INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The deep sea is known as an extreme environment with a stable low temperature and very high hydrostatic pressure. Microorganisms living at the sea bottom may have special mechanisms for adaptation to such extreme conditions. Piezophilic bacteria, formerly called barophilic bacteria, are defined as bacteria which show better growth at high pressure than at atmospheric pressure (32), and they were first isolated in 1979 (33). Still, the mechanisms involved in bacterial adaptation to high-pressure environments are not well known. We have isolated a large variety of bacteria from samples of deep-sea sediments obtained by means of manned and unmanned submersibles operated by the Japan Marine Science and Technology Center to investigate the molecular mechanisms of bacterial adaptation to high hydrostatic pressure (13, 14, 16).

Bartlett and his coworkers have studied pressure-sensing mechanisms, focusing on the expression of high-pressure marker outer membrane proteins, OmpH and OmpL, whose gene expressions are controlled by pressure, positively and negatively, respectively, in the moderately piezophilic bacterium Photobacterium profundum SS9 (2-4, 11, 30). Their results suggested that several factors are involved in gene expression controlled by pressure conditions and that biological components of the cell surface may be very important in these mechanisms (1, 15).

Recently, a pressure-regulated promoter was found in a piezophilic bacterium, strain DB6705 (17), and in another moderately piezophilic bacterium, strain DSS12 (12), subsequently identified as Shewanella benthica and Shewanella violacea, respectively (23). Near the promoter in the genomic DNA of S. violacea, an open reading frame homologous to the cydD gene of Escherichia coli was found, and the significance of this gene in bacterial growth under high hydrostatic pressure conditions was suggested (19). The cydD gene product is thought to be required for the assembly of respiratory components in E. coli (24-26). These findings suggested that the expression of the respiratory system may be regulated by hydrostatic pressure, and this regulation may play some role in adaptation to high hydrostatic pressure. Indeed, expression of cytochromes is regulated by pressure in S. benthica DB172F (27, 28) and S. violacea DSS12 (29).

S. violacea is one of the bacteria isolated in our laboratory from deep-sea sediment from the Ryukyu Trench (5,110-m depth) collected by means of the manned submersible SHINKAI 6500 (16), and this organism has been taxonomically characterized (23). It is a moderately piezophilic and psychrophilic bacterium, which shows optimal growth at a pressure of 30 MPa and at a temperature of 8°C, and it exhibits almost the same growth at pressures of 0.1 and 50 MPa. We have previously reported the spectrophotometric analysis of S. violacea membrane fractions from the cells cultivated at both high- and atmospheric-pressure conditions (29). The results suggested that the expression of a membrane-bound d-type cytochrome and the expression of a soluble c-type cytochrome(s) were regulated by hydrostatic pressure. Therefore, these cytochromes may play some role in the piezoadaptibility of this bacterium. But the molecular mechanisms of gene expression regulated by pressure in these cytochromes are still unknown.

In the present study, we purified two soluble c-type cytochromes to an electrophoretically homogeneous state from the moderately piezophilic bacterium S. violacea grown at atmospheric pressure, and the genes coding for these cytochromes were cloned and sequenced. Further, by Western and Northern blotting analyses, we investigated the expression of soluble c-type cytochromes in cells grown under different pressure conditions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Microorganism and culture conditions. S. violacea DSS12 (JCM10179), isolated from deep-sea sediment obtained from the Ryukyu Trench (24°15.23'N, 126°47.30'E) at a depth of 5,110 m by means of the SHINKAI 6500 system (16, 23), was used in this study. Cultivation of S. violacea was performed at 8°C as described previously (16) under various pressure conditions. Marine Broth 2216 (Difco Laboratories, Detroit, Mich.) autoclaved and filtered through a 0.22-µm-pore-size membrane filter was used as the culture medium. Two kinds of cultivation methods were used for cells grown under atmospheric pressure conditions: cells were grown aerobically with shaking, and cells were grown microaerobically in a sterilized package without shaking. On the other hand, some of these packages were placed in the pressure vessel and pressurized as described previously (16, 29). Under both microaerobic cultivations, the only variable factor was the applied pressure, whereas the oxygen concentrations in the medium might be the same. Cultivated cells were collected by centrifugation (10,000 × g for 15 min), washed in 10 mM Tris-HCl buffer (pH 8.0) containing 0.3 M NaCl, and stored at 80°C until use.

