Electrophoresis 1994, IS, 1559-1565 2-D PAGE of pressure-induced proteins 1559

Michael GroB" Ilona J. Kosmowsky' Rainer Lorenz** Hans Peter Molitoris* Rainer Jaenicke'

'Institut fiur Biophysik und physikalische Biochemie 'Institut fur Botanik, Universitat Regensburg

Response of bacteria and fungi to high-pressure stress as investigated by two-dimensional polyacrylamide gel electrophoresis

In an attempt to generalize previous observations (Jaenicke et al., Appl. Environ. Microbiol. 1988, 54, 2375-2380) and to find a convenient model system for studies of the pressure response, we tested the suitability of Escheri- chia coli and Therrnotoga rnaritima (bacteria), and of five different eukaryotic species including the filamentous fungi Asterornyces cruciatus and Dendiy- phiella salina, and the marine yeasts Debaiyornyces hansenii, Rhodosporidiurn sphaerocarpurn, and Rhodotomla rubra. Using two-dimensional polyacrylamide gel electrophoresis, detailed investigations on the pressure response were car- ried out with E, coli and Rhodosporidium sphaerocarpurn. In the former orga- nism, major pressure response proteins could not be detected, although there are significant differences in expression of some proteins as well as some minor components that are found in all of the high pressure cell extracts but not in extracts from cultures grown at atmospheric pressure. In Rhodospori- diurn sphaerocarpum, no change in protein expression patterns was observed between 0.1 and 20 MPa. However, approaching the limit of viability of 50 MPa, additional protein spots became detectable at 45 MPa. This finding corre- lates with the observation of abnormal growth forms of the organism at this pressure (Lorenz, R. et al. manuscript in preparation).

1 Introduction

Environmental conditions in the microbial habitats of the deep sea require adaptation to hydrostatic pressures up to 120 MPa. After more than a century of high pres- sure biology and biochemistry, dating back to the works of Certes and Regnard, details of barosensitivity, barotol- erance and barophily are still far from being understood [l]. As the adaptation to other kinds of stress or shock condition is often mediated by the expression of specific stress proteins (e.g. heat shock, cold shock or antifreeze proteins), a suitable approach to high-pressure adapta- tion would be to look for alterations in the protein pat- terns of microorganisms grown at different hydrostatic pressures, ranging from atmospheric pressure to the limit of viability. The classical way to visualize the total protein expressed by a cell is two-dimensional IEF/SDS- PAGE (2-D PAGE) as developed by O'Farrell [2]. This method can, under favorable circumstances, resolve more than 1000 proteins in one gel and needs only minimal amounts of cell extract. The technique was suc- cessfully applied to investigate the pressure response of the thermophilic archaeon Methanococcus therrnolithotro- phicus [3]. Several basic proteins in the range of 38 to 70 kDa were found to be strongly expressed in cells grown at 50 MPa, but not detectable in cells from atornospheric pressure cultures.

Because further characterization of these proteins is hampered by the difficult handling of methanogens at high pressures [4], we tried to apply the method to a more convenient system, at the same time generalizing the approach to the other two kingdoms of life, namely

to bacteria and eukarya. The present work describes a screening for both suitable systems and optimized elec- trophoresis conditions for a number of organisms including bacteria (Escherichia coli, Thermotoga mari- tima), filamentous fungi (Asteromyces cruaatus, Dendiy- phiella salina), and marine isolates of yeasts (Debaryo- myces hansenii, Rhodosporidium sphaerocarpurn, Rhodoto- rula rubra). Whereas E. coli and T maritima were chosen as well-characterized representatives of mesophilic and hyperthermophilic bacteria, respectively, the investiga- tion of the marine eukaryotes forms part of a more com- prehensive study addressing the issue of the viability of fungi in marine environments [5]. The effect of hydro- static pressure on the protein expression of E. coli and Rhodosporidium sphaerocarpurn is analyzed in detail. Another report on the pressure response of E. coli pub- lished during the preparation of this manuscript [6] will be considered in detail in the discussion section of this study.

