Department of Cell and Molecular Biology, Box 596, Biomedical Center, Uppsala University, SE-751 24 Uppsala, Sweden1
Department of Biological Sciences, PO Box 210006, University of Cincinnati, Cincinnati, OH 45221-0006, USA2
Author for correspondence: Rolf Bernander. Tel: +46 18 471 40 58. Fax: +46 18 53 03 96. e-mail: Rolf.Bernander{at}icm.uu.se
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ABSTRACT |
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Keywords: Archaea, conditional mutants, flow cytometry, hyperthermophile, Sulfolobus
Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; Ts, thermosensitive
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INTRODUCTION |
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Members of the archaeal genus Sulfolobus grow optimally at about 80 °C and pH 3 under aerobic conditions. Sulfolobus spp. have emerged as important model systems for studies of hyperthermophilic archaea, including cell-cycle analyses (Bernander & Poplawski, 1997 ; Hjort & Bernander, 1999
; Poplawski & Bernander, 1997
) and development of simple genetic techniques (Grogan, 1989
, 1996
; Grogan & Gunsalus, 1993
; Schleper et al., 1994
). Here, we report the isolation and characterization of conditional-lethal mutants of Sulfolobus acidocaldarius. At the non-permissive temperature, initiation of chromosome replication, nucleoid organization and segregation, cellular growth, and cell division could be inhibited or uncoupled in these mutants, demonstrating their usefulness as tools for investigations of essential cellular processes of hyperthermophilic archaea.
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METHODS |
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Mutant isolation.
Cells of strain DG64 from late-exponential phase were suspended in 50 mM potassium-MES buffer, pH 6·0, at a density of about 109 cells ml-1 and treated with 100 µg NTG ml-1 for 20 min at 37 °C. Typical survival rates under these conditions were 525%. Colonies of survivors grown at 70 °C were replica plated at 70 °C and 83 °C (Grogan, 1995 ). Colonies failing to grow at the higher temperature were retested on solid medium to confirm Ts growth. Those scoring Ts were streaked for isolation, and tested for growth in liquid medium at 81 °C.
Temperature-shift experiments.
Liquid cultures (15 ml in 100 ml Erlenmeyer flasks) were grown with shaking at 70 °C to stationary phase (about 2 d). The cultures were then diluted by an appropriate factor (about 103-fold in most cases) into 20 ml fresh growth medium in new 100 ml flasks, so that an optical density of 0·02 to 0·1 was reached after 2 d additional incubation. As a result, the mutant cultures had been growing undisturbed for up to 10 generations when the temperature shift was performed.
The cultures were sampled (zero time point) for flow cytometry and epifluorescence microscopy, shifted to 81 °C, and further sampled at different time points. Samples for flow cytometry were collected by pipetting aliquots from the cultures directly into ice-cold ethanol (70%, v/v, final concentration). The volume collected was adjusted to obtain approximately the same concentration of cells in each sample. For microscopy, the samples were concentrated by centrifugation before ethanol fixation.
Flow cytometry.
Sampling, preparation and staining of cells, as well as flow cytometry, were performed as described previously (Bernander & Poplawski, 1997 ). Between 2000 and 10000 cells were analysed in each sample and the data were plotted using the FCSPress software (Ray Hicks).
Phase-contrast and fluorescence microscopy.
Combined phase-contrast and fluorescence microscopy was performed as described previously (Poplawski & Bernander, 1997 ), except that a higher DAPI (4',6-diamidino-2-phenylindole) concentration was used (0·51 µg ml-1). Figures were prepared using Adobe Photoshop software.
Growth curves.
Culture growth and numbers of c.f.u. (viability) were monitored in a similar manner as for the flow cytometry and microscopy analyses, but on separate sets of cultures. Turbidity was measured as apparent OD600 in a Milton-Roy Spectronic 21D spectrophotometer, using samples diluted in growth medium as necessary to achieve an OD value of less than about 0·3. Viable titre was measured by plating serial 1:10 dilutions of duplicate culture aliquots at 70 °C.
