSUQITA, A. a n d FRAENKEL-CONRAT, H., 1962. T h e p r o t e i n s of m u t a n t s of tobacco mosaic virus. Classification of spontaneous a n d chemically evoked strains. J . Mol. Biol., 5, 292 — 300. VALENTINE, R . C., SHAPIRO, B . M . a n d STADTMAN, E . R . , 1968. R e g u l a t i o n of g l u t a m i n e s y n t h e -

tase. X I I . Electron microscopy of t h e enzyme f r o m Escherichia coli. Biochem. J . , 7, 2 1 4 3 — 2 1 5 2 . VELA, L., 1972. A s t r a i n of tobacco mosaic v i r u s f r o m Digitalis thaps. Microbiologia Espanola, 25, 2 1 1 - 2 2 4 (Rev. P l a n t P a t h o l . 53, 811). V E N A B L E , J . H . a n d COGGESHALL, R . , 1 9 6 5 . A simplified lead c i t r a t e s t a i n for use in electron microscopy. J . Cell Biol., 25, 407—408. W E B E R , K . , P R I N G L E , J . R . a n d OSBORN, M., 1 9 7 2 . M e a s u r e m e n t of molecular w e i g h t by electrophoresis on SDS-acrylamide gels. I n : COLOWICK, S. P . a n d KAPLAN, N. 0 . (Ed.), Methods in Enzymology X X V I , Academic Press New Y o r k — L o n d o n — S a n Francisco, p p . 3 — 2 7 . Mailing address: Prof. D r . F. MAYER, I n s t i t u t f ü r Mikrobiolgie d e r U n i v e r s i t ä t , Grisebachstr. 8, D-3400 Göttingen

Zeitschrift für allgemeine Mikrobiologie 24 (1984) 8, 515-523

(Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, Jena)

Modification by genetic changes of the pleiotropic interference of butyrolactone-type autoregulators with differentiation of Streptomyces griseus U . GRÄFE, I. EEITT, G. REINHARDT, D . K R E B S a n d W . F .

FLECK

Dedicated to Professor FBIEDRICH BEEGTER on occasion of his 60th birthday (Eingegangen am 4.1. 1984) Two series of aerial-mycelium-negative (Amy"), anthracycline-nonproducing (Ant~) mutants were obtained from ancestral Amy + Ant + strains of S. griseus-. a) derivatives represented by the met~ strain 39 which could not differentiate although they were still producing both the butyrolactonetype autoregulator 1_ and NADP-glycohydrolase, and b) mutants whose incapability to form spores and anthracycline pigments was apparently caused by the loss of autoregulator production. These latter mutants responded to the addition of 1 or the naturally occurring dihydro derivative 2 with complete or at least partial reconstitution of differentiation-associated functions. All of the b)-type mutant strains exhibited similar biochemical alterations in the presence of 1 or 2 regardless of the presence of additional genetic changes in the primary metabolism. Two mutants, however, displayed an altered pattern of secondary product formation. In submerged cultures the major biochemical changes observed in presence of 1 (or 2) were an increase of the lipid level in the mycelium, an alteration of the lipid composition, and a stimulation of neutral proteinase production. All of the blocked autoregulator-negative mutants were discernible from the ancestral strains and strain 39 by their lack of NADP-glycohydrolase production. This suggested the existance of a common genetic locus or a common pleiotropic regulator gene controling both gene functions. Present ideas concerning the role of butyrolactone-type autoregulator 1 as a pleiotropic effector molecule interacting with development of S. griseus are summarized in a hypothetical scheme. I n our recent p a p e r s we have reported that the formation of both aerial mycelium (Amy) and anthracycline pigments (Ant) by two strains of Streptomyces griseus required the presence of the endogenously produced, autoregulatory molecule 2-(6'methylheptanoyl)-3-hydroxymethyl-4-butanolide 1 or its derivatives which were altered in the sterochemistry at positions C2 and C3, in the length of the side chain a t C2 and in the presence of an L ' - O H group (2; E R I T T et al. 1982, 1984, G R A F E and E E I T T 1983, G R A F E et al. 1982 a, 1983). Thus, blocked A m y " A n t " m u t a n t s 86 and 15 derived from the above parental strains were induced to form both spores and the antibiotic leukaemomycin in the presence of exogenously added 1 (R = C 7 H 1 5 ) or its

