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Eukaryotic translation initiation factor 5A (eIF5A) stimulates ribosomal peptidyltransferase. The biochemical activity of eIF5A, as well as sustained growth of eukaryotic cells, requires hypusine [N¹-(4-amino-2-hydroxybutyl)lysine], a posttranslationally modified amino acid unique to eukaryotic and archaeal IF5A (eIF5A and aIF5A, respectively) (see reference 10 for a recent review). The function of aIF5A is inferred from that of eIF5A, since no reliable cell-free translation systems from archaea have been established. Hypusination of pre-IF5A proceeds in two steps. First, deoxyhypusine synthase (DHS) transfers the aminobutyryl moiety of the polyamine spermidine to the -amine of a conserved lysine in pre-IF5A to form deoxyhypusine. Next, deoxyhypusine hydroxylase adds a hydroxyl group to the -carbon to form hypusine. Deoxyhypusination occurred in all eukaryotes (4) and archaea (1) tested. N¹-guanyl-1,7-diaminoheptane (GC₇) is a very efficient inhibitor of DHS in eukaryotes. This homolog of spermidine inhibits the first step in the hypusination pathway by binding to DHS, occluding the binding site for spermidine, with an apparent K_i of 10 µM (6, 9). Hypusine, the product of the second reaction, has been found in all eukaryotes and crenarchaea tested, whereas euryarchaea seem not to perform the second modification step (1). Neither deoxyhypusine nor hypusine have been found in bacteria (4). The current model of eIF5A's involvement in protein synthesis and cell proliferation suggests that it is required for correct translation of a subset of mRNAs, possibly including ones that encode key proteins for cell growth and proliferation (7).

Members of the genus Sulfolobus are the first archaea whose cell cycle features have been determined (2). The cell cycle is characterized by a short prereplicative period (B or G1 period; 1 chromosome eq/cell) and a long postreplicative period (D or G2 period; 2 chromosome eq/cell). Another unusual feature is the presence of 2 chromosome eq/cell in stationary phase.

We have investigated the effects of the highly efficient pharmacological inhibitor of DHS, GC₇ (6), on the growth of four archaeal and one bacterial species. We have also analyzed, by flow cytometry, the detailed effects on the cell cycle of Sulfolobus acidocaldarius.

Growth inhibition by GC₇. Four different species of archaea and one bacterium were tested for their sensitivity to GC₇. The antibiotic was synthesized and separated as previously described (3), dissolved to 0.05 M in 1.0 mM acetic acid, and kept frozen. S. acidocaldarius DSM 639 and Sulfolobus solfataricus DSM 1616 were grown as previously described (2). Halobacterium halobium DSM 670 and Haloferax mediterranei DSM 1411 were grown at 37°C (11) (the low-salt medium described in reference 11 for Haloferax volcanii was used to grow H. mediterranei). Escherichia coli MG 1655 was grown at 37°C in Luria-Bertani medium. All species were grown in liquid medium in water baths at a shaking speed of 200 rpm. Aliquots taken from exponentially growing cultures were used to inoculate 25 ml of preheated liquid media. The cells were then allowed to grow one generation prior to addition of GC₇ to final concentrations of 100 and 1,000 µM. Growth was monitored by measuring the optical density at 600 nm (OD₆₀₀).

Dose-dependent inhibition of S. acidocaldarius growth. Three features of the archaeon S. acidocaldarius made it suitable to study the effect of GC₇ in more detail. (i) Its cell cycle has been characterized (2). (ii) It lends itself well to flow cytometric analyses. (iii) Its aIF5A protein has been isolated and shown to be fully hypusinated (12). S. acidocaldarius was grown in the presence of 0, 50, 100, 200, 350, 500, or 1,000 µM GC₇ to determine the concentration necessary to affect growth. The growth curves presented in Fig. 1A show that the growth rate was inversely proportional to the concentration of GC₇. The effect is thus cumulative and increases until growth ceases completely. We used the slope of the linear part of the growth curve in a logarithmic plot as an approximate measure of the growth rates. From these, the dose of drug at which the growth rate was reduced by 50% was deduced to be about 90 µM (data not shown). This concentration is similar to that required for growth arrest of eukaryotic cells (30 to 100 µM) (3, 13). The continued analysis of the effect of GC₇ on the cell cycle requires inhibited growth over an extended period (>24 h) without major morphological changes to the cells. We chose a concentration of 200 µM at which growth is inhibited sufficiently (Fig. 1A), while the cells retain normal cell and nucleoid shape as judged by phase-contrast and fluorescence microscopy (data not shown).

