J. Basic Microbiol. 32 (1992) 6 , 415-422
(National Research Centre of Antibiotics. Moscow, Russia)
Coagulation autolysis in microorganisms and its relation to coagulase production P. L. ZASLAVSKAYA, I. V. CHEKALINA, P. S. NYSand Yu. E. BARTOSHEVICH ( Receioed
-79 October 199llAccepted 16 J m e 19921
The phenomenon of coagulation autolysis was observed in two model microorganisms. i.e.. a bacterial culture and an imperfect fungus. I t was characterized by impairment of the cell membranes, followed by condensation and dehydration of the cytoplasm and long-term preservation of the cells in the form of coagulated cytoplasm. In this respect. it was similar to coagulation necrosis of human tissues. The autolysis in the microorganisms was accompanied by increase of their coagulase activity, the substrate specificity of the enzyme rather broad. The coagulase activity of the microorganisms was detected during the culture period between the lag-phase and the exponential growth phase, i.e., the phase of their active growth. It served as a signal to induce biosynthesis of peptidohydrolase and cleavage of proteins. We believe that the phenomenon of coagulation autolysis in these microorganisms is rather typical and can be considered as an adaptative reaction, inducing a cascade of events from synthesis of coagulase to overproduction of peptidohydrolases with proteolytic activity.
Coagulation necrosis of human tissues is a well known phenomenon. It is characterized by coagulation and pronounced denaturation of cytoplasmic proteins and the cytoplasm condensation and dehydration (SEROVand PAUKOV1975). Still, the mechanisms of its development are not clear and any information to the point is essential for elucidating the causes of pathological processes in living tissues. Microorganisms represent a unique model for investigating the development of many processes from both functional and morphological viewpoints. Coagulation autolysis in microorganisms has not been described so far. Optical microscopy, mainly used in studies on microorganisms, cannot reveal the succession of the autolysis events nor even distinguish between coagulated and living cells. By the use of ultrastructural analysis it was possible to observe unusual aggregation and disintegration of cells during development of the fungus producing cephatosporin C and protease, as described by us previously ( BARTOSHEVICH et nl. 1990). Investigation of microorganisms producing peptidohydrolases made it possible to reveal in them processes similar to autolysis and to raise the problem of the relationship between these processes and the enzymatic activity of microbial cells. Within the last decade a number o papers were published which showed that some microbial proteases in fungi and bacteria possess coagulating activity (SATONet a/. 1975. OTROSHKO and EGOROV1975, CHERDYNTSEVA and EGOROV1988). In some of these it was indicated that only cell extracts possessed such activity, which means that the coagulating acitivity was associated with the microbial cells. Some studies suggested a correlative relationship between the coagulating and proteolytic activity in microorganisms (SATON ef nl. 1975). though in the majority of actinomycetes, as well as in some other bacteria and in fungi, no such relationship was observed. It was also indicated that the activity spectrum of coagulase in many of the microbial cultures studied was rather broad and the cultures were shown to coagulate blood sera of humans, cows, rabbits, mice and chicken. The present paper is concerned with the morphofunctional analysis of development of two microorganisms differing in the level of their organization, i.e., a bacterial culture and
416
P. L. ZASLAVSKAYA et d .
an imperfect fungus producing peptidohydrolases. The analysis included investigation of the cell ultrastructure and enzymatic activity of the cultures. Such an approach allowed us to propose a model of coagulation autolysis i n microbial cells due to their coagulating and proteolytic activity.
Materials and methods Two organism5. i.e.. , ~ [ / / i t / i ~ j / f / ( j / i sp.. ~ / s a bacterial culture producing peptidohydrolases. and .4mwioriitrtri chr?..\onc,rrirr/i. an imperfect fungus producing cephalosporin C and extracellular protease, were used i n this study. . ~ ~ / t i r / i ~ j ~ f i sp. ~ j / /: ~The / , ~ bacterial culture n as gro\vn in ;i complex medium containing organic sources of nitrogen. monos;iccharides a n d niiner;il s:ilts ( UVAKOV ('t d.1992). The fermentation was conducted in A 1000-1 fermenter for 24 hours at a temperature of 2 1 C. .Acriwiouiirm ~ , / t ~ ~ , ~ ~ / ~ ( ~A / i imethioniner/f/; deficient strain ot' the fungus was u x d . Strain cultivation aiid determination of its enzymatic activity were described pi-eviously (BARTOSIIEYIC'H er rrl. 1990). The pH value of the fermentation broth ol' both cultures h a s measured potentiometrically. Biomass W H determined ~ nephelometricall). The respiration rate of the cultures was estimated by measuring oxygen consumption with a thermomagnetic gas analyzer. The peptidohydrolase activity was assayed LC-5000. Gel-many). The coagulase activity of the cultures with an amino acid analyzer ( BIOTKOSIC' w s determined by a modification of the assay method for staphylocoagulase (BAS (11. 1975) with a clinical congulogrnph N-334 ( USSR) . Phc modification provided a rapid and quantitative assay of the coagulating activity of the fcrmontation broths. Rabbit and human sera where used as substrates. Tests with bo\ine blood serum fibrinogen and thromboplastin dilutions ( 10- per cent) based o n the method of BAS ('I ti/. (1975) \\ere emplu)cd a s controls. Thc activity was expressed as the time rcyuircd for coagulation ol' the scruni i n tho reaction mixture. Electron microscopy of thin sections of the cultures was performed as described elsewhere (BAKTOSHEVIC'H et t i / . 1990) using a JEM- I OOB electron microscope (JEOL. Japan ). The sections w r c prepared with the OmU-3 ultramicrotome ( REICIITRT. Austi-ia) and stained with uranyl acetate (WATSON1958) or lead citrate (VENABLE and Co(;c;LsiiALi. 1965). (31
