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Maribacter dokdonensis sp. nov., isolated from sea water off a Korean island, Dokdo.

Two Gram-negative, non-spore-forming, slightly halophilic gliding bacterial strains, DSW-8(T) and DSW-9, were isolated from sea water off a Korean island, Dokdo, of the East Sea, Korea, and their taxonomic position was investigated by a polyphasic study. The two strains grew optimally at 30 degrees C and in the presence of 2-3% (w/v) NaCl. Strains DSW-8(T) and DSW-9 were characterized chemotaxonomically as containing MK-6 as the predominant menaquinone and iso-C(17:0) 3-OH, iso-C(15:0) and iso-C(15:1) as the major fatty acids. Major polar lipids were phosphatidylethanolamine, two unidentified phospholipids, an unidentified glycolipid and an amino group-containing lipid that was ninhydrin-positive. Their DNA G+C contents were 36.1 and 35.9 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains DSW-8(T) and DSW-9 fell within the genus Maribacter of the family Flavobacteriaceae. Strains DSW-8(T) and DSW-9 exhibited no difference in their 16S rRNA gene sequences and possessed a mean DNA-DNA relatedness level of 89%. Strains DSW-8(T) and DSW-9 exhibited 16S rRNA gene sequence similarity levels of 96.9-98.0% to the type strains of the four recognized Maribacter species, but their low level of DNA-DNA relatedness with these species demonstrated that they constitute a distinct Maribacter species. On the basis of phenotypic and phylogenetic data and genetic distinctiveness, strains DSW-8(T) (=KCTC 12393(T)=DSM 17201(T)) and DSW-9 were classified in the genus Maribacter as members of a novel species, for which the name Maribacter dokdonensis sp. nov. is proposed.

Bacterial Typing Techniques↗

Gaetbulimicrobium brevivitae gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from a tidal flat of the Yellow Sea in Korea.

A Gram-negative, non-spore-forming and rod-shaped gliding bacterium, designated strain SMK-19T, was isolated from a tidal flat sediment of the Yellow Sea, Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain SMK-19T grew optimally at 37 degrees C, in the presence of 2-3 % (w/v) NaCl and at pH 7.0-8.0. It contained MK-6 as the predominant menaquinone, and iso-C(17 : 0) 3-OH and iso-C(15 : 0) as the major fatty acids. Major polar lipids were phosphatidylethanolamine, unidentified phospholipids and an amino-group-containing lipid that is ninhydrin-positive. The DNA G+C content was 36.0 mol%. Phylogenetic analyses based on 16S rRNA gene sequences demonstrated that strain SMK-19T formed a distinct evolutionary lineage within the family Flavobacteriaceae. The 16S rRNA gene sequence of strain SMK-19T exhibited similarity values of <94.4 % to those of other members of the family Flavobacteriaceae. Strain SMK-19T was distinguished from phylogenetically related genera by differences in several phenotypic properties. On the basis of phenotypic, phylogenetic and chemotaxonomic data, SMK-19T (= KCTC 12390T = DSM 17196T) was classified at the type strain of a novel genus and species, Gaetbulimicrobium brevivitae gen. nov., sp. nov.

Base Composition↗

Dokdonella koreensis gen. nov., sp. nov., isolated from soil.

Two Gram-negative, motile, non-spore-forming and rod-shaped bacterial strains, DS-123T and DS-140, were isolated from soil and their taxonomic positions were investigated by a polyphasic study. Strains DS-123T and DS-140 grew optimally at 30 degrees C and pH 6.5 without NaCl. They contained Q-8 as the predominant ubiquione and iso-C(17 : 1)omega9c, iso-C(17 : 0) and iso-C(15 : 0) as the major fatty acids. Major polar lipids detected in the two strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an amino-group-containing lipid that was ninhydrin-positive. Their DNA G+C contents were 71.0 mol%. Strains DS-123T and DS-140 exhibited no difference in their 16S rRNA gene sequences and possessed a mean DNA-DNA relatedness level of 92 %. Phylogenetic trees based on 16S rRNA gene sequences showed that strains DS-123T and DS-140 formed a distinct evolutionary lineage within the Gammaproteobacteria. The 16S rRNA gene sequences of strains DS-123T and DS-140 exhibited similarity values of less than 94.1 % to members of the Gammaproteobacteria. Strains DS-123T and DS-140 were distinguished from phylogenetically related genera by differences in some phenotypic properties. On the basis of phenotypic, phylogenetic and genetic data, it is proposed that strains DS-123T (= KCTC 12396T = DSM 17203T) and DS-140 be classified in a novel genus and species, Dokdonella koreensis gen. nov., sp. nov.

