Purification and some properties of two NADP+-specific isocitrate dehydrogenases from an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-1.

Two isozymes of NADP+-specific isocitrate dehydrogenase [ICDH; EC 1.1.1.42] were confirmed to be present in an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-1, on the basis of the temperature-activity curve and electrophoretic mobilities. These isozymes were separated and purified about 170-fold for isozyme I (specific activity at 40 degrees C, 24.3 units/mg protein) and about 180-fold for isozyme II (specific activity at 20 degrees C, 59.2 units/mg protein), though the isozymes were still not homogeneous. The molecular weights of these isozymes determined by gel filtration were both about 85,000, but the properties of the isozymes were considerably different from each other. The thermostability of isozyme I resembled those of mesophiles, but isozyme II was extremely labile above 20 degrees C. NaCl affected the ICDH isozymes in different ways; the salt protected isozyme I from heat inactivation, but not isozyme II. Nevertheless it enormously enhanced the activity of isozyme II at low concentrations. Moreover, these ICDH isozymes showed different pH optima, Km values for isocitrate, susceptibilities to concerted inhibition by glyoxylate plus oxalacetate, and effects of 2-mercaptoethanol on their stabilities.     

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.     

Flocculation behavior and mechanism of an exopolysaccharide from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913

Flocculation behavior and mechanism of the exopolysaccharide secreted by Pseudoalteromonas sp. SM9913 (EPS SM9913), a psychrophilic bacterium isolated from 1855m deep-sea sediment, has been studied in this paper. EPS SM9913 showed a peak flocculating activity of 49.3 in 1g/L kaolin suspension with 4.55mmol/L CaCl2 and the optimum pH range of 5-8. It appears that the flocculating activity of EPS SM9913 was stimulated by Ca2+ and Fe2+. This study found that EPS SM9913 showed a better flocculation performance than Al2(SO4)3 at salinity of 5-100 per thousand or temperatures of 5-15 degrees C. In addition, this EPS was effective to flocculate several other suspended solids. The measured zeta-potentials, the size of flocs formed during the flocculation process and the surface profile of flocs revealed by scan electron micrograph suggest that bridging is the main flocculation mechanism of the studied EPS. Deacetylation of EPS SM9913 resulted in a significant decrease in its flocculating activity indicating that the large number of acetyl groups in EPS SM9913 played an important role in its flocculation performance.     

Characterization of the gene encoding the beta-lactamase of the psychrophilic marine bacterium Moritella marina strain MP-1

The beta-lactamase gene (mbla) of the psychrophilic marine bacterium Moritella marina strain MP-1 was identified in a previously isolated genomic DNA fragment and it was expressed in Escherichia coli cells. The mbla gene encoded a protein consisting of 287 amino acid residues. Its predicted amino acid sequence showed approximately 50% identity with that of a number of class A beta-lactamases, especially with that of CARB/PSE type of beta-lactamases (carbenicillinases). E. coli transformed with the plasmid containing mbla grew on an ampicillin-containing plate at 37 degrees C but not at 42 degrees C, suggesting that the beta-lactamase of this bacterium is heat-labile.     

Effect of temperature and growth phase on fatty acid composition of the psychrophilic Vibrio sp. strain no. 5710

Abstract The cellular fatty acid composition of the psychrophilic Vibrio sp. strain No. 5710 isolated from a deep-sea sediment sample was analyzed. The presence of docosahexaenoic acid (22:6) was demonstrated as found previously in other deep-sea bacteria, and the relative amount of 22:6 decreased as the growth temperature increased. A temperature shift from 10°C to 0°C resulted in a relative increase of 22:6, and an opposite shift led to a decrease. In addition, hexadecanoic acid (16:0) was found to increase as the growth temperature increased. Therefore, it is suggested that the adaptation of 5710 to the growth temperature was carried out by the changes in the relative amounts of 22:6 and 16:0. When 5710 was grown at low temperature, it increased the relative amount of 22:6 presumably to maintain membrane fluidity at that temperature. In contrast, 5710 grown at high temperature probably maintained the membrane fluidity by increasing the amount of a saturated fatty acid, 16:0. Furthermore, observation of the fatty acid compositions at mid-exponential phase and early stationary phase revealed the proportions of several fatty acids, including a major fatty acid, 9- cis -hexadecenoic acid (16:1c, palmitoleic acid), were affected by the growth phase which may be due to the physiological difference between the growth phases.     

High pressure nmr study of dihydrofolate reductase from a deep-sea bacterium Moritella profunda.

