INTRODUCTION

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Organisms belonging to the archaeal genus Sulfolobus are hyperthermophilic acidophiles that grow optimally at around 80°C and pH 3. Several species can grow either as chemolithoautotrophs or as heterotrophs using oxygen as a terminal electron acceptor. This has the experimental advantage that cultures may be grown aerobically, and both solid and liquid growth media have been developed (14). Auxotrophic, as well as conditional-lethal, Sulfolobus mutants have been isolated (4a, 8-10), and several cloning vectors have been constructed (1, 2, 6, 17). In addition, the complete genome sequence of Sulfolobus solfataricus is being determined (16). Thus, Sulfolobus species have the potential to become important model organisms for studies of hyperthermophiles and of archaea in general.

We have initiated studies of the cell cycle (3) of different members of the Sulfolobus genus to further increase the understanding of the biology of these organisms. During exponential growth, the cell cycle is dominated by the postreplication stage (Fig. 1A), such that the cells contain two fully replicated chromosomes during about 60% of the cellular doubling time (4). After termination of chromosome replication, nucleoid partition does not occur immediately: the cells, instead, appear to go through a G₂-like stage which lasts more than an hour before partition takes place (13).

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Exponentially growing and stationary-phase S. acidocaldarius cultures. In the first two columns, cell size and DNA content distributions obtained by flow cytometry are shown. The third column shows cell morphology and nucleoid structure as visualized by combined phase-contrast and epifluorescence microscopy after staining with the DNA-specific dye 4',6-damidino-2-phenylindole (DAPI). Bars, 2 µm. (A) Exponentially growing culture. (B) Stationary-phase culture.

In stationary phase, all cells end up with two fully replicated chromosomes, showing that the postreplication stage of the cell cycle (the D period) is the preferred resting stage for these organisms (4; Fig. 1B). Replicating cells can no longer be detected in the population at this stage. Furthermore, the cells go through a size reduction as they enter stationary phase and the nucleoid becomes unstructured and occupies a larger part of the cellular interior than in exponentially growing cells (13).

Here, we report a study of the temporal order of chromosome replication, nucleoid partition, and cell division when stationary-phase cells are diluted into fresh medium and growth is allowed to resume. Dilution experiments were also performed with exponentially growing cultures to investigate whether balanced growth was maintained or if cell cycle perturbations occurred. Finally, exponentially growing cultures were transiently shifted to room temperature or to ice-water baths and the effects on cellular growth, replication, and division were studied.

	MATERIALS AND METHODS

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Strains. S. acidocaldarius (DSM 639) and S. solfataricus (DSM 1616) type strains were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen in Braunschweig, Germany. S. shibatae B12 was a kind gift from Christiane Elie.

Culture media and growth conditions. The growth medium used for S. acidocaldarius and S. solfataricus was Allen mineral base supplemented with 0.2% tryptone (5). S. shibatae was grown in the medium described by López-García and Forterre (11), except that Ca(NO₃)₂ was omitted. Solid medium (7) was obtained by addition of 0.6% Gelrite (Merck), 10 mM MgSO₄, and 2.5 mM CaCl₂ (final concentrations). The cultures were incubated in Erlenmeyer flasks at 79°C in water baths at a shaking speed of 200 rpm, and growth was monitored by optical density (OD) measurements at 600 nm. To obtain stationary-phase cells for the dilution experiments, the cultures were incubated for a further 10 to 20 h after the OD had ceased to increase. In the dilutions, the flasks and media were always first preheated at 79°C. In the temperature shift experiments, liquid cultures were inoculated such that the strains had been growing exponentially for at least 10 generations before the shifts, which were performed at an OD of 0.2.

Sampling, preparation of cells, combined phase and epifluorescence microscopy, and flow cytometry. Sampling and preparation of cells, as well as phase-fluorescence microscopy and flow cytometry, were performed as described previously (4, 13). In the flow cytometry, cell size was measured as light scatter. We confirmed that the gradual increase in light scatter observed in the dilution experiment indeed reflected increased cellular size, by direct microscopy measurements (data not shown).

	RESULTS

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Dilution of stationary-phase cultures. The strains were grown in liquid cultures at 79°C (see Materials and Methods for strains and culture media). The cultures were considered to have reached stationary phase when the OD had ceased to increase and the cells had undergone the morphological changes described in the Introduction. They were then diluted 5- to 10-fold into fresh growth medium and sampled for phase-fluorescence microscopy and flow cytometry at different time points after the dilution.