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d e r i v a t i v e s 2 (cis or trans relative c o n f i g u r a t i o n ; R = C 7 H 1 5 , C 6 H 1 3 or C 8 H 1 7 ). R e m a r k a b l e morphological a n d biochemical c h a n g e s occurred c o n c o m i t a n t w i t h t h i s induct i o n ( E R I T T et al. 1 9 8 4 , G R A F E et al. 1 9 8 4 ) . I n detail w e o b s e r v e d a s t i m u l a t i o n of lipid s y n t h e s i s , a n a l t e r a t i o n of t h e lipid c o m p o s i t i o n a n d a partial r e c o n s t i t u t i o n of proteinase p r o d u c t i o n w h e n 1 or 2 w a s a d d e d t o the culture m e d i u m . I n t h e p r e s e n t s t u d y w e i n v e s t i g a t e d t h e biochemical properties of additional a e r i a l - m y c e l i u m - n e g a t i v e , a n t h r a c y c l i n e - n o n - p r o d u c i n g m u t a n t s in order to characterize their response to 1 or 2 in comparison w i t h t h a t of strains 86 a n d 15 described in our recent paper (GRAFE et al. 1984) a n d to elucidate t h e r e b y t h e general f e a t u r e s of t h e pleiotropic interference of a u t o r e g u l a t o r y b u t y r o l a c t o n e s w i t h t h e d e v e l o p m e n t of 8. griseus strains. Materials and methods Chemicals: NADP was supplied by Arzneimittelwerk Dresden (GDR), azocasein, phenylmethylsulfonylfluoride and standards of f a t t y acid methyl esters were purchased from SERVA (FRG), phosphatidyl-ethanolamine and cardiolipin from KOCH LIGHT (England). Strains and cultivation conditions: The anthracycline-producing, aerialmycelium-forming, parental strains 8. griseus J A 5142 and JA 3933 which had very similar properties were obtained from the strain collection of the Central Institute of Microbiology, Jena. The blocked Amy~Ant~ mutants 39, 86, 16, 6 and others were isolated after mutagenic treatment (NTG, UV, X-rays) by using the screening procedure described by ERITT et al. (1982). All cultivations were started from a stock of lyophilized conserves. Propagation and maintenance of strains were performed as described (ERITT et al. 1982, GRAFE et al. 1984) using the A153 medium. Inocula were prepared by seeding lawns of 10-days surface mycelia grown on oatmeal agar (ERITT et al. 1982) into HICKEY-TRESNER'S medium (g/1): dextrin 10; yeast extract (DIFCO) 2; CoCL • 7 H»0 0.2; p H 6.5. 48 hours inocula were transferred to 80 ml of the same medium in 500 ml flasks. All cultivations were carried out on rotary shakers (240 r.p.m., 29 °C). In the appropriate experiments the autoregulatory effector 1 (2 (lig/ml) was added a t zero time. I t was prepared from fermentor cultures of the parental strain 8. griseus J A 5142 according to GRAFE and ERITT (1983). The purity was about 20% when the biological activity towards the indicator strain 86 was taken as a measure (ERITT et al. 1982). Effector 2 (R = C 7 H 15 , trans isomer) was prepared from 8. viridochromogenes (lO^g/ml) and displayed the same effect on cytodifferentiation as 1 (GRAFE et al. 1982). Analytical procedures: Mycelial dry weight was estimated gravimetrically. Protein concentration in mycelial extracts was measured by LOWRY'S method (LOWRY et al. 1951). Disintegration of mycelia was carried out in Tris buffer p H 7.4 by a 3 X 30 sec sonic treatment (0 °C) with a labsonic 1510 instrument (BRAUN, FRG). For enzyme assays the cell debris were sedimented a t 22000 X g for 15 min and the supernatant was used for the assays. The lipid material was extracted from mycelium samples with CHCl 3 /methanol (2:1 v / v ; t w o times), the extract was washed with water, dried and weighted. The methyl esters of f a t t y acids were obtained by the treatment with 2.5% HC1 in methanol. T.l.c. of lipids was carried out on silica gel sheets (precoated,MERCK) with CHCl 3 /methanol/H 2 0 (65:25:4, v/v) as the solvent. Standards of phospholipids were run in parallel. The presence of ornithinolipid was confirmed as described earlier (GRAFE et al. 1984). Gas-chromatography of f a t t y acid methyl esters was performed with a gas chromatograph GCHF 18:3 (VEB CHROMTROV Berlin, GDR) equipped with a flame ionization detector. Glass columns (3 m x 3 mm i.d.) were filled with 3% (w/w) DEGS on Chromosorb G (80—100 mesh, SERVA) and operated isothermically a t 165 °C. The injector temperature was 200 °C and nitrogen was used as the carrier gas (35 ml/min). Peaks were identified by their retentation times as compared with those of authentic standards. Enzyme assays: NADP-glycohydrolase (EC 3.2.2.5) was measured according t o VORONINA et al. (1978) a n d t h e n e u t r a l a n d s e r i n e p r o t e i n a s e s ( E C 3.4.2.1) a c c o r d i n g t o GINTH-

HER (1979). The latter assay made use of the hydrolysis of azocasein (0.1% in 0.2 M Tris buffer; 1 m i CaCl2). Serine proteinase activity was calculated from tests with samples which had been treated with the inhibitor phenylmethylsulfonylfluoride (10~3 M, 30 min preincubation). The antibiotic concentration was assayed by the agar diffusion method using Bacillus subtilis ATCC 6633 as the test organism. Results Properties

of blocked mutants

and the effect of autoregulator

1

As it will be s h o w n elsewhere in detail, w e o b t a i n e d t w o series of aerial-myceliumn e g a t i v e , a n t h r a c y c l i n e n o n - p r o d u c i n g m u t a n t s ( A m y - A n t - ) b y m u t a g e n i c treat-

Modification of autoregulator effect on S. griseus mutants

ment of the parental strains S. griseus J A 5142 and J A 3933 blished) :

517 ( E r i t t

et al., to be pu-

a ) Mutants represented b y the asporogenous, met", derivative 39 which produced no detectable antibiotic but still induced blocked mutants of b ) - t y p e (see b e l o w ) t o cytodifferentiation.in cosynthesis experiments ( E r i t t et al. 1982). This suggested that mutant 39 and similar derivatives synthesized the autoregulator 1 regardless of the f a c t that they could not produce spores or anthracyclines. b ) Mutants such as the prototrophic strain 86, lys~ strains 6 and 15, and the polyauxotrophic strain 16 which responded to the presence of 1 ( R = C 7 H 1 5 ), 2 or similar molecules with partial or complete reconstitution of cytodifferentiation (formation of aerial mycelium and/or anthracyclines) during cultivation on agar' surfaces. T h e m a j o r i t y of mutants regained the capacity to f o r m both spores and anthracyclines as for example mutant 86 ( E r i t t et al. 1984, GRAFE.ei al. 1984) and 15. Mutant 6, however, differed in that it produced blue pigments instead of the leukaemomycin complex. T h e derivative 16 regenerated only the f o r m a t i o n of aerial mycelium but remained incapable of producing anthracyclines. This suggested that it possessed a v e r y early block in the biosynthetic p a t h w a y of the daunomycint y p e antibiotic leukaemomycin. F i g . 1 shows that during submerged cultivation on H t c k e y - T k e s n e k ' s medium mutants 86 and 15 displayed similar but slightly reduced g r o w t h as compared with the parental strains J A 5142 and 3933 while mutants 6 (lys~) and 16 (polyauxotrophic) developed poorly. I n the m a j o r i t y of cases (4 —6 independent experiments) the presence of autoregulator 1 in the medium at zero time was beneficial to g r o w t h of blocked strains and accompanied b y morphological changes ( E r i t t et al., manuscript in preparation). I n contrast to the parental strains forming filamentous hyphae blocked mutants grew as more or less dense pellets except f o r strain 86 which displayed a strongly fragmented mycelium. I n the presence of 1 or alternatively 2 ( R = C 7 H 1 5 ) pellets of strains 6 and 16 turned more filamentous, in case of strains 86 and 15 hyphae became similar as those of the t w o parental strains. A s reported elsewhere ( G r a f e et al. 1984) mutants 86 and 15 could be induced b y 1 to yield about the same amount of antibiotic (approx. 40 ¡¿g/ml leukaemomycin) as strains J A 5142 and J A 3933. Mutant 6 produced traces of antibiotic substances (mainly the blue pigments) while mutant 16 did not generate any antibiotic pigments even in submerged cultures.

