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Nitrogen starvation-induced glycogen synthesis depends on the developmental stage of Streptomyces antibioticus mycelium
FEMS Microbiology Letters 153 (1997) 57^62
Nitrogen starvation-induced glycogen synthesis depends on the developmental stage of Streptomyces antibioticus mycelium Elisa M. Migueèlez, Moènica Fernaèndez, Carlos Hardisson * èa, Facultad de Medicina and Instituto Universitario de Biotecnolog| èa de Asturias, Universidad de Oviedo, Area de Microbiolog| è n Claver| èa s/n, 33006 Oviedo, Spain c/ Julia
Received 24 April 1997; revised 9 May 1997; accepted 13 May 1997
Abstract
Experiments carried out to examine the ability of Streptomyces antibioticus to accumulate glycogen, when starved for nitrogen at different times during growth, revealed that nitrogen-starved hyphae, irrespective of the developmental time at which they were starved, accumulated glycogen only when they had acquired ultrastructural features typical of stationaryphase cultures. Oleandomycin production and trehalose accumulation were also examined during the starvation period. The observed pattern of oleandomycin production resembles that of glycogen. However, trehalose accumulates with no lag period after nitrogen starvation, regardless of the developmental phase of growth. Keywords :
Glycogen ; Oleandomycin; Trehalose;
Streptomyces
1. Introduction
The ability of streptomycetes to accumulate glycogen has been reported previously [1^5]. This polymer accumulates in a characteristic two-round pattern when streptomycetes are cultured on solid media [3,4]. The ¢rst round of accumulation occurs in the substrate mycelium, just before the emergence of the aerial hyphae, and coincides with the depletion of the nitrogen source in the culture medium. The second round of accumulation occurs in the aerial mycelium and coincides with the initiation of the sporulation process. In the present work, chemical and electron micro* Corresponding author. Tel.: +34 (85) 103557; Fax: +34 (85) 103554; E-mail: [email protected]
scopic techniques were used to investigate the pattern of glycogen accumulation in Streptomyces antibioticus during growth in a liquid culture medium. The results obtained revealed that S. antibioticus is able to respond to nitrogen starvation conditions by accumulating glycogen, but this condition although necessary is not su¤cient. Glycogen accumulation only occurs if the hyphae have attained a de¢nite developmental stage, the morphological features of which clearly coincide with those exhibited by stationary-phase hyphae.
2. Materials and methods
2.1. Microorganism, media and culture conditions
Streptomyces antibioticus
ATCC 11891 was grown
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 3 1 - 0
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E.M. Migueèlez et al. / FEMS Microbiology Letters 153 (1997) 57^62
in a modi¢ed GAE medium (MGAE) containing 0.5% (w/v) glucose, 0.1% (w/v) asparagine, 0.05% (w/v) yeast extract, 0.05% (w/v) HK2 PO4 , 0.05% (w/v) MgSO4 , 0.0001% (w/v) FeSO4 and 2.1% (100 mM) MOPS bu¡er (pH 7.0). Freshly harvested spores [6] were suspended in MGAE medium to an OD580 =0.1 and incubated in 2 l £asks, containing 400 ml of culture. For nitrogen starvation experiments, samples (400 ml) of cultures grown in MGAE medium were harvested by centrifugation (8000Ug, 8 min, 25³C), then washed with 100 mM MOPS bu¡er (pH 7.0) and resuspended in an equal volume of prewarmed nitrogen starvation medium (NSM). NSM is MGAE containing 2% (w/v) glucose, 0.01% (w/v) yeast extract and lacking asparagine. All the cultures were incubated at 35³C with shaking (250 rpm). Growth was followed by measuring dry cell weight and OD580 , and the morphological stage of the culture was monitored by phasecontrast microscopy. 2.2. Analytical procedures 2.2.1. Glucose and nitrogen measurements
Glucose and nitrogen levels in the culture media were estimated by the glucose oxidase-peroxidase method [7] and by the procedure of Rosen [8] respectively. 2.2.2. Polysaccharide determinations
For glycogen determination, samples of 40 ml of the culture were centrifuged. The resulting pellets were resuspended in water, heated at 100³C for 10 min and then disrupted by ultrasonic treatment (6 min in a 150 W MSE Ultrasonic disintegrator at 0³C). The glycogen content of the crude extracts was determined enzymatically, as previously described [2]. 2.2.3. Trehalose determinations
For trehalose determination, we used the crude extracts described above. The following mixtures were prepared: (1) 80 ml of crude extract, 65 ml of 0.1 M phosphate bu¡er (pH 6.0) and 30 ml of trehalase solution (5 mU) (Sigma); (2) 80 ml of crude extract, 65 ml of 0.1 M phosphate bu¡er (pH 6.0) and 30 ml of water. Both mixtures were incubated for 2 h at 37³C, then centrifuged, and the glucose