We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C. The on-line cell variable pressure 1H NMR spectroscopy has been used to analyze the chemical shift and signal intensity in one-dimensional 1H NMR spectra. Thermodynamic analysis based on signal intensities from protons in the core part indicates that the thermodynamic stability of Moritella profunda DHFR is relatively low over the temperature range between -5 and 30 degrees C (deltaG0=15.8 +/- 4.1 kJ/mol at 15 degrees C), but is well adapted to the living environment of the bacterium (2 degrees C and 28 MPa), with the maximum stability around 5 degrees C (at 0.1 MPa) and a relatively small volume change upon unfolding (deltaV= 66 +/- 19 ml/mol). Despite the relatively low overall stability, the conformation in the core part of the folded protein remains intact up to approximately 200 MPa, showing marked stability of the core of this protein.     

Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification, and modulation of activities by growth conditions

Each of the two isozymes, which are different in thermostability and quaternary structure, of isocitrate dehydrogenase (NADP+) [IDH: EC 1.1.1.42] was purified to an electrophoretically homogeneous state from an obligately psychrophilic marine bacterium, Vibrio sp. strain ABE-1. Hydrophobic chromatography was an efficient procedure to separate the two isozymes from each other. The isoelectric points of isozyme I (IDH-I; a dimer, Mr 88,100) and isozyme II (IDH-II; a monomer, Mr 80,500) were found to be pH 4.9 and 5.2, respectively. The two isozymes were similar in amino acid compositions, though there were slight differences in the contents of nonpolar and hydroxyl amino acids. However, their NH2-terminal amino acid sequences and immunochemical properties were clearly different from each other. The NH2-terminal amino acid sequence analysis also indicated that the subunits of IDH-I are chemically identical or highly homologous. Non-immuno-crossreactivity between the isozymes enabled us to measure the intracellular contents of the isozymes. IDH-I and -II were found to be differentially regulated in vivo by various growth conditions. IDH-I was induced by acetate, while IDH-II remained almost unchanged.     

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.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp. strain KUC-1

A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD(+)-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD(+) and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45 degrees C. The kinetic parameters of the enzyme are highly influenced by temperatures. The K(m) for L-threonine was lowest at 20 degrees C. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD(+) binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.     

A trans-unsaturated fatty acid in a psychrophilic bacterium, Vibrio sp. strain ABE-1.

A high level of a trans-unsaturated fatty acid was found in the phospholipids of a psychrophilic bacterium, Vibrio sp. strain ABE-1. This fatty acid was identified as 9-trans-hexadecenoic acid (C16:19t) by gas-liquid chromatography and infrared absorption spectrometry. C16:1(9)t accounted for less than 1% of the total fatty acids in cells grown at 5 degrees C and reached 12% of the total at 20 degrees C. We suggest that the increase in the level of the trans-unsaturated fatty acid is related to the high growth rate of this bacterium at elevated temperatures. Possible biological roles of the trans-unsaturated fatty acid in the adaptation of the microorganism to the ambient temperature are discussed.     

Genetic characterization of ompH mutants in the deep-sea bacterium Photobacterium sp. strain SS9

OmpH is an outer membrane protein produced by the deep-sea bacterium Photobacterium species strain SS9 in response to elevated hydrostatic pressure. In order to facilitate studies of the function of this protein, a series of OmpH⁺ and OmpH^- strains were obtained from SS9 by Tn5 gene replacement mutagenesis. A previously isolated ompH::lacZ strain and a derivative of this strain harboring a plasmid expressing the wild-type ompH gene were also utilized. The acridine mutagen ICR 191 preferentially inhibited the growth of OmpH⁺ over OmpH^- cells. Indeed, OmpH⁺ cultures treated with the mutagen rapidly accumulated mutants producing reduced levels of OmpH. In addition. OmpH⁺ cells took up the peptide Met-Leu-Phe approximately 15 times more rapidly than OmpH^- cells. The results are consistent with the hypothesis that OmpH functions as a relatively large, nonspecific diffusion channel.Abbreviations OMP Outer membrane protein     

Structural investigation of cold activity and regulation of aspartate carbamoyltransferase from the extreme psychrophilic bacterium Moritella profunda