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RESULTS |
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Flow cytometry and microscopy analyses
A typical DNA content distribution obtained by flow cytometry analysis of wild-type Sulfolobus cells growing exponentially at a constant temperature is depicted schematically in Fig. 1 (Bernander & Poplawski, 1997
). We screened the Ts mutants for deviations from this distribution, as well as for an altered cell-size distribution, after a shift from 70 °C to the non-permissive temperature (81 °C). First, we determined the effects of this treatment on the parental strain, DG64, an auxotroph that relies on exogenous pyrimidines for nucleic acid synthesis. The post-replication stage dominated the cell cycle in DG64 cells grown at 70 °C (Fig. 2
, top row), similar to wild-type S. acidocaldarius and Sulfolobus solfataricus strains grown at 80 °C (Bernander & Poplawski, 1997
; compare with Fig. 1
). The shift to 81 °C resulted in a slight increase in light scatter and in a somewhat reduced proportion of cells in the pre-replication (B) and replication (C) periods (Fig. 2
, second row), showing that the culture adapted to the changed growth conditions. The overnight sample (bottom row) had a higher proportion of cells with two fully replicated chromosomes, but had not yet fully entered stationary phase (Bernander & Poplawski, 1997
), as shown by persistence of the B- and C-period populations. A minor peak corresponding to cells with >2 chromosomes was also evident, and very few (<1%) DNA-less cells were observed by microscopy. Turbidity and viability measurements showed that the growth kinetics remained exponential after the shift and that, under these conditions, the parental strain reached a high cell concentration (>109 c.f.u. ml-1) and maintained viability for many hours after reaching stationary phase (Fig. 3
).
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After overnight incubation at 81 °C, most cells exhibited a dramatic reorganization of the nucleoid; the intracellular regions of fluorescence became unstructured and accumulated in a half-moon appearance (Fig. 4, bottom micrograph). In part of the population, the DNA had become degraded (Fig. 4
, cells with <1 chromosome equivalent in bottom two rows), although the cells still were intact in the microscopy analysis. In plots correlating cell size and DNA content (not shown), the DNA-less cells formed a distinct population displaying a decreased light scatter.
Mutants of class Ib similarly accumulated in the post-replication period, but with little or no cell-size increase after the shift, as represented by strain DG146. Flow cytometry (Fig. 5) showed that the DG146 cells had arrested in the D period by the 3 h time point, whereas the light-scatter distribution remained largely unaffected, indicating that chromosome replication had continued to completion while cellular growth stopped. Prolonged incubation at 81 °C (>30 h) resulted in eventual loss of cell integrity, as shown by decreased light scatter and production of cell ghosts with grainy interiors (Fig. 5
). Furthermore, massive DNA degradation occurred, such that at 48 h the frequency of cells containing
1 chromosome equivalent had decreased to about 1x10-3 (Fig. 5
, bottom row). Consequently, the viability dropped dramatically at late time points (Fig. 3
).
Growth decrease without cell-cycle arrest
Two classes of mutants (IIa and IIb) showed decreased growth after the temperature shift, but no obvious cell-cycle arrest. Mutants of class IIa showed a gradual decrease in growth rate over a 20 h period. In the flow cytometry analysis, the only significant effects detected were DNA degradation in two of the mutants. This class is therefore not exemplified in the figures.
Class IIb exhibited a more rapid halt in growth and is represented by strain DG155. Both the turbidity and the viability counts continued to increase for about two doublings after the temperature shift (Fig. 3). During this early interval, flow cytometry revealed that chromosome replication and cell division continued, although the replicating part of the population decreased in proportion, while no major effects on cell morphology or nucleoid structure were detected in the microscopy analysis (Fig. 6
). However, at later time points viability fell dramatically (Fig. 3
), accompanied by loss of DNA and decreased light-scattering signals (Fig. 6
). This early timing of culture death contrasted with the delayed effects observed for DG146 (above).
DNA increase
Class III mutants showed continued increase in cellular DNA content after the temperature shift, accompanied by slow but persistent growth. The most dramatic example of this phenotype was exhibited by strain DG134. After temperature shift, total cell mass continued to increase, whereas the increase in c.f.u. soon halted, resulting in a divergence of the turbidity and viability curves (Fig. 3). Flow cytometry and microscopy indicated that this divergence could not be attributed to an accumulation of dead cells, as was seen in several of the other Ts mutants examined. Instead, the divergence reflected continued growth in the absence of cell division, indicating that division was inhibited (Fig. 7
, first column). The large broadening of the distribution implied that cell growth ceased at different time points in different parts of the cell population.