present in the supernatants was measured as described above. The di¡erence between the glucose values obtained in mixtures 1 and 2 represents the trehalose content in each sample. Previous experiments showed that, under the above conditions, trehalose was completely degraded by trehalase. 2.2.4. Total DNA, RNA and protein content
Triplicate culture samples (1.5 ml) were taken at intervals during growth, 1.5 ml of 0.5 N perchloric acid was added and the samples were chilled in an ice bath for 30 min. After centrifugation, pellets were extracted three times with 0.5 N perchloric acid at 70³C. Supernatants were pooled and assayed either for DNA by the method of Burton [9], or for RNA by the orcinol method [10]. Pellets were dissolved in 1.0 N NaOH and protein was determined by the method of Lowry. 2.2.5. Oleandomycin determination
Samples of cultures were centrifuged (12000Ug, 10 min, 4³C) and oleandomycin content in the supernatants was estimated by bioassay, using a highly oleandomycin-sensitive Micrococcus luteus strain as described earlier [11]. Commercial oleandomycin (Sigma) was used as standard. 2.3. Electron microscopy
Samples of the culture (10 ml) were pre¢xed in 1% (w/v) osmium tetroxide for 10 min and then embedded in agar. To facilitate the longitudinal orientation of the hyphae, the procedure of bacterial resuspension in agar was improved as follows. Pre¢xed samples were ¢ltered through a moist cellulose acetate membrane ¢lter (47 mm diameter, 0.45 mm size, Oxoid) placed on a glass column (Millipore). Melted agar (5 ml; 2% w/v; 45³C) was then added and the glass column was maintained under suction for 5 min. After cooling, the agar cylinder was pushed out from the glass column, the ¢lter was removed and the bottom part of the agar cylinder (containing layered hyphae) was cut into small blocks. The blocks were ¢xed overnight in 1% (w/v) osmium tetroxide [12], washed for 2 h with 2% (w/v) uranyl acetate, dehydrated with acetone and ¢nally embedded in Epon 812. Before polymerization, the samples were properly positioned in £at molds to
FEMSLE 7659 20-10-97
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59
facilitate longitudinal sectioning of the hyphae. Ultrathin sections were stained either with uranyl acetate and lead citrate or with periodic acid, thiocarbohydrazide and silver proteinate [13]. Sections were observed in a Philips EM 300 electron microscope at 60 kV. 3. Results and discussion
3.1. Growth characteristics of S. antibioticus in MGAE medium
The growth characteristics of S. antibioticus in MGAE medium are summarized in Fig. 1. Spore germination started after about 2.5 h of incubation and was followed by a growth phase with a low growth rate (between 3 and 5.5 h of incubation) during which the culture contained a mixture of recently germinated spores with short germ tubes and spores having long unbranched germ tubes (ranging between 8 and 17 mm long). After about 5.5 h of incubation the culture entered a short phase of exponential growth (all the growth parameters: cell dry weight, DNA, RNA and protein, increased linearly). After about 7 h of incubation, there was a gradual transition into stationary phase. Glycogen accumulation was observed several hours after the beginning of the stationary phase of growth (after about 11^12 h of incubation), when only 12% of the nitrogen source initially supplied was present in the culture medium (Fig. 1b). Oleandomycin accumulation followed a pattern closely similar to that of glycogen (Fig. 1c). However, trehalose content was roughly constant regardless of the growth phase, except one peak of accumulation after 10 h of incubation, at early stationary phase. Nevertheless, the concentration of trehalose decreased quickly reaching levels similar to those found during the growth period (Fig. 1c). The ultrastructural changes exhibited by the hyphae during the growth cycle are shown in Fig. 2. Hyphae collected during the exponential period of growth (Fig. 2a; 6 h) typically showed homogeneous moderately electron-dense cytoplasm with the nuclear material dispersed loosely throughout it, perhaps re£ecting a very active state of transcription. Cross-walls and branches were only occasionally
Fig. 1. Growth characteristics of S. antibioticus in MGAE medium. Spores of S. antibioticus were suspended in MGAE medium and incubated at 35³C with shaking. At di¡erent times of incubation, samples (40 ml) were collected and used for measurements. a: OD (7), DNA (R), RNA (b) and protein (E). b: Residual glucose (Z) and ninhydrin-positive compounds (D) in the culture medium. c: Glycogen accumulation (a), trehalose content (F), oleandomycin production (O) and cell dry weight (8). The data are the mean of 5 independent experiments.