'
Results and discussion n.lolplic!firiic,ricl,lNI arici1j.si.r
sf' tk.i-rloiJiiir,rit
cf
. ~ ( i i i t / i o i } i o i i ( / . ssp
The pattern of culture development in complex niediuin for biosynthesis of peptidohydrolases was extremely specific. The lag-phase was rather long (Fig. 1) and the processess in the main part of the cells. introduced into the medium as the inoculum, could be considered as specific autolysis (Fig. 2). It was characteristic of these processes that the cell envelopes were the first to be damaged at the sites o f contact with the medium aggregates or coagulated cell clots (Fig. 2. a and b). This was followed by damage to the nucleoids, and the cells gradually converted to the destructive state (Fig. 2. c and d ) with condensation and dehydration of the cytoplasm while the shape of the cells remained unchanged (Fig. I d ) . Later. the destructed cells were adsorbed to the coagulated cells. The processes were likely to involve cells only of ii definite physiological age, i.e., the cells in the steady-state growth phase. which were introduced into the medium as the inoculum. In contrast, the young cells with higher capacity for adaptation and rehabilitation of their metabolism continued to grow and develop. Destruction and coagulation of the cell proteins. followed by formation of cell clots. were observed until the 12th or the 16th hour of the culture development, i.e., until the middle of the exponential growth phase, and required participation of specific enzyinatics systems. Oiily during that time period coagulase activity Itxs detected in the fermentation broth (Fig. 1). whereas before and after that period the activity could not be detected. The coagulase activity in the samples o r the 12- 16 hour fermentation broth was equal to 0.2 11 ;IS compared to 0.01 h in the control.
Coagulation autolysis in microorganisms
8.1
417
I
79-=. \
77-2 z
4 7 5 -
g
73-
2 50
-
40
71
30 69-
67-
10 10
65-
0
0
10
14
18 22 26 Time ( h o u r s )
30
Dynamics of the growth. respiration rate, proteolytic and coagulation activity and pH of the fermentation broth of Xcnzfhonzorzas sp. - Time of the enzyme-coagulating effect
It should be noted that the coagulase activity in the fermentation broths was detected in the experiments with blood serum of both rabbits and humans. After the beginning of the exponential growth phase, biosynthesis of peptidohydrolases, cleaving the proteins, started (Fig. 1). It could be speculated that the primary response of the culture, i.e., coagulation autolysis of the cells and formation of coagulase, serves as a stimulus for production of the enzymes, thus hydrolyzing the proteins which are then utilized by the culture to support its growth. Both processes, i.e., the culture growth and biosynthesis of peptidohydrolases, proceeded in parallels (Fig. 1). The dividing young cells could probably not serve as substrates for coagulase action, which made it possible to detect the enzyme activity in the fermentation broth. During the period preceding the culture's transition into the steady-state growth phase, when the rate of peptidohydrolase synthesis was maximum (Fig. l), the structure of the cells sharply changed and they were found to be practically in a state of shock. The cell membranes proved to be either damaged or hypertrophied, which resulted in formation of the membrane festons (invasions, invagination) and promotion of intercellular contacts (Fig. 20. Such processes were as a rule observed in microbial cultures developing under unfavourable conditions. At the transition to the steady-state growth phase the cells would actively liberate excessive amounts of the enzyme and there after synthesize it at a rather low rate (Fig. 1). This provides a balance between cell development and autolyis. The shock was accompanied by a sharp rise in the culture respiration rate (Fig. l), which was likely due to increased accessibility of substrates to the intracellular enzymes because of higher permeability of the cell envelopes. This was likely a borderline state which could result either in autolysis or in stabilization of the culture, depending on the developmental conditions. Under the conditions of our experiments the cultures stabilized, the structure of the cell envelopes normalized and the murein layer became thicker (Fig. 28). The cells remained in such a condition during the prolonged steady-state period of culture development. Therefore, the process of peptidohydrolase synthesis in Xanthomonas sp. 31) J . Basic Microbiol. 32 ( I Y Y 2 ) 6
