Shewanella violacea. Pressure regulation of quinol oxidase expression in Piezophilic. M. Hassan Qureshi

Transcription

1 Shewanella violacea M. Hassan Qureshi Moderately Shewanella violacea o c S. violacea cyoa cyob cyoc cyod cyoe S. violacea Shewanella violacea Pressure regulation of quinol oxidase expression in Piezophilic Shewanella violacea Chiaki KATO M. Hassan QURESHI Mitsunori YAMADA Kaoru NAKASONE Ron USAMI Koki HORIKOSHI We cloned and sequenced the gene for quinol oxidase enzyme, with the subsequent partial purification and characterization of an intact enzyme complex from a deep-sea moderately piezophilic bacterium, Shewanella violacea strain DSS12, grown at 50 MPa pressure. Based on the deduced amino acid sequences, the enzyme quinol oxidase seems to consist of five kinds of subunits with molecular masses of 74, 43.2, 36.6, 23.2 and 12.4 kda, respectively. The enzyme contained 0.88 mol protoheme or heme o and 0.92 mol covalently-bound heme c per mol of enzyme. Protoheme in the enzyme seems to react with carbon-monoxide and cyanide, and catalytic activity was 50 % inhibited by 5 M cyanide. Nucleotide sequence analysis of the region neighboring the subunit I gene (cyob) revealed that the genes (cyoa, cyoc, cyod and cyoe) coding for the other four subunits (II, III, IV and V) were clustered upstream and downstream of the cyob gene in order of cyoa, cyob, cyoc, cyod and cyoe, respectively. Analysis of the deduced amino acid sequence of cyo subunits showed that this oxidase is structurally related and has a high similarity with that of Escherichia coli botype quinol oxidase. Northern-blotting analysis of the RNAs, from DSS12 bacterium grown at several pressure conditions, showed that this enzyme expresses in high level when bacterium grown at elevated pressure conditions. key words : High Pressure, Piezophilic bacteria, Shewanella violacea, Respiratory system, Quinol oxidase, Gene expression, Pressure-regulation The DEEPSTAR Program, Japan Marine Science and Technology Center Department of Engineering, Toyo University 41

2 Moderate Shewanella Shewanella violacea bd cydd cydc Shewanella benthica ccb S. violacea c o S. violacea Fig. 1 Purification procedure of quinol oxidase from S. violacea strain DSS12 DEAE-Toyopearl Fig. 2 Elution profile obtained upon ion-exchange chromatography of the solubilized membrane proteins from strain DSS12 cells grown at 50 MPa pressure S. violacea S. violacea S. violacea Table 1 Purification of quinol oxidase from strain DSS12 grown at 50 MPa 42 JAMSTECR, 40 (2000)

3 S. violacea dithionite Fig. 4 Absolute absorption spectrum of the strain DSS12 quinol oxidase at room temperature. The enzyme was suspended in 10 mm Tris-HCl (ph 8.0), containing 0.5 g/l dodecyle maltoside.,oxidized state ;, reduced with dithionite SDS-PAGE Fig. 3 SDS-PAGE of the strain DSS12 quinol oxidase enzyme. The purified enzyme (33 g of protein) was treated with 2% SDS for 20 min at room temperature and then loaded on a 12% Tricine-SDS gel. Lane 1, stained with Coomassie Brilliant Blue; lane 2, stained with heme staining reagents. M, molecular mass marker proteins (Myosin [206 kda], -galactosidase [114 kda], bovine serum albumin [81 kda], ovalbumin [48 kda], carbonic anhydrase [34 kda], soybean trypsin inhibitor [29 kda], lysozyme [20 kda], and aprotinin [7.2 kda]. The molecular masses of four subunits of DSS12 quinol oxidase were estimated to be (I) 72.5, (II) 41.3, (III) 32.1 and (IV) 18.5 kda, respectively c b o c o S. violacea Fig. 5 Carbon-monoxide and cyanide binding spectra of the strain DSS12 quinol oxidase. The purified enzyme preparation (around 1.7 M heme c) was dissolved in 10 mm Tris-HCl (ph 8.0) containing 1 mm EDTA and 0.5 g/l dodecyl maltoside and was fully reduced by the addition of a small amount of solid dithionite. The reduced enzyme was incubated under a 100% CO atmosphere for 5 min and the difference spectrum, reduced +CO minus reduced, was recorded (dashed line). The cyanide-difference spectrum, reduced +CN - minus reduced, was recorded after incubating the reduced enzyme with 2 M, 4 M and 8 M cyanide for 3 min, respectively (solid line) c c JAMSTECR, 40 (2000) 43

