Area de Microbiología, Departamento de Biologia Funcional, Facultad de Medicina, 33006 Oviedo, Spain1
Author for correspondence: Jesús Sánchez. Tel: +34 985103555. Fax: +34 985103148. e-mail: JSM{at}sauron.quimica.uniovi.es
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ABSTRACT |
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Keywords: programmed cell death, substrate mycelium, chromosomal DNA degradation, viability staining, differentiation
Abbreviations: PI, propidium iodide
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INTRODUCTION |
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The hyphae of S. antibioticus undergo an orderly process of internal cell dismantling, including extensive genome digestion, that resembles the programmed cell death in animal development (Miguelez et al., 1999 ). Our group has been interested in clarifying the mechanisms that intervene in the reuse of the above-mentioned DNA constituents. With this purpose in mind we previously analysed the presence of nucleolytic activities that could play a role in such processing in S. antibioticus ATCC 11891. Initially, we detected and purified a nutritionally regulated 29 kDa periplasmic nuclease that nicks double-stranded DNA at dG/dC-rich sequences, leaving 35250 bp end products with 3'-hydroxyl and 5'-phosphate termini (Cal et al., 1995
). We then detected two main exocellular nucleases of 18 and 34 kDa, which were, as in the former, nutritionally regulated (i.e. they did not appear in rich media, which repress differentiation). Their biochemical characteristics made them suitable for the degradation and recycling of the DNA building blocks, as for example their lack of specificity on cutting DNA sequences and the formation of 5'-phosphate mononucleotides as predominant end products (Nicieza et al., 1999
). These enzymes show a dependence on Ca2+ for their activity and co-operate efficiently with the periplasmic nuclease to completely hydrolyse the DNA. In the present work we carry out such an analysis during the differentiation of S. antibioticus ETH 7451, a strain which is also currently being investigated by our group due to its remarkable capability to sporulate in submerged conditions (Novella et al., 1992
). This will facilitate the physiological analysis of the death process. We started our investigations in this strain by following the process of the mycelium death in cultures growing on agar, in order to subsequently compare this with the cell death processes taking place in submerged cultivation. To achieve this, we applied a propidium iodide viability stain technique, which allowed us to show the damage to the cytoplasmic membrane. In this way we have shown the evolution of the cell death processes in relation to the appearance of the nucleases and chromosomal DNA degradation. The main role in the substrate DNA hydrolysis is probably performed by 2022 kDa nucleases, which seem to be the equivalent of the previously described 18 kDa nuclease from S. antibioticus ATCC 119891 (Nicieza et al., 1999
). We also report a 44 kDa nuclease, not described previously, which appears when the aerial mycelium is formed. In conditions in which the nuclease activities are inhibited, such as in the presence of Zn2+, the chromosomal DNA appears less degraded. The nucleases are loosely bound to the cell wall from where they can be liberated by simple washing. The results show that the function of these enzymes is likely conserved within Streptomyces and lend further support to the possible role of the specific activities in development.
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METHODS |
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Viability assay.
A permeability assay similar to that described previously for submerged Streptomyces cultures was used (Fernández & Sánchez, 2001 ). This involves the staining of damaged (leaky) cells with a polar (cell-impermeant) stain (propidium iodide, PI) in order to detect the dead cell population of S. antibioticus. GAE plates prepared with Noble agar (Difco) and inoculated as described above were used to obtain blocks of the GAE agar with a scalpel. These were further trimmed to cubes of about 7 mm in size and introduced into a microtome (11 mm hole diameter) previously cooled to 4 °C, with the growth surface side-oriented. Sections of about 0·30·4 mm were obtained with a razor blade, deposited in the same orientation on a clean slide and covered with a SYTO 9 plus PI (LIVE/DEAD BacLight Bacterial Viability Kit; Molecular Probes, L-13152) stain mix prepared as recommended by the manufacturer (1:1, v/v). The SYTO 9 green fluorescent nucleic acid stain labels all the cells, that is, those with intact membranes and those with damaged ones. The PI red fluorescent nucleic acid stain only enters the damaged Streptomyces cells (Fernandez & Sanchez, 2001
), causing a reduction in the SYTO 9 stain because of its higher nucleic acid affinity. After staining for at least 10 min in the dark, the sample was observed under a Bio-Rad MRC600 laser confocal microscope, at 488 and 568 nm excitation and 530 (green) or 630 nm (red) emission. Both images were mixed with the Confocal Assistant version 4.02 program (Todd Clark Brelje, 19941996, freeware program distributed by Bio-Rad Laboratories) in order to make up the final dual-colour image.