Base Composition↗

Donghaeana dokdonensis gen. nov., sp. nov., isolated from sea water.

A Gram-negative, non-motile, non-spore-forming and rod-shaped bacterial strain, DSW-6T, was isolated from sea water and subjected to a polyphasic study. Strain DSW-6T grew optimally at 25 degrees C, in the presence of 2 % (w/v) NaCl and at pH 7.0-8.0. It was characterized chemotaxonomically as having MK-6 as the predominant menaquinone and iso-C(15 : 0), anteiso-C(15 : 0), iso-C(17 : 0) 3-OH and C(15 : 0) as the major fatty acids (>10 % of total fatty acids). Major polar lipids were phosphatidylethanolamine, unidentified phospholipids and amino-group-containing lipids that are ninhydrin-positive. The DNA G+C content was 36.9 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DSW-6(T) forms an independent line of descent within the family Flavobacteriaceae. The 16S rRNA gene sequence of strain DSW-6T exhibited similarity values of less than 94.7 % to the sequences of other members of the family Flavobacteriaceae. Strain DSW-6T could be distinguished from other phylogenetically related genera by differences in several phenotypic properties. Therefore, in view of the combined phenotypic and phylogenetic data, it is proposed that strain DSW-6T (= KCTC 12402T = DSM 17205T) represents a novel genus and species, Donghaeana dokdonensis gen. nov., sp. nov.

Base Composition↗

Synergistic effects of metabolically related amino acids on the growth of a multicellular plant. II. Studies of 14C-amino acid incorporation.

The synergistic inhibition of the growth of Marchantia polymorpha gemmalings by lysine and threonine and its prevention by methionine has been investigated utilizing (14)C-labeled amino acids. Experiments involving the uptake of (14)C-lysine or (14)C-threonine in the presence or absence of methionine indicated that the synergistic growth effects were not a result of altered amino acid uptake. These data, as well as direct chemical analysis, indicated that growth inhibition was correlated with an inhibition of protein synthesis. Experiments utilizing (14)C-aspartic acid revealed that the presence of lysine and threonine resulted in increased (14)CO(2) production and an accumulation of soluble (14)C-aspartic acid and labeled ninhydrin-positive compounds. These metabolic alterations were prevented when methionine was also included in the growth media. A model depicting a sequence of events which involve the interaction of regulatory mechanisms is suggested to account for the effects of specific amino acids on plant growth.

Amino Acids↗

Polyamines in soybeans.

Putrescine, spermidine, and spermine were three main polyamines isolated from soybeans and partially characterized. Occurrence of polyamines in soybeans was established by separating trichloroacetic acid extracts of soybeans by cationic exchange column chromatography, identification with thin layer chromatography, paper electrophoresis, mass spectral analysis, reactions with ninhydrin and Dragendorff reagents, and spectrophotometric characteristics. Soybeans contained a minimum of 29.0 micrograms of polyamines per gram of full-fat flour. The alcohol-soluble fraction of soybeans contained polyamines also. Resting seeds contained spermidine in higher concentration than either putrescine or spermine. Spermine appeared to be present in lowest concentration. Preliminary experiments suggested that some polyamines were possibly in bound forms.

Journal Article↗

Soybean polyamines: separation and characterization of cadaverine.

Cadaverine in soybeans was separated by ion exchange chromatography from other polyamines previously identified. Identification of cadaverine was based on ion exchange separation, thin layer chromatography, paper electrophoresis, mass and nuclear magnetic resonance spectral analyses. Since the molecules of putrescine and cadaverine are so similar, separation and identification of the two components are difficult. Their R(F) values on thin layer chromatography are close, although cadaverine produces a bluish purple color when sprayed with ninhydrin reagent, while putrescine forms a purple color. Separation likewise is poor by paper electrophoresis, gas chromatography, and gel filtration. The mass spectra of cadaverine and putrescine have m/e peaks at 30, 43, 45, 56, 73, 85, 102 and 30, 43, 59, 71, 88, respectively. The m/e peaks differentiate one compound from the other. Nuclear magnetic resonance spectra and their integration curves show that cadaverine contains two types of methylene protons (10 total) in 3:2 ratio while putrescine produces two types (8 total) in 1:1 ratio. Polyamines occur at levels of micrograms per gram of soybeans with spermidine present in the largest quantity.

