Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas

Two cytochromes c₅ (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c₅₅₁ (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.     

Thermal Stability of Cytochrome c 5 of Pressure-Sensitive Shewanella livingstonensis

Cytochrome c ₅ of pressure-sensitive Shewanella livingstonensis (SL cytc ₅) exhibits lower thermal stability than a highly homologous counterpart of pressure-tolerant Shewanella violacea. This stability difference is due to an enthalpic effect that can be attributed to the amino acid residue at position 50 (Leu or Lys). These cytc ₅ proteins are appropriate materials for understanding the protein stability mechanism.     

Correlation between the optimal growth pressures of four Shewanella species and the stabilities of their cytochromes c 5

Shewanella species live widely in deep-sea and shallow-water areas, and thus grow piezophilically and piezosensitively. Piezophilic and psychrophilic Shewanella benthica cytochrome c ₅ (SB cytc ₅) was the most stable against guanidine hydrochloride (GdnHCl) and thermal denaturation, followed by less piezophilic but still psychrophilic Shewanella violacea cytochrome c ₅ (SV cytc ₅). These two were followed, as to stability level, by piezosensitive and mesophilic Shewanella amazonensis cytochrome c ₅ (SA cytc ₅), and piezosensitive and psychrophilic Shewanella livingstonensis cytochrome c ₅ (SL cytc ₅). The midpoint GdnHCl concentrations of SB cytc ₅, SV cytc ₅, SL cytc ₅, and SA cytc ₅ correlated with the optimal growth pressures of the species, the correlation coefficient value being 0.93. A similar trend was observed for thermal denaturation. Therefore, the stability of each cytochrome c ₅ is related directly to its host’s optimal growth pressure. Phylogenetic analysis indicated that Lys-37, Ala-41, and Leu-50 conserved in piezosensitive SL cytc ₅ and SA cytc ₅ are ancestors of the corresponding residues in piezophilic SB cytc ₅ and SV cytc ₅, Gln, Thr, and Lys, respectively, which might have been introduced during evolution on adaption to environmental pressure. The monomeric Shewanella cytochromes c ₅ are suitable tools for examining protein stability with regard to the optimal growth pressures of the source species.     

Isolation of Chanoclavine-(I) and Two New Interconvertible Alkaloids,Rugulovasine A and B,from the Cultures of Penicillium concavo-rugulosum

The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme.     

Regulation of Cytochrome c- and Quinol Oxidases,and Piezotolerance of Their Activities in the Deep-Sea Piezophile Shewanella violacea DSS12 in Response to Growth Conditions

The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O₂ concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O₂ concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O₂ concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.     

Roles of a short connecting disulfide bond in the stability and function of psychrophilic Shewanella violacea cytochrome c 5*

Cys-59 and Cys-62, forming a disulfide bond in the four-residue loop of Shewanella violacea cytochrome c ₅ (SV cytc ₅), contribute to protein stability but not to redox function. These Cys residues were substituted with Ala in SV cytc ₅, and the structural and functional properties of the resulting C59A/C62A variant were determined and compared with those of the wild-type. The variant had similar features to those of the wild-type in absorption, circular dichroic, and paramagnetic ¹H NMR spectra. In addition, the redox potentials of the wild-type and variant were essentially the same, indicating that removal of the disulfide bond from SV cytc ₅ does not affect the redox function generated in the vicinity of heme. However, calorimetric analysis of the wild-type and variant showed that the mutations caused a drastic decrease in the protein stability through enthalpy, but not entropy. Four residues are encompassed by the SV cytc ₅ disulfide bond, which is the shortest one that has been proved to affect protein stability. The protein stability of SV cytc ₅ can be controlled without changing the redox function, providing a new strategy for regulating the stability and function of cytochrome c.     

Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔV_Tot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔV_Tot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔV_Tot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔV_Tot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.     