Purification of soluble cytochromes c. Frozen S. violacea cells (about 10 g [wet weight]) which had been cultivated at atmospheric pressure were thawed, and NaCl was added to a final concentration of 1.0 M. The cell suspension was treated with a sonic oscillator (20 kHz, 200 W; model UD-201; Tomy Seiko Co., Tokyo, Japan) for a total period of 45 min. Unbroken cells were removed by centrifugation (10,000 × g for 15 min). The cell-free extract obtained was centrifuged at 143,000 × g for 1 h. The supernatant obtained by ultracentrifugation was dialyzed for 12 h against 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM sodium L-(+)-ascorbate at 4°C. The dialyzed solution was applied to a diethylaminoethyl (DEAE)-Toyopearl column (TSK-gel 650M; 2.6 by 14 cm; Tosoh Co., Tokyo, Japan) equilibrated with the same buffer as used for dialysis. After washing the column with the same buffer, the cytochromes c adsorbed on the column were eluted with a 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM sodium L-(+)-ascorbate and 0.15 M NaCl. The eluted fraction was dialyzed again for 3 h against 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM sodium L-(+)-ascorbate. The fraction obtained was subjected to chromatography on a DEAE-Toyopearl column (TSK-gel 650M; 2.6 by 6 cm) equilibrated with the same buffer used for dialysis. Two types of cytochromes c (named cytochrome c_A and cytochrome c_B) adsorbed on the column were eluted with a linear gradient produced from 245 ml each of 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM sodium L-(+)-ascorbate and the buffer containing 0.3 M NaCl as shown in Fig. 1. Cytochrome c_A and cytochrome c_B were eluted at approximately 65 and 85 mM NaCl, respectively. A 1/10 volume of 1 M Tris-HCl buffer (pH 8.0) was added to each fraction. Subsequently, ammonium sulfate was added to each fraction to yield 80% saturation, and after stirring for 15 h, the resulting solution was centrifuged at 10,000 × g for 15 min. Cytochrome c_A in the supernatant was further purified as follows. The solution was applied to an EXPRESS-ION EXCHANGER D column (1 by 2 cm; Whatmann International Ltd., Kent, United Kingdom) equilibrated with 10 mM Tris-HCl buffer (pH 8.0) and 80% saturated with ammonium sulfate. The cytochrome c_A adsorbed on the column was eluted with a small volume of 10 mM Tris-HCl buffer (pH 8.0). Cytochrome c_B was obtained as a precipitate in the above-mentioned centrifugation step and was dissolved in 1 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 0.3 M NaCl. Each of these protein solutions obtained was subjected to gel filtration on a HiLoad Superdex 75 prep grade column (1.6 by 60 cm; Pharmacia Biotech. Co., Uppsala, Sweden) equilibrated with 10 mM Tris-HCl buffer (pH 8.0) containing 0.3 M NaCl. The fractionated cytochromes c were used as the purified cytochrome c_A and cytochrome c_B preparations. Purification of these cytochromes is summarized in Table 1.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Elution profiles of the soluble fraction prepared from S. violacea cells during the second DEAE-Toyopearl chromatography step of the purification procedure. The elution of heme c was monitored at 410 nm (circles). The dashed line denotes the NaCl concentration. Cytochrome cA and cytochrome cB eluted at about 65 and 85 mM NaCl, respectively.

Physical and chemical measurements. Spectrophotometric measurements were performed using a Shimadzu UV-2400PC spectrophotometer (Shimadzu Co., Kyoto, Japan) with 1-cm light path cuvettes at room temperature. The heme c content was determined on the basis of the millimolar extinction coefficient (_mM) of pyridine ferrohemochrome c having a value of 29.1 mM¹ cm¹ (8).