Journal Article↗

Oxidation of proline by plant mitochondria.

Mitochondria isolated from etiolated shoots of corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), soybean (Glycine max L. Merr.), and mung bean (Phaseolus aureus) exhibited a proline-dependent O(2) uptake subject to respiratory control. ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on O(2), but not NAD. The major ninhydrin-positive product formed via Delta(1)-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO(2), organic acids, and aspartate were recovered after [(14)C]proline feeding experiments. These results demonstrate the mitochondrial association and O(2) dependence of plant proline metabolism.

Journal Article↗

Isolation and partial characterization of an Acid endoprotease present in dormant apple shoot bark.

A major protease present in dormant bark tissues of the apple (Malus domestica Borkh. cv. "Golden Delicious") was partially purified by hemoglobin-coupled Sepharose column chromatography. The protease active at pH 4.6 and at temperatures of 30 to 50 C was found to be sulfhydryl-dependent. Phenylmercuric acetate inhibited the enzyme approximately 50% at 2 millimolar, whereas phenylmethylsulfonyl fluoride inhibited less than 30% even at 10 millimolar. Substrate specificity and the separation of the enzyme reaction products indicated that the enzyme is likely to be an endoprotease. We suggest that the storage proteins in apple bark tissue undergo some modification prior to their eventual hydrolysis to amino acids, which requires a multi-enzyme system(s). Evidence is presented that the apple bark protein extracts enzymically release ninhydrin-positive compounds upon storage at 5 C. It is concluded that the activation of the sulfhydryl-dependent acid endoprotease is associated with the rapid metabolism of storage proteins which accompanies bud break upon regrowth.

Journal Article↗

Characteristics and Activity Changes of Proteolytic Enzymes in Apple Leaves during Autumnal Senescence.

At least four different proteinases are present in senescing apple leaves (Malus domestica Borkh. cv. Golden Delicious) as determined by their pH optima, substrate specificity, and their reactivity to proteinase inhibitors. An enzyme active at pH 4.5 to 5.0 appears to be a sulfhydryl-dependent (iodoacetamide and phenylmercuric acetate-sensitive) endoproteinase, and degradation of the large subunit of ribulose bisphosphate carboxylase was observed only with this enzyme. It is tentatively concluded that this endoproteinase is responsible for the breakdown of ribulose bisphosphate carboxylase in vivo. However, the presence of more than one endoproteinase in apple leaves is suggested by the broad range of pH optima of the SH-dependent enzyme. Another enzyme active at pH 6.0 appears to be a carboxypeptidase, and was sensitive to phenylmethylsulfonylfluoride. This enzyme showed a strong hydrolytic activity against carbobenzoxyphenylalanylalanine. A sulfhydryl-dependent aminopeptidase and a second hydroxyl-dependent carboxypeptidase were active at pH 7.5Total autolytic activity (the sulfhydryl-dependent endoproteinase) as measured by the disappearance of proteins decreased during the period of protein decline. Evidence is presented that the measured proteinase activity can be dependent on assay methods and substrates. While the disappearance of protein measures most of endo-type activity, the ninhydrin assay appears to measure exo-type activity preferentially.

Journal Article↗

Quantitation and purification of quaternary ammonium compounds from halophyte tissue.

A simple and sensitive spectrophotometric assay for quaternary ammonium compounds (QACs) based on Dragendorff's reagent is described. Although not specific for a particular QAC, the assay allows for rapid survey of tissue for QAC content. A separation method for QACs in halophyte tissue containing high levels of cations was developed using ion exchange resins and ninhydrin to remove the amino acid fraction.

Journal Article↗

A technique for collection of exudate from pea seedlings.

Ethylenediaminetetraacetic acid (EDTA), at concentrations higher than 1.0 millimolar, is phytotoxic to etiolated seedlings of Pisum sativum. Substantial vascular exudation from pea epicotyls could be obtained without tissue damage at 0.5 millimolar EDTA if the solution was buffered at pH 7.5 with sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. Treated seedlings exuded 950 micrograms (leucine equivalents) of ninhydrin-positive material per day and 870 micrograms (glucose equivalents) of anthrone-positive material per day. Amino acid analysis showed the exudate to have glutamine as the major amido nitrogen containing compound and sucrose was shown to be the major sugar. Radiolabeled tryptophan and sucrose applied to cotyledons were transferred through the epicotyl and into the collection medium. The pH profile for exudation shows half maximal exudation at pH 7.2, indicating the promotion of exudation by EDTA is probably not due simply to Ca2+ chelation.