Aspartate carbamoyltransferase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allosteric regulation. The structure of the Escherichia coli enzyme has been thoroughly analyzed by X-ray crystallography, and recently the crystal structures of two hyperthermophilic ATCases of the same structural class have been characterized. We here report the detailed functional and structural investigation of the ATCase from the psychrophilic deep sea bacterium Moritella profunda. Our analysis indicates that the enzyme conforms to the E. coli model in that two allosteric states exist that are influenced by similar homotropic interactions. The heterotropic properties differ in that CTP and UTP inhibit the holoenzyme, but ATP seems to exhibit a dual regulatory pattern, activating the enzyme at low concentrations and inhibiting it in the mM range. The crystal structure of the unliganded M. profunda ATCase shows resemblance to a more extreme T state reported previously for an E. coli ATCase mutant. A detailed molecular analysis reveals potential features of adaptation to cold activity and cold regulation. Moreover, M. profunda ATCase presents similarities with certain mutants of E. coli ATCase altered in their kinetic properties or temperature relationships. Finally, structural and functional comparison of ATCases across the full physiological temperature range agrees with an important, but fundamentally different role for electrostatics in protein adaptation at both extremes, i.e. an increased stability through the formation of ion pairs and ion pair networks at high physiological temperatures, and an increased flexibility through enhanced protein solvation at low temperatures.     

Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin

A novel obligately chemolithotrophic Thiobacillus species isolated from a deep-sea hydrothermal vent is described. This organism grows lithoautotrophically on thiosulphate, tetrathionate, sulphide and sulphur which are oxidized to sulphate. The isolate is slightly halophilic and markedly halotolerant, showing optimum growth at pH 7.5 and at 35°C. The G+C content of the DNA is 67.1 mol%. The 16S rRNA sequence is distinct from any other Thiobacilli sequences. Phylogenetic analysis shows the organism to be a representative of the -group of proteobacteria and a specific relative of Thiobacillus neapolitanus. The ubiquinone is ubiquinone-8. These characters distinguish the isolate from any other Thiobacillus or Thiomicrospira species previously reported and is a new species described as Thiobacillus hydrothermalis. The type strain is isolate R3, DSM7121.     

Bacteroides sp. strain D8, the first cholesterol-reducing bacterium isolated from human feces

The microbial community in the human colon contains bacteria that reduce cholesterol to coprostanol, but the species responsible for this conversion are still unknown. We describe here the first isolation and characterization of a cholesterol-reducing bacterium of human intestinal origin. Strain D8 was isolated from a 10(-8) dilution of a fresh stool sample provided by a senior male volunteer with a high capacity to reduce luminal cholesterol to coprostanol. Cholesterol-to-coprostanol conversion by strain D8 started on the third day, while cells were in stationary phase, and was almost complete after 7 days. Intermediate products (4-cholesten-3-one and coprostanone) were occasionally observed, suggesting an indirect pathway for cholesterol-to-coprostanol conversion. Resting-cell assays showed that strain D8 could reduce 1.5 mumol of cholesterol/mg bacterial protein/h. Strain D8 was a gram-negative, non-spore-forming, rod-shaped organism identified as a member of the genus Bacteroides closely related to Bacteroides vulgatus, based on its morphological and biochemical characteristics. The 16S rRNA gene sequence of strain D8 was most similar (>99.5%) to those of two isolates of the recently described species Bacteroides dorei. Phylogenetic tree construction confirmed that Bacteroides sp. strain D8 clustered within an independent clade together with these B. dorei strains. Nevertheless, no cholesterol-reducing activity could be detected in cultures of the B. dorei type strain. Based on Bacteroides group-specific PCR-temporal temperature gradient gel electrophoresis, there was no correlation between the presence of a band comigrating with the band of Bacteroides sp. strain D8 and cholesterol conversion in 11 human fecal samples, indicating that this strain is unlikely to be mainly responsible for cholesterol conversion in the human population.     

The effect of temperature shifts on protein synthesis by the psychrophilic bacterium Vibrio sp. strain ANT-300.

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the temperature-related characteristics of protein synthesis in cells grown at 0 degrees C differed from those of cells grown at 13 degrees C. Cells grown at 0 degrees C and 13 degrees C transported amino acids at the same rates, dependent on the temperature at which rates were measured. The rates of protein synthesis in extracts of cells grown at 0 degrees C and at 13 degrees C differed, as a result of the changes in the properties of the soluble fraction involved in protein synthesis. Concurrently, levels of more than 24 polypeptides in the soluble fraction changed considerably. These results suggest that the difference in temperature dependence of protein synthesis in cells grown at various temperatures may be brought about by specific changes in the levels of a small number of polypeptides (less than 15% of the total number of proteins detected by silver-staining) in response to a change in temperature.