Although we also observed cell enlargement in other cases (see above), strain DG134 had the distinguishing characteristic that the DNA content of the cells also increased with time. Thus, replication did not cease after the temperature shift, although the replicating part of the population decreased in proportion (Fig. 7, second column). Replication was further evident as a rightward broadening of the two-chromosome peak in the DNA content distributions, and the eventual appearance of peaks corresponding to 3, 4 and even 5 chromosome equivalents per cell (Fig. 7
, bottom row). Microscopy analysis (Fig. 7
, right column) confirmed the flow cytometry results: an obvious cell-size increase over time was evident and giant multinucleated cells were present in the overnight sample. The DNA appeared as distinct and highly structured foci, which is the appearance of nucleoids in exponential-phase Sulfolobus cells. Interestingly, these nucleoids were obviously separated in some cells. This indicated that the partition (mitotic) apparatus was intact and active at the non-permissive temperature, distributing the replicated chromosomes in different directions within the giant cells. The fact that overall viability (assayed at 70 °C) did not decline over this time period suggests that the giant cells with highly structured and separated nucleoids remained intact and capable of resuming cell division at the permissive temperature.
Other classes
Several mutants did not show a major growth decrease during the shift experiments, but effects were still detectable in the flow-cytometry distributions; these were designated class IV (Table 1). In cultures of mutant DG117, for example, part of the cell population contained >2 chromosome equivalents at the overnight time point. The largest class of mutants, designated class V, showed no obvious short-term effects of a temperature shift. The fact that these mutants were clearly Ts for prolonged growth in both solid and liquid media indicates that several generation times were required before the phenotypic consequences of the mutations significantly affected cell growth and survival. Classes IV and V are not depicted in the figures.
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DISCUSSION |
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Significant phenotypes
Our previous qualitative and quantitative analyses of the cell cycle and cytology of different Sulfolobus species (Bernander, 1998 ; Bernander & Poplawski, 1997
; Hjort & Bernander, 1999
; Poplawski & Bernander, 1997
) provide a basis for interpreting several of the mutant phenotypes. In particular, the following responses were considered to be significant: i) cell-cycle arrest, ii) growth arrest or, alternatively, increasing cellular size over time, iii) increases in the number of chromosomes per cell and iv) changes in nucleoid organization. These phenotypes demonstrate that chromosome replication, cell division, nucleoid structure and distribution, and cellular growth, all of which are normally tightly controlled and co-ordinated, can be selectively inhibited or uncoupled in S. acidocaldarius.
Post-replication arrest
At the non-permissive temperature, mutants of classes Ia and Ib rapidly accumulated in the post-replication (D/G2; see Fig. 1) stage of the cell cycle. This indicates that ongoing rounds of chromosome replication were completed whereas subsequent cell division, as well as initiation of new rounds of replication, were blocked. This cell-cycle arrest was observed in a relatively large proportion of the mutants studied, including mutants that continued to grow after the temperature upshift, as well as those that stopped growing soon thereafter (Table 1
). Temporary arrest in D (G2) is observed when exponentially growing Sulfolobus cultures are diluted into fresh medium (Hjort & Bernander, 1999
), and cultures that reach stationary phase also arrest in D (Bernander & Poplawski, 1997
). Taken together, these observations suggest that the post-replication stage is a preferred cell-cycle-arrest stage in S. acidocaldarius, and that initiation of a new round of chromosome replication is prone to regulatory inhibition and particularly sensitive to cellular status.
In several mutants, including DG132 (Fig. 4) and DG134 (Fig. 7
), incubation at the non-permissive temperature supported cellular growth without concomitant cell division, producing grossly enlarged cells. These mutants provide the first examples in an archaeon of genetic uncoupling of mass increase from cell division. They further demonstrate that initiation of cell division is also prone to inhibition and constitutes another important regulatory step in the S. acidocaldarius cell cycle. The fact that these and other Ts mutants stayed viable for several hours after arrest suggests that they may be used for cell-cycle synchronization of S. acidocaldarius cultures, in addition to the recently described synchronization method based upon dilution of stationary-phase cultures (Hjort & Bernander, 1999
).