Nuclease activity gel and protein analysis.
Nuclease activities were analysed in samples obtained at the three developmental stages: substrate mycelium, aerial mycelium and sporulation. Samples were obtained in three different ways: in one of them, the bacterium was grown directly on the agar surface and the agar was extruded by forcing it through the hole of a plastic syringe (Nicieza et al., 1999 ). The resulting suspension was centrifuged at 4 °C, for 30 min at 17000 g and the supernatant used as the source of the enzyme. In other samples the mycelium was grown on the surface of cellophane disks, and was scraped out with a plain spatula, resuspended in 20 mM Tris/HCl pH 8·0, 1 mM EDTA, 7 mM 2-mercaptoethanol buffer and ruptured in an MSE Soniprep 150, in 6 cycles of 10 s, on ice. After centrifuging at 10000 r.p.m. (Eppendorf 5415C microcentrifuge) for 30 min at 4 °C, the supernatant was used as the source of the activity. Finally, in other experiments the mycelium collected from the cellophane disks as mentioned above was shaken for about 10 min in a Vortex before analysing the activity. To avoid differences in the results (due to the different sizes of the collected samples), we processed identical agar volumes in the extruded samples. For the sonicated and shaken samples, the weight/buffer volume relationships or the amount of protein were identical. The bands corresponding to the different nuclease activities were visualized in each sample by measuring the in situ DNA hydrolysis after separating the proteins in denaturing SDS-polyacrylamide gels containing denatured DNA (heated at 100 °C for 10 min and then chilled on ice) (Rosenthal & Lacks, 1977
). After the electrophoresis the proteins were renatured and the activity detected in 20 mM Tris/HCl pH 8·0, 7 mM 2-mercaptoethanol, 10 mM MgCl2, 5 mM CaCl2, 10% DMSO buffer, as reported elsewhere (Nicieza et al., 1999
). Gels were incubated for 90 min to 3 h at 37 °C. Micrococcal nuclease and bovine pancreatic DNase I (Amersham Pharmacia) were used as controls. Proteins were analysed by SDS-PAGE in 12% polyacrylamide gels (Laemmli, 1970
) after mixing the above samples with sample buffer (M. Fernandez & J. Sanchez, unpublished results) and heating at 100 °C for 5 min before loading. Proteins were stained with silver (Cal et al., 1995
). Molecular masses were estimated by their mobility with reference to marker proteins (Low Range, Bio-Rad).
DNA fragmentation assay.
DNA degradation in the mycelium of S. antibioticus ETH 7451 was analysed in samples collected from cellophane discs after a careful extraction and subsequent agarose gel electrophoresis. The mycelium was suspended in Tris/HCl pH 8·0, 1 mM EDTA (TE buffer), pH 8·0 plus 2 mg lysozyme ml-1 and incubated at 30 °C for 1 h. Then EDTA (100 mM final concn) and proteinase K (50 µg ml-1 final concn; Roche Molecular Biochemicals) were added and the sample incubated at 30 °C for 5 min. SDS (1%, final) was added and the samples incubated further for 2 h at 37 °C. An equal volume of phenol/chloroform (1:1) was added and mixed carefully. After centrifugation at 11000 g for 5 min, the aqueous phase was collected. The process was repeated until a completely clean aqueous solution was obtained. DNA was precipitated with 0·1 vol. 3 M sodium acetate and 1 vol. 2-propanol, washed by centrifugation with 70% ethanol and resuspended in TE. RNase A (100 µg ml-1) and RNase T1 (1000 U ml-1) (Sigma) were added and the solution incubated for 2 h at 37 °C. The phenol/chloroform extraction, precipitation and washing were repeated as above. The DNA was analysed by 0·8% agarose gel electrophoresis and stained with SYBR gold nucleic acid gel stain (Molecular Probes) which presents more than tenfold sensitivity over the previously used ethidium bromide (Nicieza et al., 1999 ). The gels were visualized on a 300 nm UV transilluminator and photographed with a Polaroid 667 black-and-white print film and a photographic filter (S-7569; Molecular Probes). The nature of the fluorescent band was confirmed by digestion with DNase I (0·1 µg µl-1) for 2 h at 37 °C.