View larger version (54K): [in this window] [in a new window]   FIG. 1.   (A) Dose-dependent inhibition of S. acidocaldarius growth by GC7. S. acidocaldarius was subjected to 0 (), 50 (), 100 (), 200 (), 350 (), 500 (), and 1,000 (+) µM concentrations of GC7. Growth was monitored by OD600 measurements at the indicated time points. The growth curves give approximate doubling times of 3.5 h (0 µM GC7), 4.5 h (50), 8.0 h (100), 20 h (200), 25 h (350), and 50 h (500), whereas no growth could be detected at 1,000 µM GC7. (B) Analysis of GC7 effects on the S. acidocaldarius cell cycle. Samples were taken every 15 min after addition of GC7 (200 µM), fixed in ethanol, stained with mithramycin and ethidium bromide, and analyzed by flow cytometry. The left column represents the light scatter (cell size), and the right column represents the fluorescence (DNA content) of the cell population from the same samples. (C) The effect by GC7 is cytostatic. S. acidocaldarius resumed normal cell growth (i), including cell division and replication (ii), after reduction of the GC7 concentration to noneffective levels. S. acidocaldarius cells were treated with 200 µM GC7 or left untreated, and the drug was removed after 4 h. The release was accomplished either by dilution ( and ) or by change of medium after centrifugation ( and ). (i) Growth curves were determined by OD600 measurements. The treated cultures ( and ) were compared to untreated cultures ( and ). (ii) Flow cytometric analysis of samples fixed in ethanol every 15 min after release by dilution (samples correspond to treated culture dilution curve [] in graph i).

GC₇ causes cell cycle arrest. We examined the growth arrest in more detail. GC₇ was added to exponentially growing S. acidocaldarius to a final concentration of 200 µM. Measurements of OD₆₀₀ every hour after drug addition were used to monitor growth and confirmed the growth arrest (the OD₆₀₀ changed only from 0.2 at the time of drug addition to 0.23 5 h later, while in the untreated culture the OD₆₀₀ increased from 0.13 to 0.35).

Reversion of GC₇-induced growth inhibition. The usefulness of GC₇ to study the molecular details of hypusination in archaea would be greatly enhanced if cell growth could be restored after withdrawal of the inhibitor. We therefore performed an experiment on S. acidocaldarius where the inhibitor (200 µM) was removed or reduced to a noneffective concentration after 4 h of treatment, a time sufficient to cause cessation of growth and complete cell cycle arrest (Fig. 1C, graph i). Reduction of the GC₇ concentration was achieved by two methods: (i) centrifugation (10 min at 12000 × g) followed by resuspension of the cells in preheated drug-free medium or (ii) a fivefold dilution of the culture. Samples were taken at 2-h intervals before the release from growth arrest induced by GC₇ (for OD measurements) and after the release at 1-h intervals (for OD measurements) or at 15-min intervals (for flow cytometry). Samples were prepared as described above. Both treated cultures quickly resumed growth after reduction of the GC₇ content by either method (Fig. 1C, panel i). Both untreated cultures and the treated culture after removal of GC₇ by centrifugation grew slightly faster than the diluted culture, presumably due to residual GC₇ (approximately 40 µM) (compare Fig. 1C, panel i, with Fig. 1A). The effect of GC₇ of S. acidocaldarius thus appeared to be cytostatic.

Conclusions. We have analyzed how the hypusination inhibitor GC₇ affects growth of four different archaea and the bacterium E. coli. The drug was found to be effective on all archaea tested, at concentrations comparable to those used in similar studies on eukaryotes, but to be ineffective on the bacterium. Arrest of both cellular growth and the cell cycle of the hypusine-containing archaeon S. acidocaldarius was rapid and reversible. The rapidity of the effect is in strong contrast to the slow effect seen on mammalian cells, while reversibility for eukaryotes has not been reported. The effect of GC₇ on the cell cycle of S. acidocaldarius included arrest at the end of the D period (G2) (prior to cell division), without an apparent effect on replication elongation.