seen at this stage of development. During the transition to the stationary phase of growth (after about
FEMSLE 7659 20-10-97
E.M. Migueèlez et al. / FEMS Microbiology Letters 153 (1997) 57^62
60
times transferred to NSM (see Section 2.1). There was no growth in NSM and the culture could be considered a resting cell system. The results obtained are summarized in Fig. 3. Newborn hyphae (between 2.5 and 3.5 h of incubation) were totally unable to accumulate glycogen throughout the starvation peri-
Fig. 2. Ultrastructural changes exhibited by
S. antibioticus
hy-
phae during the growth cycle. Hyphae collected during the exponential period of growth (a), during the transition to the stationary phase of growth (b) and during the stationary phase (c). Bars represent 200 nm.
9 h of incubation), the nucleoids condensed into elongated structures disposed along the major axis of the hyphae (Fig. 2b). Branching and cross-wall formation both occurred with greater frequency during this period. In stationary phase hyphae (12 h), the nuclear material undergoes physical rearrangements and appears divided into highly condensed nuclear
bodies,
processes
have
which
suggests
largely
ceased
that in
transcription
these
nucleoids
(Fig. 2c). Branching and cross-walls were very frequent.
3.2. Glycogen and secondary metabolites accumulation by S. antibioticus after nitrogen starvation
Fig. 3. Glycogen, oleandomycin and trehalose accumulation by
S. antibioticus The ability of glycogen
and
S. antibioticus hyphae to
other
secondary
accumulate
metabolites,
when
starved for nitrogen in the presence of glucose at di¡erent times during growth, was investigated. Cultures were grown in MGAE medium and at di¡erent
after nutrient stress. Cultures of
S. antibioticus
were grown in MGAE medium and at di¡erent incubation times (a, 3.5 h ; b, 5.5 h ; c, 7.5 h) resuspended in NSM medium as indicated in Section 2.1 and analyzed.
F
, trehalose content ;
O
a
, glycogen accumulation ;
, oleandomycin production ;
8
, cell dry
weight minus glycogen. The data are the mean of 5 independent experiments.
FEMSLE 7659 20-10-97
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61
unable to produce antibiotic after nutritional starvation. Hyphae collected after 5.5 h of incubation in MGAE medium were able to produce oleandomycin, but only after a lag period which progressively decreased with culture age as in the case of glycogen (Fig. 3). The capacity of starved cells of
S. antibioticus
to
synthesize trehalose was also tested. As can be observed in Fig. 3, the pattern of trehalose accumulation during starvation was di¡erent from that of glycogen. Irrespective of the growth time at which the samples were collected, trehalose was detected from the beginning with no lag period. At all the times examined, there was a sudden increase in trehalose Fig. 4. Cytochemically stained sections of nitrogen-starved cultures of
S. antibioticus.
In all the nitrogen-starved cultures, glyco-
levels followed by a period of degradation. These results revealed that
S. antibioticus
hyphae
gen accumulation only took place in hyphae exhibiting ultra-
respond to the stress imposed by nitrogen starvation
structural features typical of stationary-phase cultures (a). In no
by accumulating glycogen and secondary metabolites
case were glycogen granules seen in hyphae containing the nu-
such as oleandomycin. Starvation for nitrogen, how-
clear material dispersed throughout the cytoplasm (b). Bars rep-
ever, was not the only condition required to trigger
resent 200 nm.
glycogen synthesis. Nitrogen-starved hyphae accumulated glycogen only when they had acquired, in
od (data not shown). Hyphae collected between 3.5
the starved condition, ultrastructural features typical
and 5.5 h of incubation accumulated glycogen, but
of stationary-phase cultures. This ¢nding seems to
there was a lag period before glycogen synthesis
indicate that a developmental program, reminiscent
could be detected. The extent of this lag period
of that which is expressed in cells entering that peri-
was
od, must be accomplished for nitrogen starvation to
dependent
on
the
time
of
incubation
in
S. antibioticus.
MGAE before exposure to the nutrient stress. As
trigger glycogen accumulation in
Fig. 3a shows, the lag period for hyphae collected
cording to this, the lag period required for glycogen
after 3.5 h of growth was about 5 h and it decreased
to be accumulated in the nitrogen-starved cultures
to 3.5 h for hyphae collected after 5.5 h of growth
would re£ect the time needed for the hyphae to ac-
(Fig. 3b), and practically disappeared for hyphae
complish such a developmental program.
collected after 7.5 h or longer times of growth (Fig. 3c).