2 Materials and methods

2.1 Growth conditions

E. coli K12 was grown at 38°C on a minimal medium Am91200 [7], supplemented with vitamins, glucose and all amino acids, except Met and Cys. Sulfate was added as the only sulfur source. Thus the option to introduce a "S-radioactive marker without changing the chemical composition of the medium was maintained. To cultivate bacteria at pressures of up to 50 MPa, autoclavable silicon tubes of 12 mm ID were used, which were closed

Correspondence: Prof. Dr. Rainer Jaenicke, Institut fur Biophysik und physikalische Biochemie, Universitat Regensburg, 93040 Regensburg, Germany

Abbreviation: PIP, pressure-induced protein

* New permanent address: Oxford Centre for Molecular Sciences, New Chemistry Laboratory, South Parks Road, Oxford OX1 3QT, UK

I* New permanent address: Woods Hole Oceanographic Institulion, Biology Department, Woods Hole, MA 02543, USA

0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1994 0173-0835/94/1212-1559 $5.00+.25/0

1560 M. GroB ct a / . Olectrophoresis 1994, I;, 1559-1565

at each end by a rubber stopper. The autoclave equip- ment was as described [4], except for the electrical heating, which was replaced by thermostatted jackets, and for the pressure medium which was distilled water (saturated with nitrogen) instead of oil. The equipment can accomodate twelve tubes in four autoclaves that can be depressurized independently, requiring 1 min to take out a sample and to reestablish the initial pressure. Cell growth was monitored photometrically as turbidity at 600 nm. Preparation of bacterial cell extracts for 2-D electrophoresis included sonication of the cells, incuba- tion with nucleases and addition of urea to a final con- centration of 8 M . The samples were finally shock frozen and stored at -70°C. Crude cell extracts of Thermotoga mariitima were a kind gift from A. Wrba. Growth and har- vesting of these cells was as described [8]. Marine fungi were cultivated at various hydrostatic pressures in high pressure equipment as described elsewhere [9]. A special methodology was used to meet the requirements of aerobic organisms by providing a sufficient supply of oxygen throughout the experiment [9]. Marine yeasts were grown at 22°C on 5 X GPYA medium (5.0 g glu- cose, 2.5 g peptone, 0.5 g yeast extract, 1000 mL artificial seawater) to log phase, inoculated onto fresh medium, sealed in high density polyethylene bags and incubated at 27°C in FC-77, a fluorocarbon compound purchased from 3M (Neuss, Germany). The fungal strains used were Asteromyces cruciatus (M 149), Dendryphiella salina (M 151), Debaryomyces hansenii (M 113), Rhodotorula rubra (M 83), and Rhodosporidium sphaerocarpum (M 185) from the culture collection (Institut fur Botanik) in Regensburg. A detailed account of the cultivation methods will be published elsewhere (Lorenz, R. and Molitoris, H. P., manuscript in preparation). Protein con- centrations in all extracts were determined according to Peterson [lo].

2.2 Electrophoresis

Electrophoresis chemicals were purchased from Serva (Heidelberg, Germany). 2-D PAGE electrophoresis was carried out as described [2, 31 using the IEF conditions optimized by Duncan and Hershey [ll] and the silver stain protocol by Merril and Harrington [12], including sensitization with glutaraldehyde. Specific staining of gly- coproteins was achieved by the protocol of Dubray and Bezard [13]. Preliminary gels were run on a Hoefer Mighty Small I1 apparatus (Serva). Gels shown in the fig- ures were run on laboratory-built vertical electrophoresis equipment, in which immersion of the gels in a 2 L buffer reservoir provides good constancy of temperature. The gel size is 10.5 X 12 cm for Figs. 1 and 2, and 12 X 13.5 cm for all other gels. The carrier ampholytes used for the IEF of E. coli extracts were pH 5-7 (80%) and pH 3.5-10 (20%). For Rhodosporidium sphaerocarpum the reverse composition of carrier ampholytes was optimal. Densitometric measurements were performed with a Hirschmann Elscript 400 Densitometer (Hirsch- mann, Unterhaching, Germany) with a stepsize of 0.25 mm in each direction. For visual comparison as well as for documentation purposes, gel patterns were recorded on X-ray duplicating film (Kodak) according to the method of Harrison [14].

3 Results

3.1 Bacteria

Escherichia coli is barotolerant, indicating that it can reproduce at elevated pressures, but its growth rate decreases steadily with the increase of pressure. The steepness of the decrease, as well as the upper limit of viability, depends strongly on the solvent conditions. As shown by a systematic screening in search of an optimal medium [15], components of the growth media and tem- perature are equally important. In medium AM 9/200, which was finally used in the expression studies, 40 MPa is the highest pressure providing reasonable cell yields. The growth rate at this pressure was 49Oio of the atmos- pheric pressure rate, as compared to 20% at 50 MPa. Pro- tein extracts from cells grown at 40 MPa and at atmos- pheric pressure were subjected to 2-D electrophoresis in parallel experiments. Thus, we always obtained pairs of gels run under identical conditions differing only in the pressure applied during cell growth (Figs. 1, 2).