m m cn m t_

o

CN

CO 00

Fig. 1 Biomass formation in 48 hrs cultures of S. griseus J A 5142 and J A 3933, and their blocked mutants 86, 15, 6 and 16. ( + 1 : autoregulator 1 added at zero time)

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al.

Biochemical properties of the mutants blocked in differentiation One of the major biochemical characteristics of the autoregulator-negative, b)-type mutants was their comparably lower content of mycelial lipid material (Fig. 2). In mutants 86 and 15, stimulation of lipid synthesis has been observed up to the parental level in presence of 1 (or 2 , see G R A F E et al. 1 9 8 4 ) . A similar but far less pronounced picture emerged from experiments with strains 6 and 16. As depicted in Fig. 3, addition of autoregulator 1 to cultures of the blocked mutants caused marked alterations in the ratio of the neutral to the polar lipids. When aliquots of lipid samples were applied to t.l.c. plates, then both spot areas and staining intensity could be taken as measure for the relative amount of polar lipids within the

£

D

"ai o E a> 5-

.C

u>

X)

Fig. 2 Mycelial lipid content in 48 hrs cultures on HICKEY-TRESNER'S medium (in % of mycelial dry weight). Bars indicate the variation of the results in 6 independent experiments. 5142 and 3 9 3 3 : parental strains; 86, 15, 6 and 16: blocked mutants; + 1 : 1. added at zero time

51 86

16 +1

5186

16 + l

+l

FE CL LFE

a

b

Fig. 3 a) Aminolipids and b) phospholipids from 4 8 hrs mycelia of parental strain J A 5142 (51) and blocked mutants 86 (86), 16 (16) and 6 (6). + 1 : 1 added a t zero time. P E : phosphatidylethanolamine; C L : cardiolipin; O L : ornithinolipid; L P E : lyso-phosphatidylethanolamine. E a c h lipid sample was applied in 200 ¡xg amount. Staining was carried out using either ninhydrine (a) or ZINZADE'S r e a g e n t ( b )

519

Modification of autoregulator effect on S. griseus mutants

pool of total ipids. The pospholipid content of the parental strains J A 5142 and J A 3933 (the latter not shown here) was much lower than that of the mutant 86. This was apparently due to phosphate limitation of the medium and a higher rate of lipid synthesis in the former two strains. In the latter strain the enhancement of lipid production in presence of 1 favoured also the formation of neutral lipids at the expense of the phospholipids because phosphate supply was limiting. When we added together with 1 0.1% K H 2 P 0 4 (pH 6.5) to the medium higher portions of phospholipids but not of ornithinolipids were formed since, in general, formation of the latter lipids by streptomycetes is suppressed by excessive phosphate supply (BATRAKOV and B E R G E L SON 1978). When the slower growth rate of mutant 16 and, consequently, the incomplete use of the inorganic phosphate of medium was taken into account, the lacking formation of ornithinolipid could be readily understood. In this nutritional situation 1 seems to stimulate preferably the formation of phospholipids as observed for mutant 86 when it was grown in presence of 1 and excessive phosphate. The increase of portion of phospholipids in mutant 6 can be interpreted in a similar way. However, without the addition of 1 a lower content of phosphatidylethanolamine and cardiolipin was observed as compared with blocked mutant 86. This suggested that additional genetic changes in mutant 6 interfered with the phosphate-mediated control of lipid composition (GRAFE et al. 1982b). Other findings, too, suggested that all the blocked mutants were characterized by several, different, genetic changes in addition to the loss of autoregulator production. For instance, the relative proportions Table 1 Relative proportions (%) of fatty acids from the mycelia of strain S. griseus JA 5142 (Amy + Ant + ) and the A m y - A n t " mutants 86, 15, 16 and 6 (all values for 48 hrs cultures) strain

a/i

i l 4 : 0 14:0 i l 5 : :0 a l 5 : 0 15:0 i l 6 : 0 16:0

J A 5142 86 86 + J 15 15 + 1 16 16 + 1 6 6 + 1

1.1 2.0 2.7 1.9 1.5 3.5 3.4 1.6 1.9

4 4.7 1.4 4.4 4.5 7.4 5.2 5.2 3.4

0.7 0.6 0.9 1.4 1.6 1.5 0.7 3.3 1.1

5.4 4.7 tr. 5.8 5.3 tr. tr. 5.2 3.1

26 34.2 34.2 32 30 41.4 47 25.2 38.3

2.7 4.7 4.5 4.9 3.6 7.4 7.6 3 4.2

23.5 17.1 12.6 17 20.3 11.8 13.8 15.5 20

11 7.1 8.1 7.8 7.3 7.4 9.4 9.3 6.3

16:1

al7: 0 UI

18:0

18:1

3.7 4.1 4.5 4.8 4.1 5.9 2.8 6.7 3.2

10 10.6 13.5 8.7 11.3 8.1 8.3 10.4 15.7

1.7 1.2 3 2.9 1.6 1.3 1.4 1.5 0.5

2.3 2.3 4.6 tr. tr. 3.2 tr. 3 tr.