Ac-
On the other hand, trehalose accumulates with no lag period after nitrogen starvation, regardless of the
The observation of cytochemically stained sections
developmental phase of growth, thus probably indi-
in the electron microscope revealed that, in all the
cating two distinct regulatory mechanisms for glyco-
nitrogen-starved
accumulation
gen and trehalose biosynthesis. It has been described
only took place in hyphae with the ultrastructural
that trehalose levels can increase either as a conse-
features characteristic of the stationary phase (i.e.
quence of stationary-phase growth conditions [14] or
frequent branching and cross-wall formation and
in response to low levels of oxygen [15]. In our case,
highly condensed nuclear bodies) (Fig. 4a). In no
the decreasing levels of oxygen imposed by pelleting
case were glycogen granules seen in hyphae such as
might account for the observed pattern of trehalose
those observed during the lag period that precedes
accumulation (data not shown).
cultures,
glycogen
glycogen synthesis, which contained the nuclear material dispersed throughout the cytoplasm (Fig. 4b). The ability of starved cells to produce oleandomy-
Acknowledgments
cin is shown in Fig. 3. As can be observed, young hyphae (between 3.5 and 5.5 h of incubation) were
We would like to thank Keith F. Chater for crit-
FEMSLE 7659 20-10-97
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ically reading and correcting the manuscript. This work was supported by Grants BIOTCT910255 from the EC and BIOT94-1025 from the Comisioèn Interministerial de Ciencia y Tecnolog|èa CICYT (Spain). E.M.M. was the recipient of a Contrato de Reincorporacioèn del Ministerio de Educacioèn y Ciencia. M.F. received a pre-doctoral fellowship from the Fundacioèn Banco Herrero. References [1] Branìa, A.F., Manzanal, M.B. and Hardisson, C. (1980) Occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes. J. Bacteriol. 144, 1139^1142. [2] Branìa, A.F., Manzanal, M.B. and Hardisson, C. (1982) Characterization of intracellular polysaccharides of Streptomyces. Can. J. Microbiol. 28, 1320^1323. [3] Branìa, A.F., Meèndez, C., D|èaz, L.A., Manzanal, M.B. and Hardisson, C. (1986) Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus. J. Gen. Microbiol. 132, 1319^1326. [4] Plaskitt, K.A. and Chater, K.F. (1995) In£uences of developmental genes on localized glycogen deposition in colonies of a mycelial prokaryote, Streptomyces coelicolor A3(2): a possible interface between metabolism and morphogenesis. Phil. Trans. R. Soc. Lond. 347, 105^121. [5] Ramade, N. and Vining, L.C. (1993) Accumulation of intracellular carbon reserves in relation to chloramphenicol biosynthesis by Streptomyces venezuelae. Can. J. Microbiol. 39, 377^ 383.
[6] Hardisson, C., Manzanal, M.B., Salas, J.A. and Suaèrez, J.E. (1978) Fine structure and biochemistry of arthrospore germination in Streptomyces antibioticus. J. Gen. Microbiol. 105, 203^214. [7] Lloyd, J.B. and Whelan, W.J. (1969) An improved method for enzymic determination of glucose in the presence of maltose. Anal. Biochem. 30, 467^470. [8] Rosen, H. (1957) A modi¢ed ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 67, 10^15. [9] Burton, K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315^323. [10] Schneider, W.C. (1957) Determination of nucleic acids in tissues by pentose analysis. In: Methods in Enzymology (Colowick, S.P. and Kaplan, N.O., Eds.), Vol. 3, pp. 680^684. Academic Press, New York. [11] Vilches, C., Meèndez, C., Hardisson, C. and Salas, J.A. (1990) Biosynthesis of oleandomycin by Streptomyces antibioticus : in£uence of nutritional conditions and development of resistance. J. Gen. Microbiol. 136, 1447^1454. [12] Ryter, A. and Kellenberger, E. (1958) Etude au microscope eèlectronique de plasma contenant de l'acide deso¨xyribonucle¨ique. Z. Naturforsch. 13b, 597^605. [13] Thieèry, J.P. (1967) Mise en eèvidence des polysaccharides sur coupes ¢nes en microscopie eèlectronique. J. Microsc. 6, 987^ 1018. [14] Loewen, P.C. and Hengge-Aronis, R. (1994) The role of the sigma factor cS (KatF) in bacterial global regulation. Annu. Rev. Microbiol. 48, 53^80. [15] Hoelzle, I. and Streeter, J.G. (1990) Increased accumulation of trehalose in Rhizobia cultured under 1% oxygen. Appl. Environ. Microbiol. 56, 3213^3215.