41 8
P. L. ZASLAVSKAYA e t ril.
Fig. 2 Ultrxtructurc ol‘ . ~ . ~ / / / / / / ~ ~ / ~ /sp. / ~ / /cells ~ ~ . \ during their development in the fermentation medium. a ) The cell p(>pulation nith coagulated c!toplasni of the aiitolyzed cells, 8 h. b) Impairment of c~toplasmicmcmbraiics i i t thc sites oftheir contact with the coagulated cells, 10 h c) Further destruction 01’ the ccll orymcllcs. 10 h. d ) Diffcrcnt stages of coagulation autolysis of the cells, 12 h. c) The cell niemhriines i n thc st,itc ol.shock. I 9 h f) Stable condition of the cells in the growth steady-state phase. 2-1 11. The bciilc i i i Figs. 3 and -7 is equal t o I pin
Coagulation autolysis in microorganisms
419
Fig. 3 Coagulation autolysis in A . c/w~~.soger~imz. a) The population with the dcstructcd cells in the form of coagulated cytoplasm, 48 h. b ) Impairment of the cytoplasmic membranes and membranes of the mitochondria and vacuoles, 24 h. c ) Coagulation autolysis of thc cells with preserved shape and cell walls. 24 h. d ) Removal of the walls from the cells with impaircd membranes, 24 h. e) Condensation and dehydration of the necrotizcd cells. 48 h 30'
420
P. L. ZASLAVSKAYA et (11.
was quite logical in both the succession of the syntheses of various substrate-specific enzymes and the specific structural changes in the cells, accompanying such syntheses, which allowed the cells to adapt to the changing intracellular and extracellular conditions. Cougirlrrtioii ~riito!,~.ris i i i Acr.emoiiiiinz chrysogerizirn
Detailied structural analysis of A . c~hrj~sogenuriz morphogenesis in relation to the synthesis of cephalosporin C and protease (BARTOSHEVICH er a/. 1990) has shown that during the entire developmental period specific destructed cells were present in the culture. Such cells had coagulated cytoplasm deprived of any organelles. yet their shape remained unchanged (Fig. 3a). Analysis of autolysis in such cells revealed that it was accompanied by impairment of the cytoplasmic membranes. as well as the membranes of the mitochondria and vacuoles (Fig. 3, ii and b). Later. the cells with the impaired membranes appeared to shed their cells (Fig. 3, c and d) and condensation and dehydration of the necrotized cells (Fig. 3, a and e) was observed. Dehydration and coagulation of the cytoplasm of the cells of the imperfect fungus, as with the cells of higher organisms led to formation of stable products, not subject to hydrolytic cleavage for ii prolonged period of time. This accounts for the presence of coagulated cells in the culture throughout the entire period of its development. It should be noted that A . chrjmgeriuni was characterized by dimorphism, i.e., development of either yeast-like or mycelial cells (NASHand HUBER1971). During the developmental period the mycelial cells are transformed into the yeast-like cells, and the latter synthesized cephalosporin C and protease (BARTOSHEVICH et a / . 1990). Coagulation autolysis was also specific only for the yeast-like cells. The highest levels of autolysis were detected during the lag- and exponential growth phases (Fig. 4). During this time the physiologically mature yeast-like cells, added as the inoculum to the medium were destructed, probably because of their inability to adapt to the presence of the new proteins, to change
70 30
40
60
80
100
170
140
160
Time [ h o u r J l
Fig. 4 Dynamics of the growth, proteolytic and coagulating activity of A . chrj~so,gmuril. - Time of the enzyme-coagulating effect
Coagulation autolysis in microorganisms
42 1
their metabolism and to start synthesis of peptidohydrolases under such conditions. The coagulation autolysis in the fungus was accompanied by production of coagulase, with a maximum observed at 24 hours of the culture development (Fig. 4). The coagulase activity in the 24-hour fermentation broth was equal to 0.1 or 0.2 compared to 0.01 in the control. Thus, the yeast-like cells of the culture synthesized protease during the proliferation period (Fig. 4) and then self-destructed by synthesizing coagulase under specific conditions not favourable for their development. It could be concluded that to survive under such conditions the culture began to form mycelial cells. Therefore, coagulation autolysis in A. chrysogenum could be considered as a form of culture adaptation to the new conditions of a medium enriched with proteins, under which the physiologically younger cells survived and began to develop and synthesize peptidohydrolases with proteolytic activity. Corzclusioris