9 8.3 12 9.7 10.5 4.4 3.2 12.2 5.3

Abbreviations: a/i: ratio o f a l 5 : 0 t o i l 6 : 0 fatty acids; i l 4 : 0 , 12-methyltridecanoic acid; 14:0 tetradecanoic acid; i l 5 : 0 13-methyltetradecanoic acid; a l 5 : 0 , 12-methyltetradecanoic acid; 15:0 pentadecanoic acid; i l 6 : 0 isopalmitic acid; 16:0, palmitic acid; 16:1 hexdecenic acid; a l 7 : 0 , 14methylhexadecanoic acid; U I : unidentified; 18:0, stearic acid; 18:1, oleic acid; tr., traces.

en -t

£n or 15

Fig. 4 Extracellular proteinase levels in the culture medium (48 hrs). (one arbitrary unit represents a change of the optical density by 1.0; bars indicate the variation of the results). + 1 : Addition of 1 at zero time

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of f a t t y acids were altered in comparison with the parental strain J A 5142 (and 3933, not shown here) even when 1 had been administered to the medium (Tab. 1, see c.f. the ratio a/i fatty acids). Fig. 4 shows that blocked mutants could be further distinguished from the ancestral strains by a much lower secretion of neutral proteinases. Experiments with the inhibitor phenvlmethylsulfonylfluoride revealed that both the extra- and intracellular neutral proteinase activity consisted mainly of serine proteinases. When cultures of the blocked mutants were grown in presence of 1 (or 2), partial reconstitution was detected of both extra and intracellular levels of neutral proteinases. A further difference between the blocked mutants on the one hand and the ancestral strains on the other concerned the extracellular level of NAD(P)-glycohydrolase which has been proposed to be involved in the mechanism of action of the A-factor (1, 2S, 3S derivative, R = C5H17) in the course of stimulation of streptomycin biosynthesis by S. griseus ( V O R O N I N A et al. 1978). According to Fig. 5 extracellular (and intracellular, not shown) levels of enzyme were high in the cultures of strains J A 5142, J A 3933 (Amy + Ant + ) and the A m y - Ant" mutant 39 but the autoregulator-negative derivatives displayed only trace activities even in the presence of the autoregulatory inducer of cytodifferentiation.

< D ifi a "D -C oo >N CL Q
-5 met-2

8 13 9 3 7 3

< 0.2 < 0.2 < 1.0 < 0.1 4

48

72 96 0 tih) ——

i 2,

i 48

i 72

i 0 96

J

0

Fig. 3 Influence of a KH 2 P0 4 -solution added prior to autoclavation: on the nourseothricin biosynthesis (Fig. 3 A), on the intracellular phosphatase activity measured at pH 7.2 (Fig. 3 B), and on the growth measured as intracellular protein (Fig. 3C) of shaked-flasks cultures. Phosphate concentrations added: 0 mM (o), 0.12 mM (•), 0.235 mM (•), 0.49 nrn (•), 0.70 nrn (A), and 1.17 mM (A) a c t i v i t y seems to be more independent of the growth rate than the p H 9.2 e n z y m e activity. B o t h activities were, however, already induced intracellularly and extracellularly parallel to nourseothricin during the late of the first growth period. This pointed to the occurrence of phosphate limitation in cells after the short logarithmic growth phase.

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Q. "oi I s S-S 8 \ -c i o 2 Qj E 8-a0.02

0.01

26

50

7U

982

26

50 t(h)

74

982

26

50

74

98

_

0.10

0.05

Fig. 4 Influence of the presence of zinc ions (1.74 m i ) (filled symbols) on: the nourseothricin biosynthesis (Fig. 4A), the intracellular phosphatase activity, measured at p H 7.2 (Fig. 4B), the extracellular phosphatase activity, measured at pH 7.2 (Fig. 4C), the inctracellular protein (growth) (Fig. 4D), and the extracellular protease activity (Fig. 4 E ) in comparison to experiments where no zinc ions were added (open symbols). Fermentations were performed in shaking flasks

To a certain extent the specific activities of the intracellular phophatases seemed to be growth-correlated, because the intracellular activities were only increased during phases of limited growth indicating a partial lack of phosphate. Intracellular phosphatases were not formed during the short logarithmic growth phase and the stationary phase. A protease was also excreted simultaneously with the phosphatases, especially the 7.2 phosphatase. This suggested that lack of phosphate had also an initiating effect on protease formation. A total phosphate concentration of 4.27 mM was estimated in the medium. About 8% were found to be acid-dissolvable at the start of the fermentation after steam