Construction of a  phage library of the S. violacea chromosome and screening for the cytochrome c genes. Chromosomal DNA isolated from S. violacea was partially digested with Sau3AI. DNA fragments 15 to 23 kb in size, fractionated by electrophoresis on a 0.7% agarose gel, were inserted into the BamHI site of EMBL3 (Stratagene Co., La Jolla, Calif.). The ligated DNA was employed for in vitro packaging by using GIGAPACK III XL packaging extracts (Stratagene Co.) according to the manufacturer's instructions. The  phage library was screened by using the digoxigenin (DIG) detection system (Boehringer Mannheim Co., Mannheim, Germany). Based on the determined N-terminal amino acid sequence of cytochrome c_A (YDKAZHIZHSMG) and that of cytochrome c_B (EGNAEVGKTKAIVZS), two degenerate oligonucleotides were designed. The nucleotide sequences of these probes were 5'-TAY GAY AAR GCI TGY CAY ATH TGY CAY AGY ATG GG-3' (35-mer) specific for the cytochrome c_A gene and 5'-GAR GGN AAY GCN GAR GTN GGN AAR CAN AAR GCN ATH GTN TGY TC-3' (45-mer) specific for the cytochrome c_B gene. These oligonucleotides were labeled with digoxigenin at the 5' end for use as hybridization probes. The library was screened with both probes, and then positive plaques were purified by four serial plaque hybridization steps. The inserts in  phage amplified by long and accurate PCR were sonicated, and a shotgun library was constructed in the pUC18 vector (10). For sequencing of these fragments, the random shotgun sequencing method was used with a DNA sequencer (model 373S; Perkin-Elmer/Applied Biosystems Co., Foster City, Calif.). Assembling and editing of the determined DNA sequences were performed with AutoAssembler version 2.0 (Perkin-Elmer/Applied Biosystems Co.), and GENETYX-MAC version 10 from Software Development (Tokyo, Japan) was used for sequence analysis.

Western blot analysis. Frozen cells of S. violacea DSS12, which had been cultivated at atmospheric pressure or at 50 MPa, were thawed separately, and NaCl was added to a final concentration of 1 M. The cell suspension was treated with a sonic oscillator (20 kHz, 200 W) for a total period of 30 min. Unbroken cells were removed by centrifugation (10,000 × g for 15 min). The cell-free extract obtained was centrifuged at 143,000 × g for 1 h. The supernatant was dialyzed for 16 h against 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, and the resulting solution was used as the soluble fraction for Western blotting analyses. Western blotting after SDS-PAGE was carried out by using the NovaBlot system (Pharmacia Biotech. Co.), with 1/500 dilutions of anti-serum (rabbit) against the purified cytochromes, which was prepared by Sawady Technology Corporation (Tokyo, Japan). The antibody-cytochrome complexes were detected by means of an anti-rabbit immunoglobulin G secondary antibody coupled to alkaline phosphatase, using the Immuno-Blotting Kit according to the manufacturer's instructions (Bio-Rad Co.).

Northern blotting and primer extension. RNA was prepared from S. violacea as described previously (17). The RNA pellet was dissolved in diethyl pyrocarbonate-treated water, quantified by spectrophotometry, and stored at 80°C. Northern blotting analysis was performed by the method reported previously (17). Probes for Northern blotting analysis were constructed by the PCR DIG Probe Synthesis Kit (Boehringer Mannheim Co.) with the following synthesized primers. Primer set for the cytochrome c_A gene was as follows: forward, 5'-GTTAGCAATGACTGCAGTCG-3', and reverse, 5'-CTGTAAAACATACCACCTGG-3'. Primer set for the cytochrome c_B gene was as follows: forward, 5'-GTTAGCTCTTGCACTATCAG-3', and reverse, 5'-TAATGCTTCGATATCGTCGC-3'.

Nucleotide sequence accession numbers. The DNA sequences reported in this paper have been deposited in the DDBJ (Mishima, Japan), EMBL (Heidelberg, Germany), and GenBank (Mountain View, Calif.) nucleotide sequence databases. The accession numbers of the DNA sequences containing the genes coding for cytochromes c_A and c_B of S. violacea DSS12 are AB032404 and AB032405, respectively.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Purification of soluble cytochromes c from S. violacea. Two kinds of soluble cytochromes c were purified by several steps of column chromatography (Table 1); the one that was eluted first from the DEAE-Toyopearl column was designated cytochrome c_A and the one that was eluted later from the same column was designated cytochrome c_B. When these cytochromes c were subjected to SDS-PAGE, each showed one major band in the gel stained with Coomassie brilliant blue and a heme-staining reagent, as shown in Fig. 2. Both of the cytochromes c were purified to an electrophoretically homogeneous state. The molecular masses of the purified cytochromes as determined by SDS-PAGE were 8.3 kDa for cytochrome c_A and 23 kDa for cytochrome c_B, and both proteins had heme iron.