FEMSLE 7659 20-10-97
Nitrogen starvation-induced glycogen synthesis depends on the developmental stage of Streptomyces antibioticus mycelium Elisa M. Migueèlez, Moènica Fernaèndez, Carlos Hardisson * èa, Facultad de Medicina and Instituto Universitario de Biotecnolog| èa de Asturias, Universidad de Oviedo, Area de Microbiolog| è n Claver| èa s/n, 33006 Oviedo, Spain c/ Julia
Received 24 April 1997; revised 9 May 1997; accepted 13 May 1997
Abstract
Experiments carried out to examine the ability of Streptomyces antibioticus to accumulate glycogen, when starved for nitrogen at different times during growth, revealed that nitrogen-starved hyphae, irrespective of the developmental time at which they were starved, accumulated glycogen only when they had acquired ultrastructural features typical of stationaryphase cultures. Oleandomycin production and trehalose accumulation were also examined during the starvation period. The observed pattern of oleandomycin production resembles that of glycogen. However, trehalose accumulates with no lag period after nitrogen starvation, regardless of the developmental phase of growth. Keywords :
Glycogen ; Oleandomycin; Trehalose;
Streptomyces
1. Introduction
The ability of streptomycetes to accumulate glycogen has been reported previously [1^5]. This polymer accumulates in a characteristic two-round pattern when streptomycetes are cultured on solid media [3,4]. The ¢rst round of accumulation occurs in the substrate mycelium, just before the emergence of the aerial hyphae, and coincides with the depletion of the nitrogen source in the culture medium. The second round of accumulation occurs in the aerial mycelium and coincides with the initiation of the sporulation process. In the present work, chemical and electron micro* Corresponding author. Tel.: +34 (85) 103557; Fax: +34 (85) 103554; E-mail: [email protected]
scopic techniques were used to investigate the pattern of glycogen accumulation in Streptomyces antibioticus during growth in a liquid culture medium. The results obtained revealed that S. antibioticus is able to respond to nitrogen starvation conditions by accumulating glycogen, but this condition although necessary is not su¤cient. Glycogen accumulation only occurs if the hyphae have attained a de¢nite developmental stage, the morphological features of which clearly coincide with those exhibited by stationary-phase hyphae.
2. Materials and methods
2.1. Microorganism, media and culture conditions
Streptomyces antibioticus
ATCC 11891 was grown
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 3 1 - 0
FEMSLE 7659 20-10-97
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E.M. Migueèlez et al. / FEMS Microbiology Letters 153 (1997) 57^62
in a modi¢ed GAE medium (MGAE) containing 0.5% (w/v) glucose, 0.1% (w/v) asparagine, 0.05% (w/v) yeast extract, 0.05% (w/v) HK2 PO4 , 0.05% (w/v) MgSO4 , 0.0001% (w/v) FeSO4 and 2.1% (100 mM) MOPS bu¡er (pH 7.0). Freshly harvested spores [6] were suspended in MGAE medium to an OD580 =0.1 and incubated in 2 l £asks, containing 400 ml of culture. For nitrogen starvation experiments, samples (400 ml) of cultures grown in MGAE medium were harvested by centrifugation (8000Ug, 8 min, 25³C), then washed with 100 mM MOPS bu¡er (pH 7.0) and resuspended in an equal volume of prewarmed nitrogen starvation medium (NSM). NSM is MGAE containing 2% (w/v) glucose, 0.01% (w/v) yeast extract and lacking asparagine. All the cultures were incubated at 35³C with shaking (250 rpm). Growth was followed by measuring dry cell weight and OD580 , and the morphological stage of the culture was monitored by phasecontrast microscopy. 2.2. Analytical procedures 2.2.1. Glucose and nitrogen measurements
Glucose and nitrogen levels in the culture media were estimated by the glucose oxidase-peroxidase method [7] and by the procedure of Rosen [8] respectively. 2.2.2. Polysaccharide determinations
For glycogen determination, samples of 40 ml of the culture were centrifuged. The resulting pellets were resuspended in water, heated at 100³C for 10 min and then disrupted by ultrasonic treatment (6 min in a 150 W MSE Ultrasonic disintegrator at 0³C). The glycogen content of the crude extracts was determined enzymatically, as previously described [2]. 2.2.3. Trehalose determinations
For trehalose determination, we used the crude extracts described above. The following mixtures were prepared: (1) 80 ml of crude extract, 65 ml of 0.1 M phosphate bu¡er (pH 6.0) and 30 ml of trehalase solution (5 mU) (Sigma); (2) 80 ml of crude extract, 65 ml of 0.1 M phosphate bu¡er (pH 6.0) and 30 ml of water. Both mixtures were incubated for 2 h at 37³C, then centrifuged, and the glucose
