Effects of high hydrostatic pressure on bacterial cytoskeleton FtsZ polymers in vivo and in vitro

Akihiro Ishii1,2,{dagger}, Takako Sato2, Masaaki Wachi3, Kazuo Nagai4 and Chiaki Kato1,2

1 Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
2 Japan Agency for Marine–Earth 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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Some rod-shaped bacteria, including Escherichia coli, exhibit cell filamentation without septum formation under high-hydrostatic-pressure conditions, indicating that the cell-division process is affected by hydrostatic pressure. The effects of elevated pressure on FtsZ-ring formation in E. coli cells were examined using indirect immunofluorescence microscopy. Elevated pressure of 40 MPa completely inhibited colony formation of E. coli cells under the cultivation conditions used, and the cells exhibited obviously filamentous shapes. In the elongated cells, normal cell-division processes appeared to be inhibited, because no FtsZ rings were observed by indirect immunofluorescent staining. In addition, it was observed that hydrostatic pressure dissociated the E. coli FtsZ polymers in vitro. These results suggest that high hydrostatic pressure directly affects cell survival and morphology through the dissociation of the cytoskeletal frameworks.


{dagger}Present address: Saitama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST, Faculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Oura-gun, Gunma, 374-0193, Japan.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hydrostatic pressure is a well-known physical stress, causing various reactions in living cells, which result from physical effects obeying the principle of Le Chatelier (Abe et al., 1999). Biological processes that are accompanied by an increase in reaction complexes or in the volume of products are inhibited by elevated pressure, and, conversely, processes that are accompanied by a decrease in volume are promoted. At the molecular level, hydrostatic pressure alters the hydration of proteins. Cellular processes are also affected under high-pressure conditions; for example, DNA, RNA and protein synthesis in Eschericha coli cells is inhibited at 50, 58 and 77 MPa, respectively (Zobell & Cobet, 1962, 1964).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
E. coli K-12 strains and cultivation.
E. coli K-12 strain W3110 was used as a wild-type strain. Strain JM109 (recA1 endA1 gyrA96 thi hsdR17 e14 supE44 relA1; Takara) was used as a recA gene mutant. Both strains were cultured at 37 °C under various pressure conditions. E. coli strain M15, containing the pREP4 plasmid (Qiagen), was used as the host strain to clone a histidine-tagged ftsZ gene. Strain M15 was cultured at 30 °C in medium supplemented with 25 µg kanamycin ml–1 and 50 µg ampicillin ml–1. The strains were cultured in Luria–Bertani (LB) medium or on LB plates containing 1·5 % agar. The cultivation method under high-pressure conditions has been described previously (Kato et al., 1995). The surface of the LB plates was covered with 1 % agarose before pressurization.

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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Fig. 1. Fixation apparatus for cells cultivated under high-pressure conditions. Components of the fixation equipment are labelled in the left-hand panel. The configuration of the apparatus during pressurization and fixation is shown in the centre and right-hand panels, respectively.

 
Preparation of FtsZ protein and in vitro polymerization assay.
The E. coli ftsZ gene was cloned into the pQE-30Xa vector (Qiagen), and a 6-His tag was attached to the N-terminus of ftsZ. The cloned His-tagged FtsZ (His–FtsZ) protein was expressed in strain M15 and purified on a HiTrap Chelating HP column (Amersham). The in vitro FtsZ polymerization assay was performed using modifications of published methods (Mukherjee & Lutkenhaus, 1998; Yu & Margolin, 1997). The function of the recombinant ftsZ gene thus obtained was confirmed by ftsZ84-mutant strain JEFZ1; namely, the suppression of temperature sensitivity and altered characteristics in the in vitro sedimentation assay under our experimental conditions (data not shown). The His–FtsZ protein (1 µM) was polymerized in a solution containing 50 mM HEPES (pH 7·5), 50 mM KCl and 10 mM MgCl2, and 1 mM GTP sodium salt (Sigma-Aldrich) was added to start the polymerization reaction. All polymerization experiments were performed at room temperature. The polymerization reaction was allowed to progress for 10 min at atmospheric pressure, and pressurized at the required pressure conditions for 30 min. Then, the reaction was fixed with 3 % glutaraldehyde, and the pressure level maintained for an additional 10 min.