View larger version (56K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE gel of purified soluble cytochromes c from S. violacea cells. After electrophoresis (15% acrylamide gel), the gel was stained with Coomassie brilliant blue (B) and a heme-staining reagent (A). Contents of the purified proteins are about 5 µg in each lane.

Molecular properties of the cytochromes c. Figure 3 shows the absorption spectra of the purified cytochrome c_A (Fig. 3A) and cytochrome c_B (Fig. 3B). Cytochrome c_A showed an absorption peak at 410 nm in the oxidized form and peaks at 419, 525, and 553 nm in the reduced form (Fig. 3A). The pyridine ferrohemochrome of cytochrome c_A showed peaks at 521 and 550 nm (data not shown). This result suggested that cytochrome c_A has heme c as a prosthetic group. The millimolar extinction coefficient of the reduced cytochrome c_A at 553 nm was determined to be 19.5 mM¹ cm¹, based on that of the -peak of the pyridine ferrohemochrome c. The midpoint redox potential was determined to be 0.301 mV. The results of SDS-PAGE indicated that this cytochrome consists of one polypeptide with a molecular mass of 8.3 kDa. The mass of the cytochrome per heme c was estimated to be 9,300 Da, based on the protein content and the heme c content. Thus, cytochrome c_A of S. violacea has one heme c per molecule.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Absorption spectra of cytochromes cA (A) and cB (B) from S. violacea cells. Each of the cytochromes c was solubilized in 10 mM Tris-HCl (pH 8.0) containing 0.3 M NaCl. The protein concentration in the case of cytochrome cA was 1.43 µM and that in the case of cytochrome cB was 0.81 µM. The reduced form of each (solid line) was prepared by the addition of a small amount of Na2S2O4, and the oxidized form of each (dashed line) was prepared by adding a small amount of (NH4)2S2O8.

1

1

Cloning and sequencing of the genes coding for the soluble cytochromes c_A and c_B and primer extension analyses. The N-terminal amino acid sequences of cytochromes c_A and c_B were determined to be QEGKAVYDKAZHIZHSMGVAGA and EGNAEVGKTKAIVZSAZHGVDG, respectively. These sequences were used to design degenerate oligonucleotide probes for screening the phage library of S. violacea genomic DNA. The nucleotide sequences of the probes synthesized were as follows: cytochrome c_A, 5'-TAYGAYAARGCITGYCAYATHTGYCAYAGYATGGG-3', and cytochrome c_B, 5'- GARGGNAAYGCNGARGTNGGNAARACNAARGC NATHGTNTGYTC-3'. A few positive plaques were obtained, and the phages in these plaques were purified by a repeated plaque hybridization procedure. Finally, we identified a single positive plaque for each of the above cytochromes c gene sequences, and we determined the DNA sequence of the region including the open reading frame coding for each of these proteins.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   Nucleotide and deduced amino acid sequences of the cloned genes coding for cytochromes cA (A) and cB (B). The underlined amino acids were identified by automated Edman sequencing of the purified proteins. The possible signal sequences are shown in italic characters. A heme c binding motif (CXXCH) is shaded. The putative 35 and 10 sequences (underlined) are located upstream from the transcriptional start point, as determined by primer extension (+1).

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Primer extension analysis to determine the transcriptional start points of the genes coding for cytochromes cA (A) and cB (B). The DNA sequence ladders of these genes were obtained by the dideoxy termination method employing the same primer. The nucleotide sequence corresponding to the ladder is shown on the left. The transcriptional start points are indicated by arrows and asterisks, and the 5' end of the mRNA is also shown.

Pressure regulation of expression of the cytochromes c. The results of Western blot analysis of the soluble fraction of cells grown aerobically or microaerobically at 0.1 MPa or microaerobically at 50 MPa, using antisera raised against purified cytochrome c_A and cytochrome c_B, are shown in Fig. 6A. The results showed that cytochrome c_B is expressed only in cells grown at atmospheric pressure. In contrast, cytochrome c_A is expressed regardless of the pressure conditions during growth. Further, the pattern of expression of the cytochromes c was the same in the case of both cells grown aerobically and cells grown microaerobically at atmospheric pressure.