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Dilution of a stationary-phase S. acidocaldarius culture into fresh medium. (A) OD measurements (600 nm). (B) Flow cytometry analysis of samples removed from the culture at different time points. (C) Same as B but with more closely spaced time points. The 280-min samples were lost.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Proportion of cells with segregated nucleoids and/or visible constriction at the same time points as in Fig. 2. (A) Proportion of the cell population that showed segregated nucleoids and/or visible constriction at different time points after dilution from stationary phase. (B) Examples of cells scored as having segregated nucleoids. The right column shows two of the cells to the left illuminated by phase-contrast light alone to more clearly visualize the lack of cell constriction. Bar, 2 µm. (C) Examples of cells scored as dividing. The right column shows the same cells as to the left but with phase-contrast illumination only to more clearly visualize cell constriction. Bar, 2 µm.

Dilution of exponentially growing cultures. Dilution experiments were also performed with exponentially growing cultures to investigate the effects on chromosome replication and cell division and to determine whether balanced growth was maintained.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Dilution of an exponentially growing S. acidocaldarius culture into fresh medium. (A) Flow cytometry analysis of aliquots removed from the culture at the time points indicated. (B) OD measurements (600 nm).

Transient temperature shift experiments. Members of the genus Sulfolobus are hyperthermophiles that grow optimally at temperatures of around 80°C. We wanted to investigate the effects of shifts to lower temperatures on growth and cell cycle characteristics. Flasks containing exponentially growing cultures were therefore removed from a shaking water bath and placed in other shaking water baths either at room temperature or in ice-water. After different time periods (between 10 min and 8 h), the flasks were returned to the 79°C water bath. The OD was monitored throughout the shifts, and samples for flow cytometry were removed at regular time intervals.

View larger version (35K): [in this window] [in a new window]   FIG. 5.   Temperature shift experiments. An S. acidocaldarius culture growing exponentially at 79°C was shifted to room temperature for 5 or 8 h and then returned to 79°C. (A) OD measurements (600 nm). The culture was shifted from 79°C to room temperature at time zero, and portions were returned to 79°C at the time points indicated by the arrows. The control culture was grown at 79°C without temperature shifts. (B) Flow cytometry analysis of samples removed from the culture incubated for 5 h at room temperature. (C) Flow cytometry analysis of samples removed from the culture incubated for 8 h at room temperature. Note the nonlinear time scales.

Other Sulfolobus species. Three different Sulfolobus species, S. acidocaldarius, S. solfataricus, and S. shibatae, were studied, but due to space limitations, only the S. acidocaldarius results are illustrated. When the experiments were performed with S. solfataricus and S. shibatae, the results were similar in most respects. The only exception was that there was a larger variation in the time period from the dilution event until the first signs of cell division (usually between 4 and 6 h, occasionally longer), particularly for S. solfataricus, whereas for S. acidocaldarius the time interval of 4.5 h showed less variation between experiments. Nucleoid segregation and cell constriction times were not determined for S. solfataricus and S. shibatae.

	DISCUSSION

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In this report, we describe a series of dilution and temperature shift experiments which provide information about fundamental cell cycle characteristics and physiological responses of hyperthermophilic archaea.

The growth of a batch culture is usually divided into a series of stages, one of which is the lag phase (for discussions of batch cultures, lag phases, and shift experiments, see reference 12). In inoculation of batch cultures, the length of the lag is often estimated from OD measurements. However, the OD values are often so low after dilution that the experimental error is large and, importantly, OD measurements do not differentiate between viable and nonviable cells. This may therefore result in overestimation of the length of the lag phase, particularly since cultures that have been left in stationary phase for long time periods often contain a substantial fraction of nonviable cells. After dilution from stationary phase, the Sulfolobus cells analyzed here rapidly started to grow with little evidence of any lag. We find it likely that in the natural environment, there is selection for the ability to quickly start to grow and utilize nutrients when they become available. Thus, we believe that also in the natural habitat, Sulfolobus cells are able to initiate cellular growth rapidly when the environmental conditions become favorable.

Nucleoid segregation, cell division, and chromosome replication (in that order) did not take place until several hours after dilution, despite the rapid initiation of cellular growth. This shows that these major cell cycle events are tightly coupled to cellular mass increase, and to be initiated, they require that the cells first reach a critical size, volume, or other parameter. The fact that nucleoid segregation did not occur until after several hours of cellular growth lends support to the suggestion (13) that a G₂-like period separates termination of replication from nucleoid partition in these organisms. A strong interdependence between chromosome replication and preceding nucleoid partition and cell division was also revealed, as no cells were able to initiate replication until the preceding events had been completed.