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RESULTS |
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As previously reported for S. antibioticus ATCC 11891 (Nicieza et al., 1999 ), the development of S. antibioticus ETH 7451 is impaired in the presence of 0·51 mM Zn2+. This means that whereas in the control cultures at 15 h the aerial mycelium is completely formed, the cultures with Zn2+ are retarded in the phase of substrate mycelium. After 24 h, the control cultures have sporulated; the plates with Zn2+ are in the aerial mycelium phase, with no sign of septation within the hyphae (not shown). When the nucleases were analysed in the extruded agar samples from both media at 15 h, the control cultures showed the presence of the 34 kDa and 45 kDa bands (Fig. 2c
); however, in the cultures with 0·5 and 1 mM Zn2+ the 2022 kDa band (the 20 kDa band was not seen with 1 mM Zn2+) and the 34 kDa band were visible. The gel activity analysis was repeated with the extruded agar sample from the 0·5 mM Zn2+ plates (see Fig. 2c
) but in this case 0·5 mM and 1 mM Zn2+ were added to the incubation buffer. The results (Fig. 2d
) show that the activity of the three 2022 and 34 kDa nucleases is inhibited by Zn2+, although the 2022 kDa nucleases seem more sensitive than the 34 kDa enzyme; the 18 kDa band present in the control is not affected. It can be concluded that it is the activity, and not the synthesis of the enzymes, which is inhibited by Zn2+. This inhibition is likely to be responsible for the fact that in the 24 h cultures the chromosomal DNA appears less degraded in the presence of 0·5 and 1 mM Zn2+, when the control cultures are sporulated and the DNA is substantially degraded (Fig. 2e
; see above).
An SDS-PAGE analysis of the proteins of the washed substrate (vegetative) mycelium from S. antibioticus ETH 7451 showed a remarkably high proportion of proteins loosely bound to it (Fig. 2b). This suggests that the surface, and most likely the cell wall from the vegetative mycelium is a functionally very active structure which maintains a notable reservoir of proteins, some or most of them probably related with scavenger and/or degradative functions. These cell-wall located proteins are drastically reduced in number in the aerial mycelium and, in fact, only a few predominant proteins (33 kDa and 38 kDa) are observed at 15 h in the washed S. antibioticus ETH 7451 mycelium (Fig. 2b
). These two proteins plus another of about 86 kDa, and minor bands of 42 kDa and 36 kDa are all that can be seen in the sporulation phase gels (Fig. 2b
). This will notably facilitate the purification and biochemical characterization of the nucleases and the subsequent cloning of the genes.