present in the supernatants was measured as described above. The di¡erence between the glucose values obtained in mixtures 1 and 2 represents the trehalose content in each sample. Previous experiments showed that, under the above conditions, trehalose was completely degraded by trehalase. 2.2.4. Total DNA, RNA and protein content
Triplicate culture samples (1.5 ml) were taken at intervals during growth, 1.5 ml of 0.5 N perchloric acid was added and the samples were chilled in an ice bath for 30 min. After centrifugation, pellets were extracted three times with 0.5 N perchloric acid at 70³C. Supernatants were pooled and assayed either for DNA by the method of Burton [9], or for RNA by the orcinol method [10]. Pellets were dissolved in 1.0 N NaOH and protein was determined by the method of Lowry. 2.2.5. Oleandomycin determination
Samples of cultures were centrifuged (12000Ug, 10 min, 4³C) and oleandomycin content in the supernatants was estimated by bioassay, using a highly oleandomycin-sensitive Micrococcus luteus strain as described earlier [11]. Commercial oleandomycin (Sigma) was used as standard. 2.3. Electron microscopy
Samples of the culture (10 ml) were pre¢xed in 1% (w/v) osmium tetroxide for 10 min and then embedded in agar. To facilitate the longitudinal orientation of the hyphae, the procedure of bacterial resuspension in agar was improved as follows. Pre¢xed samples were ¢ltered through a moist cellulose acetate membrane ¢lter (47 mm diameter, 0.45 mm size, Oxoid) placed on a glass column (Millipore). Melted agar (5 ml; 2% w/v; 45³C) was then added and the glass column was maintained under suction for 5 min. After cooling, the agar cylinder was pushed out from the glass column, the ¢lter was removed and the bottom part of the agar cylinder (containing layered hyphae) was cut into small blocks. The blocks were ¢xed overnight in 1% (w/v) osmium tetroxide [12], washed for 2 h with 2% (w/v) uranyl acetate, dehydrated with acetone and ¢nally embedded in Epon 812. Before polymerization, the samples were properly positioned in £at molds to
FEMSLE 7659 20-10-97
E.M. Migueèlez et al. / FEMS Microbiology Letters 153 (1997) 57^62
59
facilitate longitudinal sectioning of the hyphae. Ultrathin sections were stained either with uranyl acetate and lead citrate or with periodic acid, thiocarbohydrazide and silver proteinate [13]. Sections were observed in a Philips EM 300 electron microscope at 60 kV. 3. Results and discussion
3.1. Growth characteristics of S. antibioticus in MGAE medium
The growth characteristics of S. antibioticus in MGAE medium are summarized in Fig. 1. Spore germination started after about 2.5 h of incubation and was followed by a growth phase with a low growth rate (between 3 and 5.5 h of incubation) during which the culture contained a mixture of recently germinated spores with short germ tubes and spores having long unbranched germ tubes (ranging between 8 and 17 mm long). After about 5.5 h of incubation the culture entered a short phase of exponential growth (all the growth parameters: cell dry weight, DNA, RNA and protein, increased linearly). After about 7 h of incubation, there was a gradual transition into stationary phase. Glycogen accumulation was observed several hours after the beginning of the stationary phase of growth (after about 11^12 h of incubation), when only 12% of the nitrogen source initially supplied was present in the culture medium (Fig. 1b). Oleandomycin accumulation followed a pattern closely similar to that of glycogen (Fig. 1c). However, trehalose content was roughly constant regardless of the growth phase, except one peak of accumulation after 10 h of incubation, at early stationary phase. Nevertheless, the concentration of trehalose decreased quickly reaching levels similar to those found during the growth period (Fig. 1c). The ultrastructural changes exhibited by the hyphae during the growth cycle are shown in Fig. 2. Hyphae collected during the exponential period of growth (Fig. 2a; 6 h) typically showed homogeneous moderately electron-dense cytoplasm with the nuclear material dispersed loosely throughout it, perhaps re£ecting a very active state of transcription. Cross-walls and branches were only occasionally
Fig. 1. Growth characteristics of S. antibioticus in MGAE medium. Spores of S. antibioticus were suspended in MGAE medium and incubated at 35³C with shaking. At di¡erent times of incubation, samples (40 ml) were collected and used for measurements. a: OD (7), DNA (R), RNA (b) and protein (E). b: Residual glucose (Z) and ninhydrin-positive compounds (D) in the culture medium. c: Glycogen accumulation (a), trehalose content (F), oleandomycin production (O) and cell dry weight (8). The data are the mean of 5 independent experiments.