Major “pressure-stress proteins” as seen in the studies on Methanococcus thermolithotrophicus could not be detected in E. coli grown at 40 MPa, but there are at least four minor components present in all gels from exponentially growing high-pressure cultures and in none of the atmospheric pressure gels (Table 1). Only two of the four proteins are still present in the stationary growth phase. A detailed comparison of thcse data with the results obtained by Welch et al. [6] is presented in Section 4.

Table 1. Pressure response in E. coli: proteins with obvious changes in exmession levels at 40 MPa as comuared to 0.1 MPaa’

Protein Log stat Corresponding location phase phase protein in [17]

1 - e 30.0 + 0 F 30.8 2 - g 21.8 + 0 G 21.0, F 21.5 3 - h 19.2 + + H 20 4 - b 15.3 + + C 15.4 = GroES

a) First column: protein location in the gel of Fig. 1. Numbers 1-4 refer to the boxes drawn in Figs. 1 and 2. Lower-case letters are used to distinguish this location (as an experimental result) from the alpha-numeric labeling system described in [17], which is used in the fourth column. Second column: enhancement (+) in loga- rithmic growth phase. Third column: enhancement (+), or not detectable (0) in stationary phase. Fourth column: tentative correla- tion of the identified proteins with PIPS from [6].

In an attempt to achieve more quantitative results, an evaluation of 15 medium-intensity spots was carried out to test the reproducibility of relative spot intensities and the statistical significance of deviations in the high-pres- sure gels. Spot intensities were normalized by referring to the sum of the integrated intensities of all 15 spots. Out of 15 spots, 10 had a reasonably good reliability (less than 20% standard deviation) in three different atmos- pheric pressure gels. Of these 10 spots there were two which were significantly out of range in the corre- sponding high pressure gels. From this statistical sample, one can extrapolate that a complete systematic evalua- tion of a statistically reliable double-set of gels should yield between 50 and 150 expression alterations in the whole protein inventory of E. coli. However, quantitative

E[ecnophotesrs 1994, 15, 159-1565

a

2-D PAGE of pressure-induced proteins 1561

b

Figure 1. Protein expression of E . coli K 12 in the logarithmic phase at 0.1 MPa (top) and at 40 MPa (bottom), 38OC; 45 pg total protein in each gel.

analysis in a strict sense would have to make use of radiolabeled proteins, since silver staining shows only a narrow range of proportionality between densitometric intensity and protein concentration.

Therrnntnga maritima cell extracts were investigated with the goal to find electrophoresis conditions yielding suf- ficient resolution for the search for stress proteins. How- ever, a tendency for horizontal (IEF direction) streaking

1562 M. GIOR er a/.

a

Electrophoresis 1994, 15, 155Y-1565

b

Figure 2. Protein expression of E. roll K12 in the stationary phase at 0.1 MPa (top) and 40 MPa (bottom), 38'C; 52 and 50 Bg total protein, respectively.

persisted with all variations of separation conditions 3.2 Eukarya applied. Prepurification of the extracts (e.g. by ultrafiltra- tion or size-exclusion chromatography) did not improve Out of the five marine fungi screened in this study, Aste- the resolution. Therefore, this organism was not romyces cruciatus, Debatyomyces hansenii and Dendly- included in further protein expression studies. phiella salina yielded only low resolution and/or relative

2-D PAUE of pressure-induced proteins 1563 Electrophoresis 1994, 15, 1559-1565

a

b

Figure 3. Protein expression of Rhodotoruh rubra in the stationary phase at 0.1 (top) and 40 MPa (bottom). Cells were grown on 5 X GPYA medium in high density polyethylene bags with FC77 as hydraulic fluid and oxygen reservoir, 2 7 T , incubation times 144 h and 143 h, respectively; 32 and 35 pg total protein, respectively.

intensity (not shown). While electrophoresis conditions were routinely varied in search for specific protocols for each organism, incompatibility of the standardized soni- cation protocol with these organisms may be the reason for the unsatisfactory results. Better separation was achieved with Rhodotorula rubra, but the results were obscured by vertical streaking, which, again, could not be abolished by any means. The vertical streaks were strictly reproducible in their width and location and they could also be detected by a periodic acidlsilver stain pro- cedure which specifically indicates glycoproteins [13]. Most surprisingly, the streaking which was consistently observed with all cell cultures obtained at 0.1 and 20 MPa did not appear when cells were grown at 40 MPa (Fig. 3).