E f f e c t of phosphate on nourseothricin biosynthesis

561

pressure sterilization for 45 min. Also, it was shown that repeated autoclaving of the medium (Fig. 2) resulted in an increase of the initial phosphate and in a decrease of nourseothricin concentration that could be reached after fermentation (Fig. 2). Since sterilization affected glucose and ammonium only in a little extent an inverse correlation may be assumed between the initial phosphate concentration and final nourseothricin concentrations. Moreover, as expected the direct addition of phosphate together with K + ions (as K H 2 P 0 4 ) strongly inhibited the formation of nourseothricin and intracellular phosphatases (Fig. 3A). As confirmed by another experiment (data not presented) the nourseothricin biosynthesis was very sensitive to increased initial phosphate concentrations. The addition of only 0.06 mM K H 2 P 0 4 lowered the yield of nourseothricin after 120 h to about 15°/0 of that obtained when no phosphate was added. I t has, however, to be mentioned that the sensitivity to added K H 2 P 0 4 varied in different experiments. These variations may have to be ascribed to the sterilization conditions which could not be precisely controlled. The intracellular phosphatase activity was also negatively influenced by K H 2 P 0 4 addition (Fig. 3C). Fig. 4 shows as comparison of kinetics of shaking flasks with and without addition of zinc ions (1.74 mM). The growth rate was lowered by the addition of zinc ions but the nourseothricin formation increased strongly (Fig. 4A). Also, the addition of zinc ions lead to an increase in the intracellular phosphatase activity (Fig. 4 B ) as well as the extracellular phosphatase (Fig. 4C). Especially the kinetics of the intracellular phosphatase indicated an influence of zinc ions on the phosphate metabolism which could be noticed already during the first hours of fermentation. Also the activity of intracellular phosphatase measured at pH 9.2 varied drastically. For example, after 24 h of cultivation an activity of 0.072 fxmoles/min • mg was measured in the presence of zinc ions while an activity of only 0.0165 ¡xmoles/min • mg was found in the absence of zinc ions. The proteolytic activity was higher in late phases of fermentation when no zinc ions were added. The total concentration of zinc in the medium was 1.75 mM, but the concentration of dissolved zinc during the fermentation was estimated to be only 0.15—0.31 mM. This difference pointed to a formation of indissolvable phosphate-containing sediments. Also, it was found to be adventageous to perform the steam pressure sterilization of fermentors in the presence of zinc ions at alkaline pH-values of 8 —9. This led to a decrease in the initial phosphate concentration to about 0.03—0.2 mM instead of 0.35 mM when sterilization was carried out at pH 6.8. The addition of equimolare amounts of F e + + + , Al + + + , or M n + + which also form undissolvable phosphates stimulated the noureseothricin biosynthesis to nearly the same extent (data not presented) as did the addition of zinc ions. These results supported our assumption that the positive effect of zinc ions on the nourseothricin biosynthesis was brought about by a decrease of the initial phosphate concentration. Discussion

Our investigations showed that limitations of phosphate, glucose and ammonium occurred during fermentations of nourseothricin. Nevertheless, the special significance of a phosphate-mediated regulation of metabolism has been demonstrated clearly. Especially the kinetics of the phosphatase activities may be regarded as an indicator for phosphate regulation. Most phosphatases are negatively regulated by phosphate in S t r e p t o m y c e t e s (MULLER et al. 1983 a).

Accordingly, the formation of phosphatase activities reflected a partial phosphate limitation which occurred already between the 2—24 t h hour. The transient increase of

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phosphate concentrations suggested release of phosphate during fermentations. A correct interpretation of the higher phosphate concentrations after initiation of nourseothricin and phosphatase synthesis has to take in acount that the analytical estimation of phosphate was carried out at acid conditions which generally leads to hydrolysis of labile phosphate ester and, consequently, to higher estimates. On the other hand it was evident from further investigations that the high dephosphorylating activity of cells observed during periods of phosphate limitation lowered the efficiency of the phosphate uptake. This resulted in higher steady state concentrations of extracellular phosphate (preliminary note: M Ü L L E R et al. 1983d). From these results it was concluded that growth was limited by phosphate and not by ammonium. The latter one was consumed up to the 36 th hour parallel to phosphate. Besides the regulatory effect of phosphate which initiated the secondary product synthesis a strong inhibitory effect was observed when high initial phosphate concentrations were present in cultures of strain Streptomyces noursei J A 3890b. As little as 0.06 m l K H 2 P 0 4 added to the medium prior to autolavation (containing in general zinc ions) inhibited the nourseothricin biosynthesis thus pointing to high sensitivity of this strain to phosphate. Therefore, the strongly negative influence of repeated autoclavation procedures on nourseothricin formation is believed to be due to an increase in the initial phosphate concentration. According to other authors (summarized by M A R T I N 1977) the range of phosphate concentrations permitting a secondary product biosynthesis varies for different secondary metabolites, it is for example 0.14—0.20 mM in tetracycline, 0.1 — 1.0 mM in bacitracin, and 0.2—0.4 mM in monamycin fermentations. The respective producer strains are known to be very sensitive to phosphate. The sensitivity of Streptomyces noursei was found to be lower in strains selected for higher productivity (preliminary note: M Ü L L E E et al. 1983e). Normally, acid-dissolvable phosphate concentrations of 0.3—0.5 mM were found in the zinc ions-containing medium autoclaved in flasks. The addition of 0.06 mM K H 2 P 0 4 leads theoretically to a total initial concentration of phosphate of 0.36—0.56 mM. The real value of dissolvable and biologically available phosphate should, however, be dependent on the real conditions of sterilization influencing the degree of formation of phosphate-containing sediments. The latter effect also seemed to be the reason for the stimulating effect of zinc ions added. The main part of added zinc was found to be undissolved in the cultures. Therefore, the formation of zinc phosphates and/or zinc ammonium phosphates has to be assumed. This assumption was supported by the finding that the stimulatory effect of zinc ions on the intracellular phosphatase appears already during the 2 nd hour. Also, other ions such as Fe + + + , Al + + + , Mn + + , and Ca + + which are known to form phosphate-containing sediments and which were added instead zinc ions stimulated the nourseothricin formation. This was in agreement with earlier assumptions ( W E I N B E R G 1 9 7 4 ) . I t is known that the formation of such sediments is favoured by higher p H values (VOLLENWEIDER 1 9 6 8 ) . Accordingly the nourseothricin biosynthesis also increased when the autoclavation was performed at higher p H values (MÜLLER et al. 1 9 8 3 C , GROSSE et al., in preparation). Taken together, the target of a biometrical optimization of the medium used for fermentation (BOCKER et al. 1 9 8 3 ) of the phosphate sensitive strain was the initial concentration of phosphate. I t has been lowered by a suitable medium composition regarding the phosphate-containing substrates, by presence of phosphate-precipitating ions and by the special condition of autoclavation or sterilization. I t could, however, not be that the medium developed in this way possessed an optimal composition. Accordingly, the addition of a certain amount of phosphate after inoculation should stimulate growth as well as the specific product formation rate