Amino Acids↗

Tumors of the pilosebaceous unit induced in the rat by the intravenous administration of 7,12-dimethylbenz(A)anthracene.

Six of 70 female Sprague-Dawley rats given a single intravenous injection of 7,12-dimethylbenz[a]anthracene (DMBA) developed 7 pilosebaceous tumors. Two of the tumors showed differentiation toward the upper portion of the pilosebaceous unit while 5 showed differentiation toward the lower portion. Each tumor was examined histochemically for the presence of inner root sheath keratin of the hair follicle using the carbamido diacetyl reaction for citrulline and for hair shaft keratin using boiling ninhydrin reagent. The 2 tumors of the upper portion of the pilosebaceous unit were sebaceous adenomas which were accompained by a keratinizing epithelim like that of the sebaceous gland duct and upper pilosebaceous canal. Histochemically, the keratin was not like that of hair shaft nor inner root sheath. The 5 tumors showing differentiation toward components of the lower pilosebaceous unit were trichoepitheliomas. They were composed of structures which, to varying degrees, recapitulated the organization of the normal hair follicle. Within these follicular structures, both inner root sheath and hair shaft type keratins were found. The occurrence of skin tumors after the intravenous administration of DMBA was unexpected since it is uncommon for skin tumors to be produced by the systematic administration of chemical carcinogens and they have never been described after the oral administration of DMBA. That the route of administration may influence tumor production with this carcinogen is suggested by the fact that the only other reported tumors, which were squamous carcinomas, also followed intravenous injection of DMBA.

9,10-Dimethyl-1,2-benzanthracene↗

Metabolic activities of pseudomonads in batch cultures in extract of minced lamb.

Pseudomonas fragi, Ps. lundensis and Ps. fluorescens were studied in axenic batch cultures growing in a lamb juice (pH 6.0) aerobically or in an atmosphere (Ps. fragi only) enriched with carbon dioxide at 4 degrees C. With all but a glucose dehydrogenase-deficient strain of Ps. fluorescens there was a sequential catabolism of glucose and lactate. Diauxic growth was observed in a nutrient-deficient meat juice supplemented with glucose and lactate. A transient peak in the concentration of gluconate and pyruvate was associated with the catabolism of glucose and lactate respectively. With Ps. fluorescens deficient in glucose dehydrogenase there was simultaneous catabolism of glucose and lactate. The stereoisomers of lactate were catabolized simultaneously in a laboratory medium. Glucose-6-phosphate was oxidized to 6-phosphogluconate by Ps. fragi and Ps. lundensis under aerobic conditions only. Creatine and creatinine were catabolized by Ps. fragi under aerobic conditions only. There was a slight decrease in the concentration of total amino acids (ninhydrin-reactive material) during the exponential phase of growth. The results suggest that the dominance of Ps. fragi in the climax populations in meat is due to catabolism of amino acid related substrates, creatine and creatinine.

Creatine↗

Myelopeptides--bone marrow mediators with immunostimulating and endorphin-like activity.

Bone marrow cells produce soluble mediators with structural and functional heterogeneity. They were found to stimulate antibody production at the peak of the immune response, owing to compounds of a peptide nature (Mr 2000-1300). Active material was isolated by means of gel chromatography and electrophoresis. This material positively reacts with ninhydrin, and has maximum absorption close to 278 nm. Its antibody-stimulating activity decreased or stopped completely after treatment with proteolytic enzymes. Apart from the immunostimulating activity, bone marrow mediators have opiate-like activity. They have an analgetic effect, and interact with the opiate receptors of brain nerve cells. After a physicochemical and functional analysis, we conclude that bone marrow produces regulatory peptides that were previously unknown. We called them myelopeptides.

Analgesics↗

Molecular design of a cyclic heptapeptide to mimic the zinc-binding site of carbonic anhydrase. Synthesis and zinc-binding studies by 13C-and 1H-N.M.R. spectroscopy.