The phenomenon of coagulation autolysis was observed on two model microorganisms, i.e., a bacterial culture and an imperfect fungus. The process was shown to be characteristic of both the prokaryote and eukaryote. It proceeded in the media fabourable for cellular synthesis of peptidohydrolases. Under such conditions coagulation autolysis may take place in cells which were unable to induce iiiiinediately the synthesis of proteolytic enzymes to cleave proteins. Coagulation autolysis in these microorganisms was characterized by impairment of the cell membranes, followed by condensation and dehydration of the cytoplasm and long-term preservation of the cells in the form of coagulated cytoplasm. In this respect it was similar to coagulation necrosis of human cells. Coagulation autolysis in these microorganisms was accompanied by synthesis of coagulase, whose activity manifested itself in blood sera of humans and animals, indicating that its substrate specificity was rather broad. The coagulase activity in these microorganisms was detected during the transition from the lag-phase to the exponential growth phase, i.e., the phase of their active growth. This served as a signal to induce biosynthesis of peptidohydrolases and cleavage of proteins. These observations are in good agreement with published data (ORTROSHKO and EGOROV 1975). It may be speculated that coagulase promoted autolysis of those cells whose metabolism was not sufficient for them to compete with the younger cells which were able to actively synthesize proteases. In this way, the differentation of the cell population in the culture is regulated, providing for active utilization of nutrients and coagulated cell clots as well as culture growth. In A . chr.vsogenur?.l biosynthesis of protease was also associated with transition of the cells from the mycelial form to the yeast-like form (BARTOSHEVICH et a/. 1990).In the latter production of pratease was connected with synthesis of the cell walls. In Xmthorizorias sp. it was associated with culture proliferation. Such an explanation of the phenomenon of coagulation autolysis in miroorganisms conforms to the logic of biochemical processes: While the actitivity of an enzyme is changing. its regulatory role often increases through a cascade mechanism, providing formation of large quantities of an active enzyme which is the last in a chain (METZLER 1988).In our experiments this was proteases. The first general response of the cells of Xunrhomoiius sp. and A . chrysogenum to coagulation autolysis was impairment of cell membranes, whereas at the time of maximum biosynthesis of intracellular peptidases there was a sharp but reversible change in the state of the cell membranes in the bacterial culture. Such responses of the cells are probably of an adaptative nature and allow the culture to adapt to new environmental conditions and to change its metabolism. The concept of KREBS(cit. from BOLDYREV 1986) suggestes that
422
P. L. ZASLAVSKAYA et ul.
biological membranes are the targets of any unfavourable effect and are the first to respond to changing environmental conditions. thus adapting to them. Therefore. we believe that the phenomenon of coagulation autolysis in microorganisms is rather typical and could be considered as an adaptative reaction, inducing a cascade of events from synthesis of coagulases to overproduction of peptidohydrolases with proteolytic activity, accompanied by changes in the culture morphogenesis at various structural levels. The results of the study on coagulation autolysis in microorganisms are of interest for medical biochemistry and could provide a model for coagulation necrosis of human or animal tissue. They could also be useful in the development of biotechnological processes for the production of peptidohydrolase. References BARTOSHFVICII. Yr. E.. ZASI.AVSKAY.A. P. L.. NOYAK.M. I . and YUDINA.0. D.. 1990. Acremonium chrysogenum : Diffcrenration and biosynthesis ofcephalosporin C. J. Basic Microbiol., 30.3 I 3 - 320. BAS. 9. U . . MPILER.A. D. and HI.MKER, H. C.. 1975. Purification and properties of Staphylocoagulase. Bioph. Acta. 379. 164-171. BOLDYRW. A. A.. 1986. Biochemistry of membranes: Introduction in Biochemistry of Membranes. Moscow. H. S . 73. CHERDYKTZEVA. T. A . and EGOROV.N. S . . 1988. Production of extracellular proteases with the activity of blood plasma coagulation and blood d o t lysis by fungi of the genera ..lspqqi//ir.s, Aciari~oriiuri~ and I ' w r i c i l l t w i i . Microbiologia. 57. 574 578. NASH.C. H. and HUBER.F. M.. 1971. Antibiotic s)nthesis and morphological differentiation of Ce~lfN/o.s~~:l,or.rirr,l ~/creufo~1iir~17. Appl. Micro biul.. 14. 446 - 453. METZ1.I.R. D. E.. 1977. Biochemistry. The Chemical Reactions of Living Cells. Academic Press. Inc.. N Y . San Francisco. London: 2. 72. OTROSIIKO. T. A. and EGOROV.N. S . , 1975, Formation of plasma coagulating coagulases by bacteria of the genus Btrcdlirs. Biol. Nauki. 7. 95-98 (Mosco\v). SATON. T.. B ~ P P YT. . and A R l h l A . K.. 1975. Microbial blood-coagulating protcase screening test and somc propertie\. .Agr. Biol.. Cheni.. 39. 1565 - 1571. SEROV.V. V. arid I'AL'KOV. V. S . . 1975. Ultrastructural Pathology. Moscow, Mcditsina. 157- 160 (in Russian). UVAROV.H. H.. KRESTYAVOVA. 1. N. and DMITRIEVA. s. V.. 1992. Relationship between morphogenesis of .~irrithurtioricisrirbr.ilirt~ririsbacterial culture and biosynthesis o f aminopeptidase during the culture growth and development. Antibiotics and Chemotherapy (Moscow).37. 16 - 19. VENAHLF.J. H. and COGGESHALL. R. A. 196s. A simplilied lead citrate strain for use in electron microscop). Cell Biol.. 5. 407-408. WATSON.M . L.. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Bioph.. Biochem. Cytol.. 4. 375-480. ~
National Research Center of Antibiotics, Nagatinskaya Mailing address: Dr. P. L. ZASI.AVSKAYA. M o s c o ~ .I 13105. Russi2i.