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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Fig. 2. Fixation apparatus for in vitro FtsZ assay under high-pressure conditions. Components of the fixation apparatus are labelled in the left-hand panel. The configuration of the apparatus during pressurization and fixation is shown in the centre and right-hand panels, respectively.

 
Electron microscopy and counting of FtsZ filaments.
The fixed FtsZ filaments, stained with uranyl acetate, were observed by electron microscopy. To analyse the length distribution of FtsZ filaments, the lengths of all filaments, of which both ends could be clearly identified, were measured in a randomly selected visual field. The lengths of 50 filaments, fixed at each pressure, were measured.


   RESULTS
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INTRODUCTION
METHODS
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DISCUSSION
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Observation of colony-forming ability and cell morphology under high-pressure conditions
To investigate cell viability and morphologic changes under pressure, E. coli cells were cultured at pressure intervals of 10 MPa in the range 0·1–60 MPa. Under our cultivation conditions (see Methods), E. coli colonies grew in the pressure range 0·1–30 MPa (Fig. 3). From 40 MPa, colonies no longer formed, and OD660 values were reduced (Fig. 3). However, the OD660 values decreased gradually from the pressure at which no colonies were formed (non-permissive pressure) in the liquid medium. At 40 and 50 MPa, E. coli cells exhibited filamentous shapes, which appeared to contribute to a slight increase in OD660. At 60 MPa, colony-forming ability and OD660 values were completely abolished, and no E. coli cells were observed under the microscope (Figs 3 and 4). A pressure of 60 MPa, under our experimental conditions, thus caused all E. coli cellular processes to cease.



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Fig. 3. Pressure sensitivity of E. coli cell growth. E. coli W3110 cells were cultured on LB plates and in LB medium at the pressures shown in the left-hand column. Colony-forming ability was assessed at 10 MPa intervals up to 60 MPa (centre). The number of E. coli cells spotted on the plates is shown at the bottom of the figure. Final OD660 values after cultivation are shown in the right-hand column.

 


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Fig. 4. Microscopy of E. coli cells at each pressure. After cultivation at the pressure shown, E. coli cells were observed by phase-contrast microscopy. Strain JM109, cultivated at 50 MPa, was also observed by differential-interference microscopy (bottom-right panel). All photomicrographs were taken at the same magnification. Bar, 25 µm.

 
One reason for E. coli cell filamentation is considered to be the SOS response (Little & Mount, 1982; Radman, 1975; Witkin, 1976), because pressure inhibits DNA synthesis (Zobell & Cobet, 1962, 1964). However, recA and sulA mutants, defective in trigger proteins for the SOS response, were filamented at 50 MPa, as in the wild-type cells (Fig. 4). This result suggests that the SOS response did not contribute to the filamentation seen at high pressure.

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. 6a, 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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Fig. 5. Microscopy of E. coli cells fixed before or after pressure release. E. coli cells were cultured at 50 MPa and fixed (a) at 50 MPa or (b) at atmospheric pressure. Cells were observed by differential-interference microscopy (DIC), DAPI staining (DAPI) and immunofluorescence microscopy (IFM).

 


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Fig. 6. Rapid restoration of FtsZ-ring formation and cell division after pressure release. E. coli cells were exposed to atmospheric pressure after cultivation at 50 MPa for 17 h. Cells were examined by differential-interference microscopy (DIC) and immunofluorescence microscopy (IFM), 10 minutes (a) and 20 minutes (b) after pressure release. Newly formed septum is indicated by the arrow. Bar, 10 µm.