The best resolution was obtained with the marine yeast Rhodosporidium sphaerocarpum, which is barotolerant to a degree comparable to E. coli [16]. This organism has the remarkable property of yielding excellent separation patterns over a wide range of electrophoresis conditions.

Preliminary results from gels obtained with 20,40 and 45 MPa cultures, as compared to samples grown at atmos- pheric pressure, revealed that no change in protein expression is observable at pressures up to 20 MPa; how- ever, at 40 to 50 MPa, approaching the threshold of com- plete growth inhibition, significant alterations occur. Hence, we focused our attention on the comparison of cultures grown at atmospheric pressure and at 40 or 45 MPa. Gels from stationary phase cultures at these three pressures are shown in Fig. 4. For the analysis of electro- phoretic patterns, an alpha-numerical labeling system was developed in analogy to the system used for E. coli [17]. For this purpose, a set of 11 internal marker pro- teins was selected, whose molecular weights were deter- mined from three different gels run with commercial broad-range molecular weight marker proteins. The internal markers are listed in Table 2 and marked with white dots in Fig. 4a. Enhanced or newly appearing spots in the 45 MPa gels, as identified by overlaying the trans- parent XRD-film exposures of silver-stained gels, are listed in Table 3. The absence or lower expression of these proteins at atmospheric pressure was verified by comparison with ca. 10 normal-pressure gels from dif- ferent experiments (not shown). Out of the four spots, only one is already enhanced at 40 MPa, indicating that the major stress response is activated between 40 and 45 MPa.

Table 2. Localization of protein spots used as internal markers in the analysis of Khodosuoridium sphuerocarpum gels

No. Location

1 E 91.3 2 D 52.3 3 D 51.7 4 E 50.5 5 B 41.9 6 E 38.8 I C 38.6 8 F 37.4 9 C 31.3

10 D 28.4 11 F 22.0

Table 3. Pressure-induced changes in protein expression in Rhodospo- ridium sphaerocarpuma)

Location 40 MPa 45 MPa

C 44.8 + + C 43.0 0 + A 29.0 0 + c 20.2 0 + a) Symbols as in Table 1.

4 Discussion

High hydrostatic pressure is an environmental condition characteristic for vast parts of the biosphere. Neverthe- less, the response of organisms toward high-pressure stress [l] is far less understood than, for instance, the heat-shock response [18]. The first attempt to charac- terize the pressure response of a unicellular organism at the protein level [3] targeted the methanogenic archaeon Methanococcus thermolithotrophicus, partly because the negative reaction volume of the methane-forming reac- tion suggested that this organism might thrive at ele- vated pressures. In spite of the positive results of this

1564 M. GIOR et a /

a

E/ectrophoresi.r 1994, 15. 1559-15b5

b d

Figure 4. Protein expression of Rhodusporidium sphaerocurpum in the stationary phase at 0.1, 40 and 45 MPa; medium and hydraulic fluid as in Fie. 3. (a) 0.1 MPa. 40 ug total protein: the internal marker Droteius listed in Table 2 are marked bv white dots. (b) 40 MPa. 35 WLR urotein. (c) 45 M i a , 2 6 pg protein. id) 45 MPa, 75 pg protein. Incubation times

study, further characterization of the detected pressure- induced proteins was hampered by the extremely diffi- cult handling of this organism. Later, a pressure-respon- sive gene was identified in a deep-sea bacterial culture [19], but, obviously, this organism was not to become a model system of general applicability either. Starting from E. coli and extending our scope into the kingdom of the eukarya, we therefore undertook a screening for suitable model systems.

In E. coli, expression changes are less spectacular than in Methanococcus thermolithotrophicus, at least under the conditions of our approach, which monitors the com- plete protein inventory of cells that have actually emerged from cell division under pressure. In contrast, a recent study in the tradition of Neidhardt’s work on stress-induced proteins in E. coli [6] made use of radioac- tive pulse labeling after application of pressure-shocks at pressures which completely inhibit cell division. Under these conditions, a set of 55 proteins is shown to be enhanced in expression relative to total protein

42 h, 42 h, 71 h and 71 h, respectively.

synthesis. However, since at these very high pressures, protein biosynthesis is severely inhibited, this set may well “include proteins whose rates of synthesis decrease to a lesser extent than those of most proteins, are unaf- fected by high pressure, or are elevated in response to high pressure” [6].