321.
(National Research Centre of Antibiotics. Moscow, Russia)
Coagulation autolysis in microorganisms and its relation to coagulase production P. L. ZASLAVSKAYA, I. V. CHEKALINA, P. S. NYSand Yu. E. BARTOSHEVICH ( Receioed
-79 October 199llAccepted 16 J m e 19921
The phenomenon of coagulation autolysis was observed in two model microorganisms. i.e.. a bacterial culture and an imperfect fungus. I t was characterized by impairment of the cell membranes, followed by condensation and dehydration of the cytoplasm and long-term preservation of the cells in the form of coagulated cytoplasm. In this respect. it was similar to coagulation necrosis of human tissues. The autolysis in the microorganisms was accompanied by increase of their coagulase activity, the substrate specificity of the enzyme rather broad. The coagulase activity of the microorganisms was detected during the culture period between the lag-phase and the exponential growth phase, i.e., the phase of their active growth. It served as a signal to induce biosynthesis of peptidohydrolase and cleavage of proteins. We believe that the phenomenon of coagulation autolysis in these microorganisms is rather typical and can be considered as an adaptative reaction, inducing a cascade of events from synthesis of coagulase to overproduction of peptidohydrolases with proteolytic activity.
Coagulation necrosis of human tissues is a well known phenomenon. It is characterized by coagulation and pronounced denaturation of cytoplasmic proteins and the cytoplasm condensation and dehydration (SEROVand PAUKOV1975). Still, the mechanisms of its development are not clear and any information to the point is essential for elucidating the causes of pathological processes in living tissues. Microorganisms represent a unique model for investigating the development of many processes from both functional and morphological viewpoints. Coagulation autolysis in microorganisms has not been described so far. Optical microscopy, mainly used in studies on microorganisms, cannot reveal the succession of the autolysis events nor even distinguish between coagulated and living cells. By the use of ultrastructural analysis it was possible to observe unusual aggregation and disintegration of cells during development of the fungus producing cephatosporin C and protease, as described by us previously ( BARTOSHEVICH et nl. 1990). Investigation of microorganisms producing peptidohydrolases made it possible to reveal in them processes similar to autolysis and to raise the problem of the relationship between these processes and the enzymatic activity of microbial cells. Within the last decade a number o papers were published which showed that some microbial proteases in fungi and bacteria possess coagulating activity (SATONet a/. 1975. OTROSHKO and EGOROV1975, CHERDYNTSEVA and EGOROV1988). In some of these it was indicated that only cell extracts possessed such activity, which means that the coagulating acitivity was associated with the microbial cells. Some studies suggested a correlative relationship between the coagulating and proteolytic activity in microorganisms (SATON ef nl. 1975). though in the majority of actinomycetes, as well as in some other bacteria and in fungi, no such relationship was observed. It was also indicated that the activity spectrum of coagulase in many of the microbial cultures studied was rather broad and the cultures were shown to coagulate blood sera of humans, cows, rabbits, mice and chicken. The present paper is concerned with the morphofunctional analysis of development of two microorganisms differing in the level of their organization, i.e., a bacterial culture and
416
P. L. ZASLAVSKAYA et d .
an imperfect fungus producing peptidohydrolases. The analysis included investigation of the cell ultrastructure and enzymatic activity of the cultures. Such an approach allowed us to propose a model of coagulation autolysis i n microbial cells due to their coagulating and proteolytic activity.
Materials and methods Two organism5. i.e.. , ~ [ / / i t / i ~ j / f / ( j / i sp.. ~ / s a bacterial culture producing peptidohydrolases. and .4mwioriitrtri chr?..\onc,rrirr/i. an imperfect fungus producing cephalosporin C and extracellular protease, were used i n this study. . ~ ~ / t i r / i ~ j ~ f i sp. ~ j / /: ~The / , ~ bacterial culture n as gro\vn in ;i complex medium containing organic sources of nitrogen. monos;iccharides a n d niiner;il s:ilts ( UVAKOV ('t d.1992). The fermentation was conducted in A 1000-1 fermenter for 24 hours at a temperature of 2 1 C. .Acriwiouiirm ~ , / t ~ ~ , ~ ~ / ~ ( ~A / i imethioniner/f/; deficient strain ot' the fungus was u x d . Strain cultivation aiid determination of its enzymatic activity were described pi-eviously (BARTOSIIEYIC'H er rrl. 1990). The pH value of the fermentation broth ol' both cultures h a s measured potentiometrically. Biomass W H determined ~ nephelometricall). The respiration rate of the cultures was estimated by measuring oxygen consumption with a thermomagnetic gas analyzer. The peptidohydrolase activity was assayed LC-5000. Gel-many). The coagulase activity of the cultures with an amino acid analyzer ( BIOTKOSIC' w s determined by a modification of the assay method for staphylocoagulase (BAS (11. 1975) with a clinical congulogrnph N-334 ( USSR) . Phc modification provided a rapid and quantitative assay of the coagulating activity of the fcrmontation broths. Rabbit and human sera where used as substrates. Tests with bo\ine blood serum fibrinogen and thromboplastin dilutions ( 10- per cent) based o n the method of BAS ('I ti/. (1975) \\ere emplu)cd a s controls. Thc activity was expressed as the time rcyuircd for coagulation ol' the scruni i n tho reaction mixture. Electron microscopy of thin sections of the cultures was performed as described elsewhere (BAKTOSHEVIC'H et t i / . 1990) using a JEM- I OOB electron microscope (JEOL. Japan ). The sections w r c prepared with the OmU-3 ultramicrotome ( REICIITRT. Austi-ia) and stained with uranyl acetate (WATSON1958) or lead citrate (VENABLE and Co(;c;LsiiALi. 1965). (31