Effect of phosphate on nourseothricin biosynthesis

563

(preliminary notes: M Ü L L E S et al. 1983c, d, H A U B O L D et al. and G R O S S E et al. in preparation). Other limitations, especially those of C- and N-sources, should be avoided. This strategy proved to be useful both in preventing a too high initial phosphate concentration and in permitting a b u n d a n t growth of a biomass being capable of producing nourseothricin. Moreover, the feeding of phosphate did not interupt the special physiological state which appeared after the transient depletion of phosphate and which was characterized by high intracellular activities of dephosphorylating enzymes ( M Ü L L E E et al. 1983a). From the data presented here it was concluded t h a t the specific phosphatase activities were subjected to phosphate-mediated regulation in early phases of the fermentation like the nourseothricin biosynthesis. A clear interpretation of the late kinetics of phosphatases was, however, more difficult since glucose and ammonium limitation overlapped the fermentation process. Recent results show t h a t a t conditions where phosphate was the only limiting substrate the nourseothricin formation was partially growth associated ( H A U B O L D etal. in preparation), if the specific growth rate remained below a critical value. The intracellular phosphatase activity increased especially during phases of reduced growth rates thus pointing also to a coupling of the phosphatase biosynthesis to growth. Extracellular protease like the extracellular phosphatases was formed simultaneously with the nourseothricin synthesis. I t s synthesis was also initiated by phosphate limitation as it was the case during turimycin and streptomycin fermentations ( M Ü L L E E et al. 1983a, b). During late phases of fermentations the extracellular alkaline phosphatase exhibited a different behaviour, because its formation stopped during stationary and second growth period. The alkaline phosphatase is probably an ectocellular enzyme which likely to be bonded to the glycocalyx of the hyphae ( H O S T Ä L E K 1981). Therefore, it seems likely t h a t the extracellular occurrence dependend on the integrity of the cells. An alternative interpretation would be a pH-dependent adsorption of the enzyme to cells at lower p H values ( O Z E G O W S K I and M Ü L L E E 1984). The presence of both ectocellular and extracellular phosphatases were a prerequisite to assure a sufficient supply of phosphate for the cells during the fermentation ( M Ü L L E E etal. 1983a). Together with proteases, nucleases and amylolytic activities the phosphatases hydrolyze the phosphate-containing soya-meal and potato starch. Our results have to be viewed together with recently published observations on generally increased activities of dephosphorylating enzymes (CTJEDOVÄ et al. 1 9 8 2 , J E C H O V A et al. 1 9 8 2 ) and in connection with uncoupling of respiration ( E F F E N B E E G E E et al. 1 9 8 3 ) as well as protein synthesis ( M Ü L L E E et al. 1 9 8 3 A , R Ö M E E and M Ü L L E E 1 9 8 3 ) resulting from onset of phosphate limitation. Nevertheless, the regulatory mechanism of phosphate sensitivity remains obscure and demands f u r t h e r investigations.

References AHARANOWITZ, Y., 1980. Nitrogen metabolite regulation of antibiotic biosynthesis. Ann. Rev. Microbiol., 34, 2 0 9 - 2 3 3 . ARIMA, K . , Ytr, J . a n d IWASAKI, S . , 1 9 7 0 . M i l k - c l o t t i n g e n z y m e f r o m Mucor pusila var. LIJTDT. I n : Methods of Enzymologie X I X , (Editors: G. E. PERLMANN and L . LOB AND), pp. 446—459.

Academie Press New York-London.

G., SCHICHT, G. und NOACK, D . , 1980. Verfahren zur Herstellung eines Antibiotikums der Streptothricin-Gruppe. DDR-WP A 61K/223321, 14. 8. 1980. BOCKER, H . and RECKNAGEL, R . D . , 1 9 8 3 . A new biometrical screening method as applied to nutrient optimization in fermentation. Acta biotechnol., 3, 89—92. BRADLER, G. und THRUM, H., 1963. Nourseothricin A und B, zwei neue antibakterielle Antibiotica einer Streptomyces noursei-Variante. Z. allg. Mikrobiol., 3, 105—112. BOCKER, H . , GRÄFE, U . , THRUM, H . , BRADLER,

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dl.

CURDOVÄ, E . , JECHOVÄ, V. a n d HOSTÄLEK, Z., 1982. P r o p e r t i e s of a g y r a s e a n d i n o r g a n i c p y r o -

phosphatase in Streptomyces cmreofaciens. Folia microbiol., 27, 159—166. W., M Ü L L E R , P . J . and B O C K E R , H . , 1 9 8 3 . Uncoupling of respiration in turimycin fermentations. Z. allg. Mikrobiol., 23, 557 — 564. GRÄFE, U., BOCKER, H. and THRUM, H., 1981. Nitrogen catabolite regulation of nourseothricin biosynthesis by Streptomyces noursei J A 3890 b. I n : Advances in Biotechnology, Vol. I l l (Editors : M. Moo-YOUNG, C. V E Z I N A and K . S I N G H ) . Proc. 6th I n t e r n . Fermentations Symp., London, Canada, Juli 2 0 - 2 5 , 1980. Pergamon Press, p. 193 — 198. H O S T A L E K , Z . , 1 9 8 0 . Catabolite regulation of antibiotic biosynthesis. Folia Microbiol., 2 5 , 4 4 5 to EFFENBERGER,

450. J E C H O V Ä , V . , CURDOVÄ, E .

and H O S T Ä L E K , Z . , 1982. Role of phosphatases in t h e biosynthesis of Chlortetracycline in Streptomyces aureofaciens. Folia microbiol., 27, 153 — 158. MARTIN, J . F., 1977. Control of antibiotic synthesis by phosphate. Adv. Biochem. Eng., 6, 105 to 127. M A R T I N , J . F. and D E M A I N , A . / L . , 1 9 8 0 . Control of antibiotic biosynthesis. Microbiol. Rev., 4 4 , 230-251. M Ü L L E R , P. J . , H I L L I G E R , H . , M E N N E R , M .

and F O R B E R G , W . , 1 9 8 2 . Phosphate release in turimycin fermentation. Z. allg. Mikrobiol., 21, 267 — 272. M Ü L L E R , P . J . , O Z E G O W S K I , J . H . and B O C K E R , H . , 1 9 8 3 A . Regulation of hydrolases formation and phosphate relase in turimycin fermentations. Z. allg. Mikrobiol., 23, 173 — 180.