View larger version (38K): [in this window] [in a new window]   FIG. 6.   (A) Western blot analysis of soluble fractions from S. violacea cells grown at 0.1 and 50 MPa. Whole protein present in the gel was transferred to a polyvinylidene difluoride membrane and then treated with antiserum raised against either cytochrome cA or cytochrome cB. The protein concentration in each soluble fraction was 1.67 mg/ml. Lanes 1, proteins from cells grown aerobically at 0.1 MPa; lanes 2, proteins from cells grown microaerobically at 0.1 MPa; lanes 3, proteins from cells grown microaerobically at 50 MPa. (B) Northern blot analysis of mRNAs from S. violacea cells grown at 0.1 and 50 MPa. Total RNA (30 µg) was loaded onto an agarose gel containing formaldehyde and transferred to a nylon membrane. PCR products described in the Materials and Methods section were used as probes. Lanes 1, RNA from cells grown aerobically at 0.1 MPa; lanes 2, RNA from cells grown microaerobically at 0.1 MPa; lanes 3, RNA from cells grown microaerobically at 50 MPa. DNA molecular weight marker III digoxigenin-labeled (Boehringer Mannheim Co.) was used as the size marker, and the sizes are indicated at the right side.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Two soluble c-type cytochromes were purified to an electrophoretically homogeneous state from a deep-sea piezophilic bacterium, S. violacea, grown at a pressure of 0.1 MPa, and the genes coding for these cytochromes c were cloned and sequenced. The deduced amino acid sequence of one of these cytochromes (cytochrome c_B) showed homology with those of the cytochromes c₄ group, and the other (cytochrome c_A) was homologous to the cytochromes c₅ group (Table 2). Cytochromes c₄- and c₅-related cytochromes are found in many bacteria (31) and are considered to be involved in aerobic respiration (21). In the case of Azotobacter vinelandii, it is thought that both cytochromes c transfer electrons to an o-type terminal oxidase in parallel (22).

As shown in Fig. 6, the level of expression of cytochrome c_A was found to be the same in cells grown at either 0.1 or 50 MPa, but cytochrome c_B was expressed only in cells grown at 0.1 MPa. Further, the pattern of expression of the cytochromes c was same in the case of both cells grown aerobically and cells grown microaerobically at a pressure of 0.1 MPa. These results suggest that the expression of cytochrome c_B is regulated by hydrostatic pressure, regardless of the oxygen concentration. These observations are consistent with recent results obtained by spectrophotometric analyses (29). Those results suggested that the respiratory system of this bacterium, when grown at high pressure, is organized in a manner different from that in cells grown at atmospheric pressure. In the case of A. vinelandii, cytochrome c₄ seems to act as a bypass for consumption of oxygen to protect the nitrogen fixation system from oxygen (22). Cytochrome c_B of S. violacea may act as bypass of the electron transport system to overcome the atmospheric pressure stress, because this bacterium is one of piezophiles that are usually found growing in a high-pressure environment, like deep-sea bottoms. This speculation is similar as in the case of cytochrome c₄ of A. vinelandii at oxygen stress; thus, such bypass may have a role to overcome from such stress conditions. But further studies are necessary to explain such phenomena.

These observations were very similar to results that we reported previously about regulation of the expression of two different membrane-bound cytochromes c by pressure in another piezophilic bacterium, Shewanella sp. strain DB-172F (27). In these piezophiles, the respiratory chain seems to be more compact and "short-cut" than that under normal atmospheric pressure conditions. This may be intimately related to the bacterial piezophilic property (15). It is possible that the regulation of expression of the respiratory system by hydrostatic pressure is one of the important mechanisms allowing these bacteria to adapt to an environment in which the hydrostatic pressure is low relative to that in the deep sea.

The putative promoter sequence of the cytochrome c_B gene, [35; CTTACA]-16 bp-[10; TAAAAT] (Fig. 4B), is similar with that of ompL of another piezophilic bacterium, P. profundum SS9, [35; CTAACA]-18 bp-[10; TAAAAA] (30). Expression of ompL was found to be regulated negatively at the transcription level under elevated pressure conditions (30), similar to the regulation of the cytochrome c_B gene. We have also reported that the promoter region of the pressure-regulated operon from deep-sea piezophilic strain DB6705, identified as S. benthica (23), is very similar to that of the ompH gene of P. profundum SS9 (3), and both of these genes are positively regulated by elevated pressure (18). Therefore, it seems possible that such deep-sea strains may have similar RNA polymerases which recognize these promoters involved in pressure regulation.