'
Results and discussion n.lolplic!firiic,ricl,lNI arici1j.si.r
sf' tk.i-rloiJiiir,rit
cf
. ~ ( i i i t / i o i } i o i i ( / . ssp
The pattern of culture development in complex niediuin for biosynthesis of peptidohydrolases was extremely specific. The lag-phase was rather long (Fig. 1) and the processess in the main part of the cells. introduced into the medium as the inoculum, could be considered as specific autolysis (Fig. 2). It was characteristic of these processes that the cell envelopes were the first to be damaged at the sites o f contact with the medium aggregates or coagulated cell clots (Fig. 2. a and b). This was followed by damage to the nucleoids, and the cells gradually converted to the destructive state (Fig. 2. c and d ) with condensation and dehydration of the cytoplasm while the shape of the cells remained unchanged (Fig. I d ) . Later. the destructed cells were adsorbed to the coagulated cells. The processes were likely to involve cells only of ii definite physiological age, i.e., the cells in the steady-state growth phase. which were introduced into the medium as the inoculum. In contrast, the young cells with higher capacity for adaptation and rehabilitation of their metabolism continued to grow and develop. Destruction and coagulation of the cell proteins. followed by formation of cell clots. were observed until the 12th or the 16th hour of the culture development, i.e., until the middle of the exponential growth phase, and required participation of specific enzyinatics systems. Oiily during that time period coagulase activity Itxs detected in the fermentation broth (Fig. 1). whereas before and after that period the activity could not be detected. The coagulase activity in the samples o r the 12- 16 hour fermentation broth was equal to 0.2 11 ;IS compared to 0.01 h in the control.
Coagulation autolysis in microorganisms
8.1
417
I
79-=. \
77-2 z
4 7 5 -
g
73-
2 50
-
40
71
30 69-
67-
10 10
65-
0
0
10
14
18 22 26 Time ( h o u r s )
30
Dynamics of the growth. respiration rate, proteolytic and coagulation activity and pH of the fermentation broth of Xcnzfhonzorzas sp. - Time of the enzyme-coagulating effect
It should be noted that the coagulase activity in the fermentation broths was detected in the experiments with blood serum of both rabbits and humans. After the beginning of the exponential growth phase, biosynthesis of peptidohydrolases, cleaving the proteins, started (Fig. 1). It could be speculated that the primary response of the culture, i.e., coagulation autolysis of the cells and formation of coagulase, serves as a stimulus for production of the enzymes, thus hydrolyzing the proteins which are then utilized by the culture to support its growth. Both processes, i.e., the culture growth and biosynthesis of peptidohydrolases, proceeded in parallels (Fig. 1). The dividing young cells could probably not serve as substrates for coagulase action, which made it possible to detect the enzyme activity in the fermentation broth. During the period preceding the culture's transition into the steady-state growth phase, when the rate of peptidohydrolase synthesis was maximum (Fig. l), the structure of the cells sharply changed and they were found to be practically in a state of shock. The cell membranes proved to be either damaged or hypertrophied, which resulted in formation of the membrane festons (invasions, invagination) and promotion of intercellular contacts (Fig. 20. Such processes were as a rule observed in microbial cultures developing under unfavourable conditions. At the transition to the steady-state growth phase the cells would actively liberate excessive amounts of the enzyme and there after synthesize it at a rather low rate (Fig. 1). This provides a balance between cell development and autolyis. The shock was accompanied by a sharp rise in the culture respiration rate (Fig. l), which was likely due to increased accessibility of substrates to the intracellular enzymes because of higher permeability of the cell envelopes. This was likely a borderline state which could result either in autolysis or in stabilization of the culture, depending on the developmental conditions. Under the conditions of our experiments the cultures stabilized, the structure of the cell envelopes normalized and the murein layer became thicker (Fig. 28). The cells remained in such a condition during the prolonged steady-state period of culture development. Therefore, the process of peptidohydrolase synthesis in Xanthomonas sp. 31) J . Basic Microbiol. 32 ( I Y Y 2 ) 6