Division inhibition
The phenotype of DG134 (continued growth and chromosome replication without cell division) is particulary noteworthy, as it suggests a block in the division process itself. In Sulfolobus, this phenotype is analogous to that of fts (filamentation temperature sensitive) mutants of E. coli (Hirota et al., 1968 ), upon which much of the genetic analysis of bacterial cell division has been based. Particular attention has been focussed on the ftsZ gene, whose product initiates bacterial cell division and participates in the constriction process (Lutkenhaus & Addinall, 1997
). Homologues of ftsZ are found in all bacteria, except Chlamydia species, as well as in all archaea within the Euryarchaeota branch that have been analysed (Baumann & Jackson, 1996
; Bult et al., 1996
; Kawarabayasi et al., 1998
; Klenk et al., 1997
; Margolin et al., 1996
; Smith et al., 1997
; Wang & Lutkenhaus, 1996
). However, no ftsZ gene has yet been identified in a crenarchaeote, despite the availability of the complete genome sequence of Aeropyrum pernix (Kawarabayasi et al., 1999
). The crenarchaeotal cell-division apparatus is therefore likely to be quite divergent from that of other prokaryotes, which focusses particular interest on the defect in strain DG134.
Growth inhibition
The majority of the mutants continued to grow after the temperature shift, suggesting that most cellular functions were unaffected at the non-permissive temperature. However, some mutants ceased growth rapidly, indicating that central cellular processes, e.g. protein synthesis, energy production or important biosynthetic pathways were affected. Our identification of several such mutants in a relatively small collection is consistent with the complexity of these processes and the large number of genes involved. The DNA degradation observed in several mutants, e.g. DG146 and DG155, may be a consequence of cell death and the concomitant acidification of the cytoplasm. This is supported by observations that other stresses, i.e. addition of non-ionic detergent (S. L. Strini & D. W. Grogan, unpublished observations) or transiently shifting cultures to a low temperature (Hjort & Bernander, 1999 ) also result in degradation of DNA in Sulfolobus cells.
Changes in nucleoid structure
In several of the mutants, the characteristic organization of the nucleoids in exponential phase (Poplawski & Bernander, 1997 ) was lost after several hours incubation at the non-permissive temperature. The fluorescence foci became more diffuse and the nucleoids often accumulated in a half-moon appearance near the cell periphery (exemplified by mutant DG132). Although the mechanistic basis for these structural changes remains to be investigated, we note that they may be associated with general loss of cellular function and integrity, as they were often accompanied by onset of DNA degradation in other cells of the same culture.
Nucleoid segregation and chromosome replication
Strain DG134 maintained cell viability for a long time after the shift to the non-permissive temperature, accompanied by retained exponential-phase nucleoid structure and continued chromosome replication. Individual nucleoids could be distinguished even when as many as five were present within a single cell (Fig. 7). This showed that active segregation of replicated genomes continued to take place, i.e. that the partition (mitotic) machinery continued to operate at the non-permissive temperature. In contrast, mutant DG132 showed little nucleoid segregation at the non-permissive temperature, and the level of chromosome re-replication was no higher than in the DG64 parental strain (compare overnight samples in Figs 2
and 4
). Tight coupling between nucleoid partition, cell division and chromosome replication is observed in wild-type strains: when a stationary phase culture, in which all cells are in the post-replication stage of the cell cycle, is diluted into fresh medium, initiation of replication does not occur until the preceding nucleoid partition and cell division events have been completed (Hjort & Bernander, 1999
). However, the results obtained with strains DG132 and DG134 suggest that only chromosome partition is required before a new round of replication can be initiated, whereas cell division is not essential. Thus, alternating rounds of replication and chromosome segregation is a key regulatory feature of the crenarchaeal cell cycle.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 August 1999;
revised 17 November 1999;
accepted 24 November 1999.
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