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DISCUSSION |
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Nuclease production, chromosomal DNA degradation and differentiation are co-ordinated events
The impairment of the aerial mycelium formation by Zn2+ could be due to additional effects of this cation on other enzymes, for example the serine proteases. A trypsin-like enzyme has been described in S. antibioticus ATCC 11891 (Nicieza et al., 1999 ) and a similar protease has been also detected in S. antibioticus ETH 7451 (M. Fernandez, unpublished results). Its role has been related to proteolytic processing of a hypothetical inactive precursor of the 18 kDa and/or the 34 kDa nucleases (Nicieza et al., 1999
; J. Huergo & J. Sanchez, unpublished results). The 2022 kDa nucleases seem to be equivalent to the 18 kDa nuclease detected previously in the surface-sporulating strain S. antibioticus ATCC 11891. In our previous work (Nicieza et al., 1999
) we reported that the 18 kDa nuclease peaks in the aerial mycelium phase (about 36 h). This apparent difference with respect to the results obtained in the ETH 7451 strain can be readily explained by the difference in the growth kinetics between the two strains. The development of S. antibioticus ATCC 11891 is slower than the ETH 7451 strain and thus, the phase of substrate (vegetative) mycelium in the ATCC 11891 strain lasts about 24 h. It is conceivable that both vegetative and aerial mycelium coexist at 48 h, whereas in the ETH 7451 strain, which develops significantly more rapidly, only vegetative or aerial mycelium would predominate at each phase (as suggested by the photographs in Fig. 1e
, f
). This could explain the higher intensity of the 18 kDa nuclease band in the activity gels from the aerial mycelium phase of S. antibioticus ATCC 11891. The similarity between the 18 kDa and the 2022 kDa nucleases is further supported by the fact that the ETH 7451 enzyme shows, as occurred with the ATCC 11891 18 kDa nuclease (Nicieza et al., 1999
), a strict requirement for Ca2+. The activity of the 44 kDa and 34 kDa nucleases, by contrast, is not noticeably impaired in the absence of that cation [this is also true for the S. antibioticus ATCC 11891 34 kDa nuclease, which was previously claimed to need Ca2+ (Nicieza et al., 1999
; J. Huergo & J. Sánchez, unpublished results)]. Moreover, the 2022 kDa nucleases are synthesized only in the vegetative (substrate) mycelium (plates incubated for 9 h) and could play, as postulated in the above strain for the 18 kDa nuclease (Nicieza et al., 1999
), a preferential role in the DNA lytic processes which conceivably take place in the substrate mycelium just before and during the course of emergence of the aerial mycelium. This hydrolysis is likely facilitated by the disorganization of the cytoplasmic membrane, as mentioned above, and shown by the viability stain (Fig. 1c
, d
). The role of these nucleases and possibly the 34 kDa enzyme, in the substrate mycelium DNA degradation, is further supported by the delaying of the chromosomal DNA hydrolysis in the presence of Zn2+ (Fig. 2e
). The 44 kDa nuclease appears specifically at the aerial mycelium phase; when the appearance of the aerial mycelium is impaired by Zn2+, the nuclease is not visible (but the 2022 kDa enzymes are present; Fig. 2c
), thus supporting the relationships between the synthesis of the enzyme at the differentiation phase and its potential role in the lytic processes which take place in the aerial mycelium before spore formation. A nuclease of similar molecular mass has been recently detected in the previously studied S. antibioticus ATCC 11891 strain (M. Fernández & J. Sánchez, unpublished results). This enzyme, as shown here, appears specifically when the aerial mycelium is forming (M. Fernández & J. Sánchez, unpublished results). The 34 kDa nuclease could be related to both lytic processes, probably contributing to the concerted activity of the mentioned nucleases to a rapid and extremely efficient degradation of the DNA, as has been shown previously with the 18 kDa and 34 kDa enzymes from S. antibioticus ATCC 11891 (Nicieza et al., 1999
).
Chromosomal DNA appears partially degraded when aerial mycelium starts to form in S. antibioticus ATCC 11891 growing on plates (Nicieza et al., 1999 ). In the sporulation phase the DNA degradation is much more intense (Nicieza et al., 1999
). These kinetics are also observed in S. antibioticus ETH 7451 (Fig. 2e
). When the aerial mycelium is completely formed (15 h) the DNA is extensively degraded, with the exception of a small slowly migrating fraction visible as a defined band on the agarose gel. This likely represents the DNA of the few spores present at this time, which are partially sensitive to lysozyme (Novella et al., 1992
). This band, although more intense, is also seen in the 24 h sporulated cultures. The remaining DNA visible on the gel is completely degraded to 100250 kb fragments; the smaller oligonucleotides and mononucleotides conceivably formed in the hydrolysis (Nicieza et al., 1999
) will not be recovered by the 2-propanol/sodium acetate step and thus are not visible on the gel. As already pointed out, S. antibioticus ETH 7451 has been previously used in our group to analyse differentiation, as it has an exceptional capability to sporulate synchronously after a nutritional down-shift under submerged conditions (Novella et al., 1992
). The bacteria harbour a NaeI-isoschizomer restrictionmodification system (Fernández et al., 1998
; A. Godany, unpublished results) and is remarkably resistant to actinophages (J. Sánchez, unpublished data). We are currently performing a study of the appearance and biochemical characteristics of the nucleases during submerged development, in order to analyse the physiological significance of both surface and submerged differentiation processes. The results available show that the biochemical characteristics of the 2022 kDa and 34 kDa nucleases (NaCl, KCl or Zn2+ inhibition, preference for single or double-stranded DNA, Mg2+ and Ca2+ requirements) are identical to the surface nucleases and the 18 kDa and 34 kDa nucleases previously described in S. antibioticus ATCC 11891 (M. Fernández & J. Sánchez, unpublished results).