41 8
P. L. ZASLAVSKAYA e t ril.
Fig. 2 Ultrxtructurc ol‘ . ~ . ~ / / / / / / ~ ~ / ~ /sp. / ~ / /cells ~ ~ . \ during their development in the fermentation medium. a ) The cell p(>pulation nith coagulated c!toplasni of the aiitolyzed cells, 8 h. b) Impairment of c~toplasmicmcmbraiics i i t thc sites oftheir contact with the coagulated cells, 10 h c) Further destruction 01’ the ccll orymcllcs. 10 h. d ) Diffcrcnt stages of coagulation autolysis of the cells, 12 h. c) The cell niemhriines i n thc st,itc ol.shock. I 9 h f) Stable condition of the cells in the growth steady-state phase. 2-1 11. The bciilc i i i Figs. 3 and -7 is equal t o I pin
Coagulation autolysis in microorganisms
419
Fig. 3 Coagulation autolysis in A . c/w~~.soger~imz. a) The population with the dcstructcd cells in the form of coagulated cytoplasm, 48 h. b ) Impairment of the cytoplasmic membranes and membranes of the mitochondria and vacuoles, 24 h. c ) Coagulation autolysis of thc cells with preserved shape and cell walls. 24 h. d ) Removal of the walls from the cells with impaircd membranes, 24 h. e) Condensation and dehydration of the necrotizcd cells. 48 h 30'
420
P. L. ZASLAVSKAYA et (11.
was quite logical in both the succession of the syntheses of various substrate-specific enzymes and the specific structural changes in the cells, accompanying such syntheses, which allowed the cells to adapt to the changing intracellular and extracellular conditions. Cougirlrrtioii ~riito!,~.ris i i i Acr.emoiiiiinz chrysogerizirn
Detailied structural analysis of A . c~hrj~sogenuriz morphogenesis in relation to the synthesis of cephalosporin C and protease (BARTOSHEVICH er a/. 1990) has shown that during the entire developmental period specific destructed cells were present in the culture. Such cells had coagulated cytoplasm deprived of any organelles. yet their shape remained unchanged (Fig. 3a). Analysis of autolysis in such cells revealed that it was accompanied by impairment of the cytoplasmic membranes. as well as the membranes of the mitochondria and vacuoles (Fig. 3, ii and b). Later. the cells with the impaired membranes appeared to shed their cells (Fig. 3, c and d) and condensation and dehydration of the necrotized cells (Fig. 3, a and e) was observed. Dehydration and coagulation of the cytoplasm of the cells of the imperfect fungus, as with the cells of higher organisms led to formation of stable products, not subject to hydrolytic cleavage for ii prolonged period of time. This accounts for the presence of coagulated cells in the culture throughout the entire period of its development. It should be noted that A . chrjmgeriuni was characterized by dimorphism, i.e., development of either yeast-like or mycelial cells (NASHand HUBER1971). During the developmental period the mycelial cells are transformed into the yeast-like cells, and the latter synthesized cephalosporin C and protease (BARTOSHEVICH et a / . 1990). Coagulation autolysis was also specific only for the yeast-like cells. The highest levels of autolysis were detected during the lag- and exponential growth phases (Fig. 4). During this time the physiologically mature yeast-like cells, added as the inoculum to the medium were destructed, probably because of their inability to adapt to the presence of the new proteins, to change
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Fig. 4 Dynamics of the growth, proteolytic and coagulating activity of A . chrj~so,gmuril. - Time of the enzyme-coagulating effect
Coagulation autolysis in microorganisms
42 1
their metabolism and to start synthesis of peptidohydrolases under such conditions. The coagulation autolysis in the fungus was accompanied by production of coagulase, with a maximum observed at 24 hours of the culture development (Fig. 4). The coagulase activity in the 24-hour fermentation broth was equal to 0.1 or 0.2 compared to 0.01 in the control. Thus, the yeast-like cells of the culture synthesized protease during the proliferation period (Fig. 4) and then self-destructed by synthesizing coagulase under specific conditions not favourable for their development. It could be concluded that to survive under such conditions the culture began to form mycelial cells. Therefore, coagulation autolysis in A. chrysogenum could be considered as a form of culture adaptation to the new conditions of a medium enriched with proteins, under which the physiologically younger cells survived and began to develop and synthesize peptidohydrolases with proteolytic activity. Corzclusioris
