Reconstitution and characterization of NtrC protein in a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.     

Structural analysis of the ntrBC genes of deep-sea piezophilic Shewanella violacea

The ntrBC genes coding for the bacterial signal-transducing protein NtrB and the bacterial enhancer-binding protein NtrC of deep-sea piezophilic Shewanella violacea were cloned and their nucleotide sequences were analyzed. The conserved regions of NtrB and those of NtrC are well conserved in the case of the ntrBC products of S. violacea.     

Purification of a ccb-type quinol oxidase specifically induced in a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F

We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state. The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15 kDa, and it contained 0.96 mol of protoheme and 1.95 mol of covalently bound heme c per mol of enzyme. Only protoheme in the enzyme reacted with CO and CN⁻, and the catalytic activity of the enzyme was 50% inhibited by 4 μM CN⁻. The isoelectric point of the native enzyme complex was determined to be 5.0. This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa. The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity. These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F. Received: November 25, 1997 / Accepted: December 25, 1997     

Comparative study on stabilization mechanism of monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea

Monomeric cytochrome c₅ from deep-sea piezophilic Shewanella violacea (SVcytc₅) was stable against heat and denaturant compared with the homologous protein from shallow-sea piezo-sensitive Shewanella livingstonensis (SLcytc₅). Here, the SVcytc₅ crystal structure revealed that the Lys-50 side chain on the flexible loop formed a hydrogen bond with heme whereas that of corresponding hydrophobic Leu-50 could not form such a bond in SLcytc₅, which appeared to be one of possible factors responsible for the difference in stability between the two proteins. This structural insight was confirmed by a reciprocal mutagenesis study on the thermal stability of these two proteins. As SVcytc₅ was isolated from a deep-sea piezophilic bacterium, the present comparative study indicates that adaptation of monomeric SVcytc₅ to high pressure environments results in stabilization against heat.     

Piezotolerance of the respiratory terminal oxidase activity of the piezophilic Shewanella violacea DSS12 as compared with non-piezophilic Shewanella species

The facultative piezophile Shewanella violacea DSS12 is known to alter its respiratory components under the influence of hydrostatic pressure during growth, suggesting that it has a respiratory system that functions in adaptation to high pressure. We investigated the pressure- and temperature-dependencies of the respiratory terminal oxidase activity of the membrane of S. violacea relative to non-piezophilic Shewanella species. We observed that the activity in the membrane of S. violacea was more resistant to high pressure than those of non-piezophilic Shewanella even though DSS12 was cultured under atmospheric pressure. On the other hand, the temperature dependency of this activity was almost the same for all of the tested strain regardless of optimal growth temperature. Both high pressure and low temperature are expected to lower protein flexibility, causing a decrease in enzyme activity, but the results of this study suggest that the mechanism maintaining enzyme activity under high hydrostatic pressure is different from that at low temperature. Additionally, the responses of the activity to the pressure- and temperature-changes were independent of membrane lipid composition. Therefore, the piezotolerance of the respiratory terminal oxidases of S. violacea is perhaps dependent on the properties of the protein itself and not on the lipid composition of the membrane. Our observations suggest that S. violacea constitutively express piezotolerant respiratory terminal oxidases that serve adaptation to the deep-sea environment.     

Piezoresponse of the cyo-Operon Coding for Quinol Oxidase Subunits in a Deep-sea Piezophilic Bacterium,Shewanella violacea

We have isolated the genes for quinol oxidase from a deep-sea piezophilic bacterium, Shewanella violacea. Analysis of the deduced amino acid sequences of the cyo subunits showed that this oxidase has high similarity to Escherichia coli bo-type quinol oxidase. Northern blot analysis showed that these genes are expressed at a high level when the bacterium is grown at elevated pressure. Upstream in the cyo-operon, a σ⁵⁴-binding motif and an octamer sequence unit were found, suggesting that these elements may play a role in regulation of expression of the cyo-operon in response to changes in pressure.     

Isolation of the rpoD gene encoding the principal sigma factor of the deep-sea piezophilic bacterium Shewanella violacea strain DSS12 and its overexpression in Escherichia coli

The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively. Comparison with E. coli sigma70 and P. putida sigma70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S. violacea sigma70 factor was overexpressed in E. coli and successfully purified to near homogeneity.     

The narQP genes for a two-component regulatory system from the deep-sea bacterium Shewanella violacea DSS12.