Amino acid substitutions in malate dehydrogenases of piezophilic bacteria isolated from intestinal contents of deep-sea fishes retrieved from the abyssal zone

To examine the occurrence in other deep-sea bacteria of two amino acid substitutions (Ala-180 and His-229) in malate dehydrogenase (MDH) found previously in the deep-sea piezophilic Moritella sp. strain 2D2, we cloned and sequenced MDH genes of deep-sea piezophilic Moritella and Shewanella strains isolated from intestinal contents of deep-sea fishes, as well as other Moritella species from deep-sea water and sediments: M. marina, M. japonica, and M. yayanosii. The piezophilic Moritella strains had a Val residue or an Ala residue at position 180 and all the Moritella strains except for one had a His residue at position 229. However, four piezophilic-strain-specific substitutions at positions 103, 111, 229, and 283 were found to be completely conserved in the MDH of the intestinal Moritella strains of deep-sea fishes, indicating the substitutions may be habitat-specific. The piezophilic Shewanella strains had a Val residue and a Gln residue at positions 180 and 229, respectively. However, the MDHs of the Shewanella strains had five piezophilic-strain-specific substitutions at positions 61, 65, 107, 161, and 202. Therefore, the enzymatic strategies for responding to deep-sea high pressure environments of the MDHs between the genera Moritella and Shewanella are potentially different. Moreover, homology modeling shows these substitutions found in the MDHs of both genera except for position 229 in the subunit interface are located on the exposed region of the MDH molecules, indicating the substitutions may be related to the hydration state of the molecules.     

Biosynthesis and dietary uptake of polyunsaturated fatty acids by piezophilic bacteria

The biochemistry of piezophilic bacteria is unique in that piezophiles produce polyunsaturated fatty acids (PUFAs). A pertinent question is if piezophilic bacteria synthesize PUFA de novo, through dietary uptake, or both. This study was undertaken to examine the biosynthesis and cellular uptake of PUFAs by piezophilic bacteria. A moderately piezophilic (Shewanella violacea DSS12) and two hyperpiezophilic bacteria (S. benthica DB21MT-2 and Moritella yayanosii DB21MT-5) were grown under 50 MPa (megapascal) and 100 MPa, respectively, in media containing marine broth 2216 supplemented with arachidonic acid (AA, sodium salt) and/or antibiotic cerulenin. There was active uptake and cellular incorporation of AA in the hyperpiezophilic bacteria DB21MT-2 (14.7% of total fatty acids) and DB21MT-5 (1.4%), but no uptake was observed in DSS12. When cells were treated with cerulenin, all three strains incorporated AA into cell membranes (13–19%). The biosynthesis of monounsaturated fatty acids was significantly inhibited (10–37%) by the addition of cerulenin, whereas the concentrations of PUFAs increased by 2–4 times. These results suggest that piezophilic bacteria biosynthesize and/or incorporate dietary polyunsaturated fatty acids that are important for their growth and piezoadaptation. The significance of these findings is also discussed in the context of phenotypic classification of piezophiles.     

Difference in NaCl tolerance of membrane-bound 5′-nucleotidases purified from deep-sea and brackish water Shewanella species

Shewanella species are widely distributed in sea, brackish, and fresh water areas, growing psychrophilically or mesophilically, and piezophilically or piezo-sensitively. Here, membrane-bound 5′-nucleotidases (NTases) from deep-sea Shewanella violacea and brackish water Shewanella amazonensis were examined from the aspect of NaCl tolerance to gain an insight into protein stability against salt. Both NTases were single polypeptides with molecular masses of ~59 kDa, as determined on mass spectroscopy. They similarly required 10 mM MgCl₂ for their activities, and they exhibited the same pH dependency and substrate specificity for 5′-nucleotides. However, S. violacea 5′-nucleotidase (SVNTase) was active enough in the presence of 2.5 M NaCl, whereas S. amazonensis 5′-nucleotidase (SANTase) exhibited significantly reduced activity with the same concentration of the salt. Although SVNTase and SANTase exhibited high sequence identity (69.7%), differences in the ratio of acidic to basic amino acid residues and the number of potential salt bridges maybe being responsible for the difference in the protein stability against salt. 5′-Nucleotidases from these Shewanella species will provide useful information regarding NaCl tolerance, which may be fundamental for understanding bacterial adaptation to growth environments.

     

Correlation between phylogenetic structure and function: examples from deep-sea Shewanella

The genus Shewanella is one of the typical deep-sea bacterial genera. Two isolated deep-sea Shewanella species, Shewanella benthica and Shewanella violacea, were found to be able to grow better under high hydrostatic pressure conditions than at atmospheric pressure. These species are not only piezophilic (barophilic), but also psychrophilic. Many psychrophilic and psychrotolerant Shewanella species have been isolated and characterized from cold environments, such as seawater in Antarctica or the North Sea. Some of these cold-adapted Shewanella were shown to be piezotolerant, meaning that growth occurs in a high-pressure habitat. In this review, we propose that two major sub-genus branches of the genus Shewanella should be recognized taxonomically, one group characterized as high-pressure cold-adapted species that produce substantial amounts of eicosapentaenoic acid, and the other group characterized as mesophilic pressure-sensitive species.     