MÜLLER, P . J . , CHRISTNER, A. a n d OZEGOWSKI, J . H . , 1 9 8 3 b . S e q u e n t i a l p r o c e s s e s of p h o s p h a t e

limitation and phosphate release in streptomycin fermentations. Z. allg. Mikrobiol., 23, 269 to 273. M Ü L L E R , P . J . , H A U B O L D , G . , B O C K E R , H., G R O S S E , H. H . , M E N N E R , M. und H E L L E R , I . , 1983c. Verfahren zur Herstellung von Sekundär-Metaboliten durch mikrobielle Fermentation. DDRW P C12 -/247 370 7, 20. 2. 83. M Ü L L E R , P. J., O Z E G O W S K I , J . H . and R Ö M E R , W . , 1983d. Parallel regulation of secondary metabolite- and protein biosynthesis in Streptomyces by sequential processes of phosphate limitation and phosphate release. I n : Abstracts of FEMS International Symposium on "Environmental Regulation of Microbiol Metabolism", 1—7 J u n i 1983, Pushchino, USSR. OZEGOWSKI, J . H. und MÜLLER, P. J., 1984. Untersuchungen zum Stoffwechsel Phosphat-limitierter Streptomycetenkulturen. I. Reinigung und Charakterisier u r g einer alkalischen Phosphatase aus Streptomyces hygroscopicus. Zbl. B a k t . Hyg., I. Abt. Orig. A, 23, 203—206. R Ö M E R , W . and M Ü L L E R , P. J . , 1 9 8 3 . Parallel regulation of cAMP phosphodiesterase and phosphatase activities in turimycin fermentations. Z. allg. Mikrobiol., 23, 2 0 3 — 2 0 6 . VOLLENWEIDER, R. A., 1968. Scientific fundamentals of t h e eutrophication of lakes and flowing waters, with particular reference to nitrogen and phosphorus as factor in eutrophication. OECD Technical Report DAS/CSI/68.27, 155 pp. Revised 1971. WEINBERG, E. D., 1974. Secondary metabolism: Control b y t e m p e r a t u r e and inorganic phosphate. Develop. I n d . Microbiol., 15, 70—81. WEINBERG, E. D., 1978. Secondary metabolism: Regulation by phosphate and trace elements. Folia Microbiol., 23, 4 9 6 - 5 0 4 . Mailing address: Dr. P. J . MÜLLER, Zentralinstitut f ü r Mikrobiologie und experimentelle Therapie der AdW, DDR-6900 Jena, Beutenbergstraße 11

Zeitschrift für allgemeine Mikrobiologie 24 (1984) 8, 565 — 574

(Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, Jena)

Substructural study of sporogenesis in Streptomyces griseus C. STRUNK

Devoted to Professor Dr. M. GIKBARDT on occasion of his 65th birthday (Eingegangen am

23.12.1983)

A Streptomyces griseus strain deficient in the formation of aerial mycelium and arthrospore develop ment (Amy - ) was studied by electron microscopy and compared with the sporulating parental strain (Amy + ). The investigations were performed with colonies grown on solid medium. The substructural characteristics of the essential events of sporogenesis could clearly been demonstrated in the aerial mycelium of the Amy + colonies. The mycelium of the surface region of the Amy" colonies showed altered features. The most pronounced alteration was the absence of the surface sheath, normally present on the aerial hyphae of the parental strain. The cross wall type II, characteristic of sporogeneous hyphae, was not discernible in the Amy" hyphae. Some substructural features resembling the essential events of normal sporogenesis were evident in the Amy" strain, albeit diminished and interfered with by abnormalities. The resulting propagative cells were of different size and feature.

Substructural studies of sporulating aerial mycelium of different Streptomyces species have revealed various modes of sporogenesis (references in H E N S E N et al. 1 9 8 1 , K A L A K O U T S K I I and A G R E 1 9 7 6 , S T R U N K 1 9 7 8 , V O B I S 1 9 8 1 ) . Their common features, which are essential for sporogenesis of streptomycetes, are: 1. The development of an additional outer cell wall layer on the aerial hyphae, the surface sheath (surface layer, fibrous layer); 2. the increased development of cross walls, resulting in the formation of closely spaced hyphae; 3. the formation of a special cross wall (sporulation septum), the type I I cross wall (WILLIAMS et al. 1973); 4. cell separation by autolytic activities; 5. secondary cell wall thickening during the process of arthrospore maturation. The sequence of these events appears to be strictly regulated. The impairment of any of these developmental steps, e.g. by mutagenic treatment, causes serious deviations of sporogenesis (CHATER 1 9 7 2 , CHATER and M E R R I C K 1 9 7 9 , HOPWOOD et al. 1 9 6 9 , 1 9 7 0 , 1 9 7 3 , M C V I T T I E 1 9 7 4 ) or even the total absence of arthrospore differentiation (CHATER a n d MERRICK 1 9 7 9 , KALAKOUTSKII a n d AGRE 1 9 7 6 , MERRICK

1976).

Streptomyces strains with altered phenotypes have been found during continuous cultivation and as a result of spontaneous segregation ( R O T H and NOACK 1982, R O T H et al. 1982 b). These derivatives differ from their parental strains by some features ( G R A F E E J A Z . 1981, 1982a, 1982b, R O T H et al. 1982a, 1982b). One of them is the deficiency in the production of aerial mycelium and arthrospores when grown on solid medium. The aim of the present paper is a comparative substructural analysis of a non-sporulating strain of Streptomyces griseus (Amy - ) LM 1 and its parental strain (Amy + ) H P (ROTH et al. 1982b). The attention was concentrated on the essential events of sporogenesis defined above. Materials and methods Strains: Streptomyces griseus HP (Amy + ) formed aerial mycelium and arthrospores when grown on solid agar medium. S. griseus LM 1 (Amy - ) was deficient in aerial mycelium and arthrospore formation under similar cultivation conditions (ROTH et al. 1981b).