seen at this stage of development. During the transition to the stationary phase of growth (after about
FEMSLE 7659 20-10-97
E.M. Migueèlez et al. / FEMS Microbiology Letters 153 (1997) 57^62
60
times transferred to NSM (see Section 2.1). There was no growth in NSM and the culture could be considered a resting cell system. The results obtained are summarized in Fig. 3. Newborn hyphae (between 2.5 and 3.5 h of incubation) were totally unable to accumulate glycogen throughout the starvation peri-
Fig. 2. Ultrastructural changes exhibited by
S. antibioticus
hy-
phae during the growth cycle. Hyphae collected during the exponential period of growth (a), during the transition to the stationary phase of growth (b) and during the stationary phase (c). Bars represent 200 nm.
9 h of incubation), the nucleoids condensed into elongated structures disposed along the major axis of the hyphae (Fig. 2b). Branching and cross-wall formation both occurred with greater frequency during this period. In stationary phase hyphae (12 h), the nuclear material undergoes physical rearrangements and appears divided into highly condensed nuclear
bodies,
processes
have
which
suggests
largely
ceased
that in
transcription
these
nucleoids
(Fig. 2c). Branching and cross-walls were very frequent.
3.2. Glycogen and secondary metabolites accumulation by S. antibioticus after nitrogen starvation
Fig. 3. Glycogen, oleandomycin and trehalose accumulation by
S. antibioticus The ability of glycogen
and
S. antibioticus hyphae to
other
secondary
accumulate
metabolites,
when
starved for nitrogen in the presence of glucose at di¡erent times during growth, was investigated. Cultures were grown in MGAE medium and at di¡erent
after nutrient stress. Cultures of
S. antibioticus
were grown in MGAE medium and at di¡erent incubation times (a, 3.5 h ; b, 5.5 h ; c, 7.5 h) resuspended in NSM medium as indicated in Section 2.1 and analyzed.
F
, trehalose content ;
O
a
, glycogen accumulation ;
, oleandomycin production ;
8
, cell dry
weight minus glycogen. The data are the mean of 5 independent experiments.
FEMSLE 7659 20-10-97
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61
unable to produce antibiotic after nutritional starvation. Hyphae collected after 5.5 h of incubation in MGAE medium were able to produce oleandomycin, but only after a lag period which progressively decreased with culture age as in the case of glycogen (Fig. 3). The capacity of starved cells of
S. antibioticus
to
synthesize trehalose was also tested. As can be observed in Fig. 3, the pattern of trehalose accumulation during starvation was di¡erent from that of glycogen. Irrespective of the growth time at which the samples were collected, trehalose was detected from the beginning with no lag period. At all the times examined, there was a sudden increase in trehalose Fig. 4. Cytochemically stained sections of nitrogen-starved cultures of
S. antibioticus.
In all the nitrogen-starved cultures, glyco-
levels followed by a period of degradation. These results revealed that
S. antibioticus
hyphae
gen accumulation only took place in hyphae exhibiting ultra-
respond to the stress imposed by nitrogen starvation
structural features typical of stationary-phase cultures (a). In no
by accumulating glycogen and secondary metabolites
case were glycogen granules seen in hyphae containing the nu-
such as oleandomycin. Starvation for nitrogen, how-
clear material dispersed throughout the cytoplasm (b). Bars rep-
ever, was not the only condition required to trigger
resent 200 nm.
glycogen synthesis. Nitrogen-starved hyphae accumulated glycogen only when they had acquired, in
od (data not shown). Hyphae collected between 3.5
the starved condition, ultrastructural features typical
and 5.5 h of incubation accumulated glycogen, but
of stationary-phase cultures. This ¢nding seems to
there was a lag period before glycogen synthesis
indicate that a developmental program, reminiscent
could be detected. The extent of this lag period
of that which is expressed in cells entering that peri-
was
od, must be accomplished for nitrogen starvation to
dependent
on
the
time
of
incubation
in
S. antibioticus.
MGAE before exposure to the nutrient stress. As
trigger glycogen accumulation in
Fig. 3a shows, the lag period for hyphae collected
cording to this, the lag period required for glycogen
after 3.5 h of growth was about 5 h and it decreased
to be accumulated in the nitrogen-starved cultures
to 3.5 h for hyphae collected after 5.5 h of growth
would re£ect the time needed for the hyphae to ac-
(Fig. 3b), and practically disappeared for hyphae
complish such a developmental program.
collected after 7.5 h or longer times of growth (Fig. 3c).