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.     

Pressure response in deep-sea piezophilic bacteria

Several piezophilic bacteria have been isolated from deep-sea environments under high hydrostatic pressure. Taxonomic studies of the isolates showed that the piezophilic bacteria are not widely distributed in terms of taxonomic positions, and all were assigned to particular branches of the Proteobacteria gamma-subgroup. A pressure-regulated operon from piezophilic bacteria of the genus Shewanella, S. benthica and S. violacea, was cloned and sequenced, and downstream of this operon another pressure regulated operon, cydD-C, was found. The cydD gene was found to be essential for the bacterial growth under high-pressure conditions, and the product of this gene was found to play a role in their respiratory system. Results obtained later indicated that the respiratory system in piezophilic bacteria may be important for survival in a high-pressure environment, and more studies focusing on other components of the respiratory chain have been conducted. These studies suggested that piezophilic bacteria are capable of changing their respiratory system in response to pressure conditions, and a proposed respiratory chain model has been suggested in this regard.     

Conformational States of the Water-Soluble Fragment of Cytochrome b5. I. pH-Induced Denaturation

Cytochrome b ₅ is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water–alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of a negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b ₅ as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b ₅ functioning is discussed.     

Kinetics of the electron transfer reaction of Cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>552</Subscript> adsorbed on biomimetic electrode studied by time-resolved surface-enhanced resonance Raman spectroscopy and electrochemistry

Cytochrome c ₅₅₂ (Cyt-c ₅₅₂) and its redox partner ba ₃ -oxidase from Thermus thermophilus possess structural differences compared with Horse heart cytochrome c (cyt-c)/cytochrome c oxidase (CcO) system, where the recognition between partners and the electron transfer (ET) process is initiated via electrostatic interactions. We demonstrated in a previous study by surface-enhanced resonance Raman (SERR) spectroscopy that roughened silver electrodes coated with uncharged mixed self-assembled monolayers HS–(CH₂) _n –CH₃/HS–(CH₂) _{n + 1}–OH 50/50, n = 5, 10 or 15, was a good model to mimic the Cyt-c ₅₅₂ redox partner. All the adsorbed molecules are well oriented on such biomimetic electrodes and transfer one electron during the redox process. The present work focuses on the kinetic part of the heterogeneous ET process of Cyt-c ₅₅₂ adsorbed onto electrodes coated with such mixed SAMs of different alkyl chain length. For that purpose, two complementary methods were combined. Firstly cyclic voltammetry shows that the ET between the adsorbed Cyt-c ₅₅₂ and the biomimetic electrode is direct and reversible. Furthermore, it allows the estimation of both the density surface coverage of adsorbed Cyt-c ₅₅₂ and the kinetic constants values. Secondly, time-resolved SERR (TR-SERR) spectroscopy showed that the ET process occurs without conformational change of the Cyt-c ₅₅₂ heme group and allows the determination of kinetic constants. Results show that the kinetic constant values obtained by TR-SERR spectroscopy could be compared to those obtained from cyclic voltammetry. They are estimated at 200, 150 and 40 s⁻¹ for the ET of Cyt-c ₅₅₂ adsorbed onto electrodes coated with mixed SAMs HS–(CH₂) _n –CH₃/HS–(CH₂) _{n + 1}–OH 50/50, n = 5, 10 or 15, respectively. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

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.     

Differences in biochemical properties of two 5′-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions

Deep-sea Shewanella violacea 5′-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions.

Abbreviations: NTase: 5′-nucleotidase; SANTase: Shewanella amazonensis 5′-nucleotidase; SVNTase: Shewanella violacea 5′-nucleotidase; CD: circular dichroism     

Unexpected Elevated Production of Aquifex aeolicus Cytochrome c 555 in Escherichia coli Cells Lacking Disulfide Oxidoreductases

Mutant strains of Escherichia coli lacking DsbA, DsbB, or DsbD (proteins required for disulfide bond formation in the periplasm) did not produce mitochondrial or chloroplast cytochromes c, as previously observed for bacterial ones. Unexpectedly, however, cytochrome c ₅₅₅ (AA c ₅₅₅) from a hyperthermophile, Aquifex aeolicus, was produced in the E. coli periplasm without Dsb proteins, three times more than with them. These results indicate that the Dsb proteins are not necessarily required for AA c ₅₅₅ production in E. coli, possibly because of hyperthermophilic origin compared with the others.