 
In vitro analysis of FtsZ polymerization under atmospheric- and high-pressure conditions
To investigate the physical effects of pressure on FtsZ-protein polymerization, we developed a fixation system for the FtsZ polymerization reaction in the vessel used for pressurization. This system was similar to the cell-fixation system above (Fig. 2). The manipulations employed are described in Methods. We cloned the His-FtsZ protein for the in vitro polymerization assay. For the pressure experiments, the reaction mixture was fixed for 10 min at each pressure, after 30 min pressurization. Then the pressure was released, and the fixed His-FtsZ samples were observed by electron microscopy with uranyl acetate staining. At atmospheric pressure, His-FtsZ proteins polymerized to form filaments (Fig. 7a) longer than 5 µm (Fig. 8). At the permissive pressure level of 30 MPa, FtsZ filaments were polymerized but partially dissociated. However, the number of filaments observed was smaller, and they were shorter in length than those observed at 0·1 MPa, as shown in Figs 7(b) and 8. In this study, at the non-permissive pressure of 50 MPa, no FtsZ filaments were observed, and we believe that the filaments were completely dissociated (Fig. 7c). To analyse the distribution of filament length, the lengths of 50 filaments at both 0·1 and 30 MPa were measured (Fig. 8). Most FtsZ filaments formed at 0·1 MPa were longer than 5 µm, but those formed at 30 MPa were less than 3 µm in length, and many aggregates were also observed.



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Fig. 7. Electron microscopy of FtsZ-filament assembly at three different pressures. FtsZ filaments were fixed at (a) 0·1 MPa, (b) 30 MPa or (c) 50 MPa.

 


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Fig. 8. Distribution of lengths of FtsZ filaments at atmospheric (black bars) and high pressure (30 MPa; white bars).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The present study showed that elevated pressures of 40 and 50 MPa inhibited E. coli colony formation and led to elongation of cell shape. These results strongly suggest that elevated pressure inhibits E. coli cell-division processes. Immunofluorescence microscopy showed that no FtsZ-ring formation occurred in filamentous cells at 50 MPa. In addition to the in vivo observations, we found that elevated pressure directly inhibited the polymerization of the FtsZ protein in vitro. The FtsZ pressure sensitivity thus corresponds to the in vivo non-permissive pressure level. In other studies, two effects on the inhibition of FtsZ polymerization in E. coli cells, the SOS response and chromosome block, have been reported (Sun et al., 1998; Witkin, 1976). These effects were suspected to have led to the elongation of E. coli cells in this study, because inhibition of DNA synthesis has been reported (Zobell & Cobet, 1962, 1964), and, under pressure, nucleoids were not clearly partitioned (Fig. 5). However, recA-mutant cells were filamentous under high-pressure conditions (Fig. 4), and thus the SOS response was not considered to contribute to filament formation under pressure. The effect of pressure on the nucleoids is still unclear. One possibility is that the lack of clear partitioning between nucleoids is due to compression, rather than the inhibition of segregation, because the nucleoids quickly segregated after pressure release (Fig. 5). In this case, the inhibitory effect on the uncompressed nucleoids, as in the mukB mutant, would be less than that on the unsegregated nucleoids, as in the topoisomerase mutant (Sun et al., 1998). These results suggest that hydrostatic pressure directly affects FtsZ function and, in addition, may interfere with nucleoid structure. As a result, E. coli cell morphology and growth are greatly altered.

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.


   ACKNOWLEDGEMENTS
 
We thank Ms C. Yenches for assistance in editing the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas: Single-Cell Molecular Technology (area number 736) and a grant from the 21st Century COE Program, Ministry of Education, Culture, Sports, Science and Technology of Japan.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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deBoer, P., Crossley, R. & Rothfield, L. (1992). The essential bacterial cell-division protein FtsZ is a GTPase. Nature 359, 254–256.[CrossRef][Medline]

Hirota, Y., Ryter, A. & Jacob, F. (1968). Thermosensitive mutants of E. coli affected in the processes of DNA synthesis and cellular division. Cold Spring Harbor Symp Quant Biol 33, 677–693.[Medline]

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Received 5 December 2003; revised 26 February 2004; accepted 3 March 2004.



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