1 Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
2 Japan Agency for MarineEarth Science and Technology, 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan
3 Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
4 Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan
Correspondence
Akihiro Ishii
akihiro-i{at}toyonet.toyo.ac.jp
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ABSTRACT |
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INTRODUCTION |
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In vivo phenomena, such as morphologic changes in eukaryotic and bacterial cells, under high-pressure conditions, have been studied. Elevated pressure alters the distinctive cell shapes of eukaryotic cells into simple round ones, and the structures of cytoskeletal proteins, such as microtubules and actin and myosin fibres, are depolymerized in vivo and in vitro (Bourns et al., 1988; Crenshaw et al., 1996
; Salmon, 1975a
, b
). Among bacterial cells, rod-shaped bacteria, including E. coli, exhibit diminished viability, and become filamented, without septum formation, under high-pressure conditions (Marquis, 1976
; Zobell & Cobet, 1962
, 1964
). The filamentous cells formed under high-pressure conditions resemble E. coli filament-forming temperature-sensitive (fts) mutants, in which cell division is defective at non-permissive temperatures. The defective genes in these mutants, designated fts (Hirota et al., 1968
), are known to be important for cell division. One of them, ftsZ, encodes the protein FtsZ, which polymerizes to a cytoskeletal ring, localizes at the division site and plays the most important role in the cell-division process (Bi & Lutkenhaus, 1991
; Lutkenhaus & Addinall, 1997
). The FtsZ protein is a GTPase with weak sequence homology to tubulins (deBoer et al., 1992
; Mukherjee et al., 1993
; RayChaudhuri & Park, 1992
) and, in fact, its crystal structure is similar to that of alpha- and beta-tubulin (Lowe & Amos, 1998
). Therefore, this bacterial cytoskeleton appears to be sensitive to elevated pressure, as are eukaryotic microtubules or other cytoskeletons. We assumed that FtsZ-ring formation is a critical step in survival under high-pressure conditions, and that, in E. coli, it is inhibited by the physical effects of pressure. Here, we show that filamentous E. coli cells cannot form colonies at a pressure of around 50 MPa, and that FtsZ polymerization is inhibited in vivo and in vitro at the same pressure.
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METHODS |
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Preparation for microscopy.
The conditions for E. coli strain W3110 cultivation and the fixation method were described previously (Sato et al., 2002), and the apparatus is shown in Fig. 1
. The top of a 15 ml centrifugation tube was removed and filled with the culture medium. The remaining body of the tube was filled with 80 % methanol as a fixation reagent, and a weighted needle was placed within the tube. Both sections were sealed with Parafilm and then put back together. The equipment was placed in a titanium vessel for pressurization, and incubated under the required pressure and temperature conditions. The pressure vessel was then inverted. The Parafilm separating the culture medium from the 80 % methanol was ruptured, allowing the contents of the two sections of the tube to mix. The fixed cells were used for microscopy. The immunofluorescence microscopy method has been described previously (Ishii et al., 2002
).
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To analyse the effects of pressure on FtsZ polymerization, the apparatus shown in Fig. 2 was used. This apparatus combines two 1 ml syringes, with one syringe cut at the top, separated by an O ring. First, 100 µl 10 % glutaraldehyde (fixation reagent) was placed in the lower syringe, a weighted plunger inserted, and a steel ball placed on the O ring to separate the two syringes completely. Next, a plunger was inserted in the upper syringe and 250 µl of the polymerization reaction mixture described above injected into the upper syringe through the injection port. The injection port was sealed, and the combined syringes were placed in a pressure vessel. Finally, to mix the reaction mixture and fixation reagent, the pressure vessel was inverted.