Streptomyces as a developmental model
These and our previous data (Nicieza et al., 1999 ) point to the existence of a series of co-ordinated and notably conserved biochemical events, related to chromosomal DNA degradation during the lytic processes that accompany development and differentiation in Streptomyces. In the hypothetical sequence for cell death induction, a change in the nutritional environment, growth rate or both, will transmit a still uncertain signal to the Streptomyces cells which in turn could activate a cytoplasmic effector (perhaps with the co-operation of Ca2+, protein kinases or even caspase-like proteases (Smith, 1995
; Zhang, 1996
; Aravind et al., 1999
). This could trigger mycelial cell death and the lytic processes. The formation of the nucleases which intervene in the DNA degradation process is conceivably under a strict control, part of which could be the induction of the above-mentioned proteolytic processing of an inactive high molecular mass precursor which would give rise to the active 18 and 34 kDa nuclease forms (Nicieza et al., 1999
; J. Huergo & J. Sánchez, unpublished results). A second round of DNA hydrolysis could take place in the aerial mycelium, when spores are forming. In this, the 44 kDa enzyme plus the 34 kDa nuclease could play the role. We have investigated the existence of nucleases with the characteristics and location of those described here, in several other species of Streptomyces, such as Streptomyces coelicolor, Streptomyces albus, Streptomyces achromogenes or Streptomyces lividans; our initial results show the presence, in all of them, of 34 kDa and 1823 kDa cell-wall located nucleases (no activity could be detected within the cytoplasm) with similar biochemical characteristics to those described here and in S. antibioticus ATCC 11891 (J. Huergo & J. Sánchez, unpublished results).
It has been suggested that micro-organisms which present a complex developmental life (such as Streptomyces, Bacillus, Anabaena, Caulobacter, Rhizobium or the myxobacteria) would have programmed cell death mechanisms which could be considered as the phylogenetic precursors of the eukaryotic programmed cell death (Yarmolinsky, 1995 ; Hochman, 1997
). This has been shown for S. antibioticus, in which an orderly process of internal cell dismantling, rather than an uncontrolled autolytic process, takes place (Miguelez et al., 1999
). However, as shown in this study and in our work, a remarkable difference between this mechanism and the programmed cell death of higher organisms is that dead hyphae from Streptomyces do not completely disappear, but remain to form part of the colony structure, thus allowing the passage of the recycled nutrients. On the other hand, several eukaryotic protein kinases and apoptotic proteins have homologues in Streptomyces (Zhang, 1996
; Aravind et al., 1999
) and some of the apoptotic nucleases, as also occurs with the 18 kDa and the 2023 kDa Streptomyces enzymes, have Mg2+ and Ca2+ requirements and are inhibited by high NaCl or KCl concentrations, Zn2+ or aurin tricarboxylic acid (Peitsch et al., 1994
; Montague & Cidlowski, 1996
; Hale et al., 1996
; Hughes & Cidlowski, 1999
; Widlak & Garrard, 2001
). It seems that the study of the biochemical and genetic basis of the programmed cell death in this bacterium could contribute to a better understanding of the role and evolution of this important process in eukaryotic cells.
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ACKNOWLEDGEMENTS |
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Received 27 June 2001;
revised 14 September 2001;
accepted 4 October 2001.