The phenomenon of coagulation autolysis was observed on two model microorganisms, i.e., a bacterial culture and an imperfect fungus. The process was shown to be characteristic of both the prokaryote and eukaryote. It proceeded in the media fabourable for cellular synthesis of peptidohydrolases. Under such conditions coagulation autolysis may take place in cells which were unable to induce iiiiinediately the synthesis of proteolytic enzymes to cleave proteins. Coagulation autolysis in these microorganisms was characterized by impairment of the cell membranes, followed by condensation and dehydration of the cytoplasm and long-term preservation of the cells in the form of coagulated cytoplasm. In this respect it was similar to coagulation necrosis of human cells. Coagulation autolysis in these microorganisms was accompanied by synthesis of coagulase, whose activity manifested itself in blood sera of humans and animals, indicating that its substrate specificity was rather broad. The coagulase activity in these microorganisms was detected during the transition from the lag-phase to the exponential growth phase, i.e., the phase of their active growth. This served as a signal to induce biosynthesis of peptidohydrolases and cleavage of proteins. These observations are in good agreement with published data (ORTROSHKO and EGOROV 1975). It may be speculated that coagulase promoted autolysis of those cells whose metabolism was not sufficient for them to compete with the younger cells which were able to actively synthesize proteases. In this way, the differentation of the cell population in the culture is regulated, providing for active utilization of nutrients and coagulated cell clots as well as culture growth. In A . chr.vsogenur?.l biosynthesis of protease was also associated with transition of the cells from the mycelial form to the yeast-like form (BARTOSHEVICH et a/. 1990).In the latter production of pratease was connected with synthesis of the cell walls. In Xmthorizorias sp. it was associated with culture proliferation. Such an explanation of the phenomenon of coagulation autolysis in miroorganisms conforms to the logic of biochemical processes: While the actitivity of an enzyme is changing. its regulatory role often increases through a cascade mechanism, providing formation of large quantities of an active enzyme which is the last in a chain (METZLER 1988).In our experiments this was proteases. The first general response of the cells of Xunrhomoiius sp. and A . chrysogenum to coagulation autolysis was impairment of cell membranes, whereas at the time of maximum biosynthesis of intracellular peptidases there was a sharp but reversible change in the state of the cell membranes in the bacterial culture. Such responses of the cells are probably of an adaptative nature and allow the culture to adapt to new environmental conditions and to change its metabolism. The concept of KREBS(cit. from BOLDYREV 1986) suggestes that
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biological membranes are the targets of any unfavourable effect and are the first to respond to changing environmental conditions. thus adapting to them. Therefore. we believe that the phenomenon of coagulation autolysis in microorganisms is rather typical and could be considered as an adaptative reaction, inducing a cascade of events from synthesis of coagulases to overproduction of peptidohydrolases with proteolytic activity, accompanied by changes in the culture morphogenesis at various structural levels. The results of the study on coagulation autolysis in microorganisms are of interest for medical biochemistry and could provide a model for coagulation necrosis of human or animal tissue. They could also be useful in the development of biotechnological processes for the production of peptidohydrolase. References BARTOSHFVICII. Yr. E.. ZASI.AVSKAY.A. P. L.. NOYAK.M. I . and YUDINA.0. D.. 1990. Acremonium chrysogenum : Diffcrenration and biosynthesis ofcephalosporin C. J. Basic Microbiol., 30.3 I 3 - 320. BAS. 9. U . . MPILER.A. D. and HI.MKER, H. C.. 1975. Purification and properties of Staphylocoagulase. Bioph. Acta. 379. 164-171. BOLDYRW. A. A.. 1986. Biochemistry of membranes: Introduction in Biochemistry of Membranes. Moscow. H. S . 73. CHERDYKTZEVA. T. A . and EGOROV.N. S . . 1988. Production of extracellular proteases with the activity of blood plasma coagulation and blood d o t lysis by fungi of the genera ..lspqqi//ir.s, Aciari~oriiuri~ and I ' w r i c i l l t w i i . Microbiologia. 57. 574 578. NASH.C. H. and HUBER.F. M.. 1971. Antibiotic s)nthesis and morphological differentiation of Ce~lfN/o.s~~:l,or.rirr,l ~/creufo~1iir~17. Appl. Micro biul.. 14. 446 - 453. METZ1.I.R. D. E.. 1977. Biochemistry. The Chemical Reactions of Living Cells. Academic Press. Inc.. N Y . San Francisco. London: 2. 72. OTROSIIKO. T. A. and EGOROV.N. S . , 1975, Formation of plasma coagulating coagulases by bacteria of the genus Btrcdlirs. Biol. Nauki. 7. 95-98 (Mosco\v). SATON. T.. B ~ P P YT. . and A R l h l A . K.. 1975. Microbial blood-coagulating protcase screening test and somc propertie\. .Agr. Biol.. Cheni.. 39. 1565 - 1571. SEROV.V. V. arid I'AL'KOV. V. S . . 1975. Ultrastructural Pathology. Moscow, Mcditsina. 157- 160 (in Russian). UVAROV.H. H.. KRESTYAVOVA. 1. N. and DMITRIEVA. s. V.. 1992. Relationship between morphogenesis of .~irrithurtioricisrirbr.ilirt~ririsbacterial culture and biosynthesis o f aminopeptidase during the culture growth and development. Antibiotics and Chemotherapy (Moscow).37. 16 - 19. VENAHLF.J. H. and COGGESHALL. R. A. 196s. A simplilied lead citrate strain for use in electron microscop). Cell Biol.. 5. 407-408. WATSON.M . L.. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Bioph.. Biochem. Cytol.. 4. 375-480. ~
National Research Center of Antibiotics, Nagatinskaya Mailing address: Dr. P. L. ZASI.AVSKAYA. M o s c o ~ .I 13105. Russi2i.
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