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RESULTS |
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Observation of FtsZ-ring formation in vivo under atmospheric and high-pressure conditions
E. coli cell filamentation is believed to involve a defect in cell-division processes (Hirota et al., 1968). Studies of cell-division processes have shown that the first step is construction of a cytoskeletal FtsZ ring (Bi & Lutkenhaus, 1991
; Lutkenhaus & Addinall, 1997
). The assembly functions of cytoskeletal proteins are possibly sensitive to high-pressure conditions. To investigate the effect of pressure on the cell-division process at the non-permissive pressure of 50 MPa, we observed in vivo FtsZ-ring formation by immunofluorescence microscopy. We previously developed a fixation system in the pressurization vessel that allowed observation of pressurized cells without pressure release (Sato et al., 2002
). The pressure vessel is a completely closed system, and no manipulations can be performed from outside.
E. coli cells were cultured overnight at 50 MPa in the fixation system, and then fixed, with or without pressure release (details described in Methods.) The cells fixed with or without pressure release were stained with DAPI and anti-FtsZ antibody to observe nucleoid structure and FtsZ localization, respectively. Cells fixed before pressure release exhibited unclear partitioning nucleoids and no FtsZ localization (Fig. 5a). On the other hand, after pressure release, nucleoids were clearly segregated, and several FtsZ rings were observed (Fig. 5b
). Cell-division processes then resumed normally, because septum formation was observed within 10 min (Fig. 6
a, arrow), and cells of normal length were generated within 20 min (Fig. 6b
). This rapid restoration of cell-division steps suggests that sufficient cell components had been synthesized. At any rate, sufficient FtsZ protein to polymerize at potential division sites was synthesized under high-pressure conditions, because Western blot analysis comfirmed that the amount of FtsZ was stable (data not shown). These observations suggest that E. coli cell growth, in terms of the increase in colony numbers, was limited at 40 MPa, in spite of the increase in cell mass. Therefore, we believe that the growth inhibition was caused by the inhibition of cell division, rather than the cessation of DNA, RNA and protein biosynthesis at a pressure of around 50 MPa (see Introduction).
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DISCUSSION |
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Morphologic change in eukaryotic cells, i.e., cell rounding, is dependent on individual cells, and shows a heterogeneous response even in a single culture. Unaltered cytoskeletal structures, such as microtubules, myosins, vinculins and vimentins, remain in both rounded and non-rounded cells under high-pressure conditions (Bourns et al., 1988; Crenshaw et al., 1996
). Therefore, eukaryotic cell rounding is considered to be associated with yet-to-be-clarified cellular processes (one candidate is mitosis), rather than with dissociation of the cytoskeleton (Bourns et al., 1988
; Crenshaw et al., 1996
). Our results suggest that the E. coli cytoskeletal protein FtsZ directly contributes to cell morphology and growth under high-pressure conditions. The difference in pressure sensitivity is considered to be due to the variety of cell cytoskeletons and differences in the levels of their components and their maintenance proteins. Bacteria contain only two cytoskeletal proteins, FtsZ and MreB (and their several homologues), and one of these, FtsZ, plays an important role in cell division (Bi & Lutkenhaus, 1991
; Jones et al., 2001
; Lutkenhaus & Addinall, 1997
). We think that the increase in volume between the monomer form and the polymer form inhibits the polymerization reaction of FtsZ under high-pressure conditions, as for eukaryotic cytoskeletal proteins. Therefore, in bacteria, inhibition of FtsZ-polymerization activity during cell division might cause the filamentous form to appear.
Interestingly, the polymerization activity of actin proteins extracted from deep-sea fish is promoted in vitro by elevated pressure, since there is a decrease in volume in the polymer form (Swezey & Somero, 1985), although cell rounding appears to be a secondary effect in response to cell-rounding signals. Deep-sea organisms are adapted to extremely high-pressure conditions. Cytoskeletal protein adaptation to pressure is an important factor determining whether cells survive under high-pressure conditions. In future, comparison between a deep-sea bacterial cytoskeleton and a terrestrial one will help to reveal pressure adaptation mechanisms.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 December 2003;
revised 26 February 2004;
accepted 3 March 2004.
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