A cyclic heptapeptide [cyclo-(Gly-L-His-Gly-L-His-L-Gly-L-Gly)] was designed to mimic the Zn(II)-binding site of carbonic anhydrase. The cyclic heptapeptide was synthesized from the linear heptapeptide, Gly-L-His-Gly-L-His-Gly-L-His-Gly-OH, which in turn was obtained by coupling of the fragments, viz. BOC-Gly-L-His-Gly-N3 and L-His-GLy-L-His-Gly-OBzlNO2 followed by deblocking of amino and carboxyl protecting groups. Conversion of the linear heptapeptide to the azide by treatment with diphenylphosphoryl azide was followed by cyclization in high dilution. A homogeneous material was isolated by counter-current distribution followed by gel filtration. It was found to be ninhydrin negative. The n.m.r. spectrum of the material upon integration indicated the proper ratios of various kinds of protons to be expected of the cyclic heptapeptide. A detailed 13C- and 1H-n.m.r. investigation was undertaken to determine the Zn(II)-binding ligands of the cyclic heptapeptide. The assignments for all the resonances were attempted by spin-decoupling method, pH and solvent effects, and by comparison of resonances of similar protons and carbons of model peptides. The n.m.r. titration results of the Zn(II) bound form of the cyclic peptide showed the presence of a 1:1 complex. Upon Zn(II)-binding, the changes in the chemical shift of the imidazole protons were relatively large, indicating that this ring is involved in the complexation. All the peptide -NH-resonances were observable and unaffected; consequently, none of these nitrogens can serve as a ligand. In the case of 13C resonances, addition of 1 equiv. of Zn(II) to the cyclic heptapeptide, te C(2), C(4), and C(5) carbon resonances of this group were dramatically affected and showed a very large change in chemical shift upon complexation. The results demonstrate that Zn(II) binds to all three imidazole residues of the designed cyclic heptapeptide.

Binding Sites↗

Similarities and differences of five peroxidases from turnip and horseradish. Peptide mapping studies on glycoproteins.

Four isoperoxidases of turnip root and isoperoxidase C of horseradish root were digested with trypsin, and their peptide maps, prepared by high-voltage paper electrophoresis, were compared. All five tryptic digests were completely soluble at pH 8. The maps were developed with a variety of general and specific reagents: ninhydrin, histidine, tyrosine, tryptophan and arginine reagents. Cystine peptides and cysteic acid derivatives have also been characterized. All detected half-cystine residues seemed engaged in disulfide bridges. For each individual peroxidase the number of specifically staining peptides agreed very well with the amino acid composition. The two most acidic peroxidases of turnip, P1 and P2, only differ significantly in one peptide. The P2 gene is tentatively proposed to have developed from the P1 gene by a single base mutation, changing an asparagine residue to alysine residue. A less acidic turnip peroxidase, P3, is distinct, although related to peroxidases P1 and P2. Horseradish isoperoxidase C also belongs to this group which appears to be closely related in the amino acid sequences around four disulfide bridges. Peroxidase P7 differs from this group, at least around two of its disulfide bridges, and therefore, may differ from the other four in parts of its three dimensional structure. Sequences of particular importance to peroxidase function must be present in all peroxidases. From the peptide mapping studies we only find two highly homologous sequences present in all five examined peroxidases. Both contain histidine. This finding corroborates previous suggestions of two histidine sequences near the peroxidase heme prosthetic group. The rules applied in relating peptides of different proteins are outlined, and the sources of errors in mapping of glycoproteins of high carbohydrate content (about 20%) are discussed in detail.

Amino Acids↗

Lipopeptidophosphoglycan from Trypanosoma cruzi. Amide and ester-linked fatty acids.

Lipopeptidophosphoglycan, extracted from whole cells of epimastigote forms of Trypanosoma cruzi, has now been shown to contain 12.6% of fatty acids in addition to the previously identified content of neutral sugars (60%), glucosamine (0.8%), peptide (9.5%) and acid-hydrolyzable phosphate (2%). The main fatty acids are palmitic (6.9%) and lignoceric (4.6%) acids. Stearic (0.55%), oleic (0.15%) and myristic (0.18%) acids were also found. One third of the fatty acids are bound in the lipopeptidophosphoglycan as esters (14 mmol%) and two thirds as amides (28 mmol%). Lignoceric acid was found to be bound only as amide. Two ninhydrin-positive compounds, obtained by chloroform extraction of a total acid hydrolysate of the lipopeptidophosphoglycan, were tentatively identified as sphingosine bases.

Amides↗