Ac-
On the other hand, trehalose accumulates with no lag period after nitrogen starvation, regardless of the
The observation of cytochemically stained sections
developmental phase of growth, thus probably indi-
in the electron microscope revealed that, in all the
cating two distinct regulatory mechanisms for glyco-
nitrogen-starved
accumulation
gen and trehalose biosynthesis. It has been described
only took place in hyphae with the ultrastructural
that trehalose levels can increase either as a conse-
features characteristic of the stationary phase (i.e.
quence of stationary-phase growth conditions [14] or
frequent branching and cross-wall formation and
in response to low levels of oxygen [15]. In our case,
highly condensed nuclear bodies) (Fig. 4a). In no
the decreasing levels of oxygen imposed by pelleting
case were glycogen granules seen in hyphae such as
might account for the observed pattern of trehalose
those observed during the lag period that precedes
accumulation (data not shown).
cultures,
glycogen
glycogen synthesis, which contained the nuclear material dispersed throughout the cytoplasm (Fig. 4b). The ability of starved cells to produce oleandomy-
Acknowledgments
cin is shown in Fig. 3. As can be observed, young hyphae (between 3.5 and 5.5 h of incubation) were
We would like to thank Keith F. Chater for crit-
FEMSLE 7659 20-10-97
62
E.M. Migueèlez et al. / FEMS Microbiology Letters 153 (1997) 57^62
ically reading and correcting the manuscript. This work was supported by Grants BIOTCT910255 from the EC and BIOT94-1025 from the Comisioèn Interministerial de Ciencia y Tecnolog|èa CICYT (Spain). E.M.M. was the recipient of a Contrato de Reincorporacioèn del Ministerio de Educacioèn y Ciencia. M.F. received a pre-doctoral fellowship from the Fundacioèn Banco Herrero. References [1] Branìa, A.F., Manzanal, M.B. and Hardisson, C. (1980) Occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes. J. Bacteriol. 144, 1139^1142. [2] Branìa, A.F., Manzanal, M.B. and Hardisson, C. (1982) Characterization of intracellular polysaccharides of Streptomyces. Can. J. Microbiol. 28, 1320^1323. [3] Branìa, A.F., Meèndez, C., D|èaz, L.A., Manzanal, M.B. and Hardisson, C. (1986) Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus. J. Gen. Microbiol. 132, 1319^1326. [4] Plaskitt, K.A. and Chater, K.F. (1995) In£uences of developmental genes on localized glycogen deposition in colonies of a mycelial prokaryote, Streptomyces coelicolor A3(2): a possible interface between metabolism and morphogenesis. Phil. Trans. R. Soc. Lond. 347, 105^121. [5] Ramade, N. and Vining, L.C. (1993) Accumulation of intracellular carbon reserves in relation to chloramphenicol biosynthesis by Streptomyces venezuelae. Can. J. Microbiol. 39, 377^ 383.
[6] Hardisson, C., Manzanal, M.B., Salas, J.A. and Suaèrez, J.E. (1978) Fine structure and biochemistry of arthrospore germination in Streptomyces antibioticus. J. Gen. Microbiol. 105, 203^214. [7] Lloyd, J.B. and Whelan, W.J. (1969) An improved method for enzymic determination of glucose in the presence of maltose. Anal. Biochem. 30, 467^470. [8] Rosen, H. (1957) A modi¢ed ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 67, 10^15. [9] Burton, K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315^323. [10] Schneider, W.C. (1957) Determination of nucleic acids in tissues by pentose analysis. In: Methods in Enzymology (Colowick, S.P. and Kaplan, N.O., Eds.), Vol. 3, pp. 680^684. Academic Press, New York. [11] Vilches, C., Meèndez, C., Hardisson, C. and Salas, J.A. (1990) Biosynthesis of oleandomycin by Streptomyces antibioticus : in£uence of nutritional conditions and development of resistance. J. Gen. Microbiol. 136, 1447^1454. [12] Ryter, A. and Kellenberger, E. (1958) Etude au microscope eèlectronique de plasma contenant de l'acide deso¨xyribonucle¨ique. Z. Naturforsch. 13b, 597^605. [13] Thieèry, J.P. (1967) Mise en eèvidence des polysaccharides sur coupes ¢nes en microscopie eèlectronique. J. Microsc. 6, 987^ 1018. [14] Loewen, P.C. and Hengge-Aronis, R. (1994) The role of the sigma factor cS (KatF) in bacterial global regulation. Annu. Rev. Microbiol. 48, 53^80. [15] Hoelzle, I. and Streeter, J.G. (1990) Increased accumulation of trehalose in Rhizobia cultured under 1% oxygen. Appl. Environ. Microbiol. 56, 3213^3215.
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