We have previously estimated the lengths of the cell cycle periods in different Sulfolobus species by simulating DNA distributions obtained in flow cytometry analyses of exponentially growing cultures (4). The partial population synchrony that was obtained in the dilution experiments reported here allows an independent estimation of the lengths of these periods to be performed by monitoring the "leading edge" of the population. By this method, the postreplication period was found to be the longest cell cycle stage (although it should be pointed out that in the dilution experiments the starting population consisted of small resting cells, instead of newborn exponentially growing cells) and the prereplication period was found to be short (about 30 min), in agreement with previous estimates. The length of the chromosome replication period (C period, S phase) could not be estimated by the dilution approach.

Our understanding of the S. acidocaldarius life cycle is summarized in Fig. 6. The indicated relative lengths of the cell cycle periods represent our best current estimates based on the experiments reported in this article, as well as previously reported results (4, 13). Entry into stationary phase occurs in early G₂ and is accompanied by nucleoid restructuring. Stationary-phase cells are smaller than exponentially growing cells in early G₂ and often appear to be even smaller than newborn exponentially growing cells (osmotic differences between fresh medium and depleted medium may influence these size comparisons to some extent). The smaller size in stationary phase might be the result of an increased division frequency relative to the mass increase during the final cell cycles at the end of the exponential growth phase, as is the case in many other prokaryotes, although this remains to be experimentally confirmed. Dilution into fresh medium results in the response described in this report: the cells re-enter the cell cycle in early G₂ at a small cell size and with two fully replicated genomes. A long period of mass increase precedes nucleoid partition, which is followed by cell division. Nucleoid partition (mitosis) and/or cell division is a prerequisite for subsequent initiation of chromosome replication.

View larger version (16K): [in this window] [in a new window]   FIG. 6.   Life cycle of S. acidocaldarius. Thick lines represent stages distinguishable by flow cytometry analysis (prereplication, replication, and postreplication), and thin lines represent stages identified by microscopy (nucleoid segregation and cell division). The relative sizes of the cells as they progress along the cell cycle are indicated, but the circles are not intended to reflect the actual shape of the cells (lobed spheres) or the nucleoids (highly structured, except in stationary phase). Nucleoids are drawn as filled (nonreplicating DNA) or striped (replicating DNA) circles. Stationary-phase cells contain two fully replicated chromosomes (2N) in a single, unsegregated nucleoid.

Dilution of exponentially growing cells resulted in an unexpected transient inhibition of growth (30 to 90 min after the dilution) and cell division (50 to 90 min after the dilution), whereas initiation and elongation of chromosome replication continued. The mechanisms behind these effects, as well as their physiological relevance, are unknown. However, in experiments in which balanced growth is desirable, it is important to be aware of the fluctuations that are induced in the population as a result of a dilution event.

When cultures growing exponentially at 79°C were shifted to room temperature or ice-water, they appeared to freeze in mid-growth and no further cellular mass increase or division occurred. Also, ongoing rounds of replication were not terminated, indicating that the replisomes halted in mid-replication, presumably due to loss of protein function at low temperatures. Room temperature represents a downshift of 55 to 60°C from the optimal growth temperature of Sulfolobus species, which accounts for the rapid cessation of cellular activity. More surprising was the apparent ease with which cultures held at room temperature resumed growth and cell cycle progression upon a return to 79°C. Temperature shifts of this magnitude are seldom, if ever, encountered in the natural habitats of Sulfolobus species, and the cells may have few or no mechanisms with which to respond to such shifts and therefore freeze in their exponential state. Room temperature was less detrimental to the cells than transfer to ice, and short-term survival in the laboratory may, thus, be enhanced by simply transferring exponentially growing cultures to room temperature instead of ice-water. For long-term survival, it is also necessary to raise the pH of the medium to prevent acidification of the cytoplasm (15), which results in DNA degradation (4a). Importantly, in current efforts to establish transformation procedures for Sulfolobus species, the number of transformants might be increased by simply avoiding incubation on ice.

Finally, the partial synchrony obtained in the dilution of the stationary-phase cultures provides a simple protocol by which cell cycle-specific events may be studied in Sulfolobus species. In combination with the S. solfataricus complete genome sequence (16), this should, e.g., open up possibilities for a genome-wide analysis of differential gene expression during the Sulfolobus cell cycle.