Shewanella violacea DSS12 is facultative piezophile isolated from the deep-sea. The expression of cydDC genes (required for d-type cytochrome maturation) of the organism is regulated by hydrostatic pressure. In this study, we analyzed the nucleotide sequence upstream of cydDC in detail and found that there are putative binding sites for the NarL protein which is part of a two-component regulatory system also containing the sensor protein NarX. Furthermore, we identified the narQP genes (homologues of narXL) from S. violacea DSS12 and demonstrated the heterologous expression of narP in Escherichia coli. These results will be helpful in examining pressure regulation of gene expression in S. violacea at the molecular level.     

Bacterial adaptation to high pressure: a respiratory system in the deep-sea bacterium Shewanella violacea DSS12

Shewanella violacea DSS12 is a psychrophilic facultative piezophile isolated from the deep sea. In a previous study, we have shown that the bacterium adapted its respiratory components to alteration in growth pressure. This appears to be one of the bacterial adaptation mechanisms to high pressures. In this study, we measured the respiratory activities of S. violacea grown under various pressures. There was no significant difference between the cells grown under atmospheric pressure and a high pressure of 50 MPa relative to oxygen consumption of the cell-free extracts and inhibition patterns in the presence of KCN and antimycin A. Antimycin A did not inhibit the activity completely regardless of growth pressure, suggesting that there were complex III-containing and -eliminating pathways operating in parallel. On the other hand, there was a difference in the terminal oxidase activities. Our results showed that an inhibitor- and pressure-resistant terminal oxidase was expressed in the cells grown under high pressure. This property should contribute to the high-pressure adaptation mechanisms of S. violacea.     

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.     

Favourable effects of eicosapentaenoic acid on the late step of the cell division in a piezophilic bacterium, Shewanella violacea DSS12, at high-hydrostatic pressures

Shewanella violacea DSS12, a deep-sea bacterium, produces eicosapentaenoic acid (EPA) as a component of membrane phospholipids. Although various isolates from the deep sea, such as Photobacterium profundum SS9, Colwellia psychrerythraea 34H and various Shewanella strains, produce EPA- or docosahexaenoic acid-containing phospholipids, the physiological role of these polyunsaturated fatty acids remains unclear. In this article, we illustrate the physiological importance of EPA for high-pressure adaptation in strain DSS12 with the help of an EPA-deficient mutant (DSS12(pfaA)). DSS12(pfaA) showed significant growth retardation at 30 MPa, but not at 0.1 MPa. We also found that DSS12(pfaA) grown at 30 MPa forms filamentous cells. When an EPA-containing phospholipid (sn-1-oleoly-sn-2-eicosapentaenoyl phosphatidylethanolamine) was supplemented, the growth retardation and the morphological defect of DSS12(pfaA) were suppressed, indicating that the externally added EPA-containing phospholipid compensated for the loss of endogenous EPA. In contrast, the addition of an oleic acid-containing phospholipid (sn-1,2-dioleoyl phosphatidylethanolamine) did not affect the growth and the morphology of the cells. Immunofluorescent microscopic analysis with anti-FtsZ antibody revealed a number of Z-rings and separated nucleoids in DSS12(pfaA) grown at 30 MPa. These results demonstrate the physiological importance of EPA for the later step of Z-ring formation of S. violacea DSS12 under high-pressure conditions.     

Cloning and overproduction of the rpoZ gene encoding an RNA polymerase omega subunit from a deep-sea piezophilic Shewanella violacea strain DSS12.

We have cloned the rpoZ gene, encoding RNA polymerase omega protein, by PCR approach from the deep-sea piezophilic and psychrophilic bacterium, Shewanella violacea strain DSS12. The cloned gene, 285bp in length, was found to encode a protein consisting of 94 amino acid residues with a molecular mass of 10,327 Da. Significant homology was evident comparing the RpoZ protein of S. violacea with that of Shewanella oneidensis (69% identity), Vibrio cholerae (65% identity), Escherichia coli K-12 (64% identity) and Haemophilus influenzae (61% identity). From the Northern blot analysis, S. violacea rpoZ gene was expressed constitutively under pressure conditions of 0.1, 30 and 50MPa. We constructed expression plasmid to overproduce the RpoZ protein and transformed into E. coli JM109 as a host of overproduction. Upon induction, the recombinant protein encoded by plasmid pQrpoZ was overexpressed and purified using Ni2+ affinity column.