The four “pressure-induced proteins” (PIPs) detected in this study are listed in Table 1. Their locations in the gel shown in Fig. 1 are labeled with lower-case letters plus apparent molecular mass in kilodalton (e.g. “e 30.0”) and are tentatively correlated with PIPs identified in the pres- sure-shock labeling study [6] designated following the alpha-numeric labeling system applied by Neidhardt [17], where the isoelectric points are labeled with capital let- ters from A (acidic) to B (basic) and the apparent molec- ular weight is indicated in kDa. It is noteworthy that, although our set of five includes the heat-shock protein GroES, we did not detect enhancement of GroEL, DnaK and GrpE, which were shown to be transiently induced by pressure shock [6].

Electrophoresis 1994, 15, 1559-156s 2-D PAGE of pressure-induced proteins 1565

The apparent repression of the ribosomal protein L12 (B 13.0), which migrates in close proximity to L7 and can be clearly seen to lose intensity with pressure in compar- ison to the other ribosomal protein, is due to the well- known interconversion between the two, which has been shown to be growth-rate dependent [20]. Whereas at higher growth rates, synthesis of L12 outperforms the acetylation step yielding L7, slower growth rates as ob- served under high pressure favor the acetylated version L7, which is also listed among the 55 PIPs reported by Welch et al. [6]. Some strongly basic PIPs may have escaped our analysis, due to the well-known short- coming of the classical O’Farrell technique at the basic end. Welch et al. also used nonequilibrium methods [21] and thus found the strongest induction effect of all in an extremely basic protein with a p1 of 11.0 (PIP 115.9) [6]. The fact that pressure-shock induces heat- and cold- shock proteins [6], combined with the observation that ribosomes appear to play a role as sensors for these two stress conditions [22], strongly suggests a similar role for ribosomes in response to hydrostatic pressure. Indeed, the pressure sensitivity of ribosomes and functional ribo- somal complexes has been characterized in detail [23, 241, with the post-translocational ribosome emerging as the most sensitive state.

In contrast to E. coli, whose protein patterns have been studied in great detail, with over 600 spots being unam- biguously identified up to now [25], the marine isolate of Rhodosporidium sphaerocarpum is only poorly character- ized from a biochemical point of view. However, the fact that this organism does give excellent 2-D gels, even at high protein concentrations, allowing a combined 2-D PAGE/microsequencing approach [26 ] , clearly suggests that Rhodosporidium sphaerocurpum is a suitable model for the investigation of the stress response in eukarya. Little is known about this topic, except for the fungicidic effect of high-pressure sterilization methods [27]. Thus, the availability of convenient model systems may help to close this gap. A detailed discussion of high-pressure effects on protein expression in the context of morpho- logical and physiological changes is being published else- where (Lorenz, R., Grofl, M., Jaenicke, R. and Molitoris, H. P.; manuscript in preparation). Thus, characterization of the pressure response in nonadapted organisms will lay the foundation for future studies addressing the mechanisms of adaptation in true barophiles.

This work was supported by the Deutsche Forschungsge- meinschaft, by the Fonds der Chemischen Industrie and the research funds of Regensburg University. During his doc- toral thesis, M. G. received a bursary of the Friedrich-Ebert- Stifung; R. L. was supported by a scholarship from the University of Regensburg. Cultures of marine fungi were kindly supplied by S. Crow (Atlanta, GA, USA; M 113), J. Fell (Miami, FLA, USA; M 83), H. Huber (Regensburg; M

185) and S. Rohrmann (Regensburg; M 149, M 151), and cell extracts of Thermotoga maritimu by A . Wrba. Help and advice from Drs. G. Bernhardt, M. Groitl, H.-D. Liidemann and R. Seckler are gratefully appreciated. Numerous stu- dents have had their hands in the “ripening” of our electro- phoresis protocol, which in the course of fouryears led to a remarkable degree of foolhardiness. Some of the gels shown in this paper were run by Ines Meyer, Arne Hengerer and Anja Miiller.

Received April 26, 1994

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