566

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Substructure of sporogenesis in S. griseus

567

Cultivation: The investigations were performed with colonies grown at 28 °C on AL 53-agar (ROTH a n d NOACK 1 9 8 2 ) or o n Y S A (GRAFE et al.

1981).

Electron microscopy: Specimens were prepared as described previously (STRUNK 1978) and examined by using the following electron microscopes: SEM 3-2 (WF, Berlin-Oberschoneweide), EM 400 T (PHILIPS, Eindhoven), BA 540 (TESLA, Brno).

Results Surface mycelium of S. griseus Amy+ colonies Amy + colonies grown on a complex agar medium at 28 °C initially appeared shiny and smooth. After about 3 days of cultivation they became white and hairy indicating the emergence of aerial mycelium. The substructural investigation of the colony surface at this stage of development revealed all stages of sporogenesis. They may be summarized with reference the essential events of sporogenesis proposed at the outset of this paper. The surface sheath was present on all hyphae of the aerial mycelium (Figs. 1—5), even on those that had not developed spores yet (Fig. 1). The cross walls were present in more or less regular and short spatial intervals (Fig. 2) distinctly designed from the outset as a double ingrowth from the inner part of the lateral wall (Fig. 3). The light space between the two cross wall layers was locally filled with a dark substance keeping up a reticulated continuity between the two layers (Figs. 3 a—d). Hence, depending on the intersecting plane, the edge of the ingrowing cross wall disk was "open" or "closed". The autolytic cross wall splitting seemed to begin before the cross walls had been completed (Fig. 3). The separated spore cells, more or less spherical in shape, remained surrounded by the surface sheath during the process of secondary cell wall thickening (Fig. 4). The mature arthrospores were characterized by the condensed cytoplasm, the thickened cell wall, and the overlaying surface sheath not completely covering the spore (Fig. 5). A variation of the culture conditions caused some morphological deviations from this main way of sporogenesis, which, however, is irrelevant to the problem studied here. In general, the observations agreed with the results previously reported on another sporulating S. griseus strain ( W I L L I A M S and S H A R P L E S 1 9 7 0 ) . The mode of the S. griseus sporogenesis was consistent with the first of the three basic types of sporo-

Figs. 1 - 6 Sections obtained from the surface area of Amy+ colonies. Symbols: B = Branch, CM = Cytoplasmic Membrane, N = Nucleus, SS = Surface Sheath Fig. 1 Branching hypha surrounded by the surface sheath; sporogenesis not yet initiated. Magn. 66000 X Fig. 2 Early stage of sporogenesis. The formation of cross walls (arrows) in short spatial intervals has started. Magn. 32500 x Fig. 3 Four serial sections of a cross wall of type II. Simple arrows point to the dark substance between the ' two cross wall layers. Depending on the intersection plane, "open" (O) and "closed" (C) edges of the ingrowing cross wall disk are seen. Double arrow: cross wall splitting. Magn. 91200 X Fig. 4 Detail of Figure 6. Spore chain in an early stage of development. The immature spores are still enclosed by the surface sheath. Magn. 60000 X

568

C. S T R U N K

•*>•*> 0p 9 Vi

• «AI A

Ä

569

Substructure of sporogenesis in 8. griseus

genesis classified by MANZANAL and and HARDISSON 1978).

HAKDISSON (HARDISSON

and

MANZANAL

1976,

MANZANAL

Surface mycelium of 8. griseus Amy

colonies

Colonies of the Amy" strain cultivated under conditions similar to those of the Amy + strain retained the shiny and smooth feature over a period of several days. Later on the colonies got a sculptured surface but no aerial mycelium emerged. Figs. 6 and 7 illustrate the different appearance of the two colony types. On the surface of the sporulating Amy + colony spore chains and cytoplasm-rich hyphae were recognizable (Fig. 6), whereas the surface of the Amy" colony of similar age gave the overall impression of degeneration (Fig. 7). B y using higher magnifications, the feature of the Amy" hyphae became evident. No distinct surface sheath was discernible (Figs. 8 — 14). No distinct double character could be found in the cross walls, either in the early developmental stages (Fig. 8) or in later stages (Figs. 9 and 10). On the other hand, certain substructural features of the Amy" hyphae resembled those associated with the essential events of normal sporogenesis. An increased tendency to form cross walls occurring in small intervals like those seen in sporulating hyphae could be observed. However, these cross walls were more irregular spaced (Figs. 9 — 11 and Fig. 13). The development of the resulting cell chains may proceed into two extreme directions: 1. The cells, delimated by simple and small cross walls, suggested a vegetative character. Each cell was able to branch (Fig. 9). Cell chains of this kind had no direct contact with the aerial space. 2. The cells got sporal character by rounding up, condensation of the cytoplasm and secondary cell wall thickening (Fig. 10). Branching did not occur. However, these cell chains differed considerably from true spore chains by several irregularities, e.g. aberrant thickening of both the cross walls and parts of the lateral wall, irregular spacing and frequent malformations (Fig. 13). Furthermore, closely spaced hyphae of intermediate character were discernible. They were composed of cytoplasm-rich cells of different length and empty cells indicating an untimely decay (Fig. 11). Although no double character was found in the cross walls of the Amy" hyphae, cell separation was possible (Fig. 11 and Fig. 12). However, the process of cross wall splitting seemed often to be stopped before completion, apparently caused by abnormalities of the cross walls (Fig. 13) or/and as a consequence of limited cell viability (Fig. 14).