4 S. violacea S. violacea Stratagene Dash aa3 bo Primer Primer S. violacea bo Fig. 6 Gene structure and subunits of quinol oxidase enzyme cloned from the strain DSS12, and % identity of the subunits deduced amino acids sequences compared with E. coli bo type quinol oxidase Stratagene LA- Taq cyoa E bo S. violacea identity S. violacea S. violacea cyo S. violacea Pressure Box? 44 JAMSTECR, 40 (2000)

5 S. benthica c S. violacea S. violacea Fig. 7 Analysis of the transcription controlled by elevated pressure in S. violacea strain DSS12. (A) Northern-blotting. (B) Primer extension analysis S. violacea Fig. 8 Nucleotide sequence of the promoter region of the cyo operon in strain DSS12. Transcriptional start points are shown as "+1" Moderately Shewanella violacea co bo S. benthica ccb co bo cyo C. Kato, T. Sato, and K. Horikoshi "Isolation and properties of barophilic and barotolerant bacteria from deepsea mud samples. " Biodiv. Conserv. 4, 1-9. (1995) L. Li, C. Kato, Y. Nogi, and K. Horikoshi "Distribution of the pressure-regulated operons in deep-sea bacteria. " FEMS Microbiol. Lett., 159, (1998) Y. Nogi, C. Kato, and K. Horikoshi "Taxonomic studies of deep-sea barophilic Shewanella strains and description of Shewanella violacea sp. nov." Arch. Microbiol., 170, (1998) C. Kato, A. Ikegami, M. Smorawinska, R. Usami, and K. Horikoshi "Structure of genes in a pressure-regulated operon and adjacent regions from a barotolerant bacterium strain DSS12. " J. Mar. Biotechnol., 5, (1997) K. Nakasone, A. Ikegami, C. Kato, R. Usami, and K. Horikoshi "Mechanisms of gene expression controlled by pressure in deep-sea microorganisms. "Extremophiles, 2, (1998) C. Kato, H. Tamegai, A. Ikegami, R. Usami, and K. Horikoshi "Open reading frame 3 of the barotolerant bacterium strain DSS12 is complementary with cydd in Escherichia coli: cydd functions are required for cell stability. " J. Biochem., 120, (1996). H. Tamegai, C. Kato, and K. Horikoshi "Pressure-regulated respiratory system in barotolerant bacterium, Shewanella sp. strain DSS12. " J. Biochem. Mol. Biol. Biophys., 1, (1998) C. Kato, N. Masui, and K. Horikoshi "Properties of obligately barophilic bacteria isolated from a sample of deepsea sediment from the Izu-Bonin trench. " J. Mar. Biotechnol., 4, (1996) M. H. Qureshi, C. Kato, and K. Horikoshi "Purification of a novel ccb-type quinol oxidase specifically induced in a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. " Extremophiles, 2, (1998) C. Kato, and M. H. Qureshi: Pressure response in deepsea piezophilic bacteria. J. Mol. Microbiol. Biotechnol., 1, (1999) J. Sambrook, E. F. Fritsch, and T. Maniatis "Molecular Cloning: a laboratory manual. " Cold Spring Harbor JAMSTECR, 40 (2000) 45

6 Laboratory Press, New York. (1989) V. Chepuri, L. Lemieux, J. Hill, J. O. Alben, and R. B. Gennis "Recent studies of the cytochrome o terminal oxidase complexes of Escherichia coli." Biochim. Biophys. Acta., 1018, (1990) NVVV U S 46 JAMSTECR, 40 (2000)