Phospholipids of marine proteobacteria of the genusPseudoalteromonas

The study of the phospholipid composition of 14 type strains of marine proteobacteria of the genusPseudoalteromonas showed that phospholipids are the main polar lipid constituents of membranes in these proteobacteria. The phospholipid patterns of the strains studied were found to be similar and involved five phospholipids typical of gram-negative bacteria, namely, phosphatidylethanolamine, phosphatidylglycerol, bisphosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid. The major phospholipids were phosphatidylethanolamine and phosphatidylglycerol, which add up to 89–97% of the total phospholipids; bisphosphatidic acid was dominant among minor phospholipids. The prevalence of phosphatidylethanolamine (62–77% of the total phospholipids) and the absence of diphosphatidylglycerol are the characteristic features of most bacteria of this genus. As inEscherichia coli, the phospholipid composition of the marine proteobacteria depended on the presence of magnesium in the medium.     

Isoprenoid quinones and fatty acids ofZoogloea

NineZoogloea strains including the type strain ofZ. ramigera (IAM 12136=ATCC 19544=N.C. Dondero 106) and newly isolated strains were investigated for isoprenoid quinone composition and whole-cell fatty acid profiles. Seven of the tested strains, having phenotypic properties typical ofZoogloea, were characterized by their production of both ubiquinone-8 and rhodoquinone-8 as major quinones, whereas the remaining two strains,Z. ramigera IAM 12669 (=K. Crabtree I-16-M) and IAM 12670 (=P.R. Dugan 115), formed ubiquinone-10 and ubiquinone-8, respectively, as the sole quinone. All rhodoquinone-producing strains contained palmitoleic acid and 3-hydroxy-decanoic acid as the major components of nonpolar and hydroxylated fatty acids, respectively. Marked differences were noted in the fatty acid composition between the strains with and without rhodoquinones. The chemotaxonomic data suggested that the rhodoquinonelacking strains should be excluded from the genusZoogloea. Since there have been no reliable taxonomic tools forZoogloea, rhodoquinone analysis may provide a new criterion of great promise for identifyingZoogloea strains.     

Purification and Partial Identification of Novel Antimicrobial Protein from Marine Bacterium <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> Species Strain X153

A marine bacterium, X153, was isolated from a pebble collected at St. Anne du Portzic (France). By 16S ribosomal DNA gene sequence analysis, X153 strain was identified as a Pseudoalteromonas sp. close to P. piscicida. The crude culture of X153 was highly active against human pathogenic strains involved in dermatologic diseases, and marine bacteria including various ichthyopathogenic Vibrio strains. The active substance occurred both in bacterial cells and in culture supernatant. An antimicrobial protein was purified to homogeneity by a 4-step procedure using size-exclusion and ion-exchange chromatography. The highly purified P-153 protein is anionic, and sodium dodecylsulfate polyacrylamide gel electrophoresis gives an apparent molecular mass of 87 kDa. The X153 bacterium protected bivalve larvae against mortality, following experimental challenges with ichthyopathogenic Vibrio. Pseudoalteromonas sp. X153 may be useful in aquaculture as a probiotic bacterium.     

Antibiotic Susceptibility as a Taxonomic Characteristic of Proteobacteria of the Genera Alteromonas, Pseudoalteromonas, Marinomonas, and Marinobacter

Type strains of bacteria of the genera Alteromonas, Pseudoalteromonas, Marinomonas, and Marinobacterand symbiotrophic bacteria isolated from the Far Eastern bivalve mollusks Crenomytilus grayanusand Mizuhopecten yessoensisand identified as Pseudoalteromonas nigrifaciensand P. citreawere tested for susceptibility to antibiotics. Of the ten antibiotics used in this study, benzylpenicillin, polymyxin, lincomycin, and oleandomycin were valuable for the polyphasic taxonomy of marine aerobic proteobacteria. Marine bacteria of the genera studied exhibited characteristic antibiotic susceptibility profiles, which were useful for the differentiation of the genera.     

Effects of Culture Method and Growth Phase on Free Lipid Composition of Yersinia pseudotuberculosis

The influence of culture method (free-floating cells in liquid nutrient broth or bacteria attached to agar surface on solid agarized medium of the same formulation) and bacterial age on the composition of free lipids in Yersinia pseudotuber-culosis (O:Ib serovar, strain KS 3058) grown in the cold (5°C) has been investigated. The specific growth rate of the bacteria on solid medium was about threefold less than that in liquid medium. The qualitative composition of phospholipids and fatty acids only slightly depended on the bacterial culture method. At the same time, the colonially growing cultures contained somewhat more total lipids, they synthesized more phospholipids, in the linear growth phase they contained more lysophosphatides, and they had higher fatty acid unsaturation index and higher pathogenic potential than their planktonic counterparts grown in otherwise identical conditions. The bacterial growth phase influenced the amount of 3-hydroxytetrade-canoic acid and, indirectly, that of lipopolysaccharide. The dynamics of changes in the amount of this acid with bacterial age was opposite in the surface and broth cultures.     

Changes in the Lipid Composition of Mucor hiemalis Sporangiospores Related to the Age of the Spore-Forming Culture

Analysis of sporangiospore lipids of the fungus Mucor hiemalis F-1156 showed that alterations occur in the content of fatty acids and individual classes of lipids during long-term cultivation (for about 20 days). The changes in the lipid composition related to the age of the spore-forming mycelium suggest an important role of sporangiospore lipids in spore germination and in further development of the spherical cells formed in this processes. The M. hiemalis F-1156 sporangiospores with a lipid pool exhausted during long-term cultivation can give rise to both mycelial and yeastlike growth.     

Occurrence and diversity of mesophilic Shewanella strains isolated from the North-West Pacific Ocean

Although bacteria of the genus Shewanella belong to one of the readily cultivable groups of "Gammaproteobacteria", little is known about the occurrence and abundance of these microorganisms in the marine ecosystem. Studies revealed that of 654 isolates obtained from marine invertebrates (ophiuroid Amphiopholis kochii, sipuncula Phascolosoma japonicum, and holothurian Apostichopus japonicus, Cucumaria japonica), seawater and sediments of the North-West Pacific Ocean (i.e. the Sea of Japan and Iturup Is, Kurile Islands), 10.7% belonged to the genus Shewanella. The proportion of viable Shewanella species varied from 4% to 20% depending on the source of isolation. From the isolation study, representative strains of different phenotypes (from seventy presumptive Shewanella strains) were selected for detailed characterization using phenotypic, chemotaxonomic, and phylogenetic testing. 16S rDNA sequence-based phylogenetic analysis confirmed the results of tentative identification and placed the majority of these strains within only a few species of the genus Shewanella with 98-99% of 16S rDNA sequences identity mainly with S. japonica and S. colwelliana, suggesting that the strains studied might belong to these species. Numerically dominant strains of S. japonica were metabolically active and produced proteinases (gelatinases, caseinases), lipases, amylases, agarases, and alginases. Shewanella strains studied demonstrated weak antimicrobial and antifungal activities that might be an indication of their passive role in the colonization on living and non-living surfaces.     

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7–8 and at temperature close to 35°C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40–45°C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.     

Taxonomic Composition and Biological Activity of Microorganisms Associated with a Marine Ascidian Halocynthia aurantium

The taxonomy and biological activity of 176 heterotrophic microorganisms associated with the ascidian Halocynthia aurantiumfrom the coastal waters of Peter the Great Bay (Sea of Japan) were studied. In the microbial community, bacteria of the genera Pseudoalteromonas(38% of all strains studied) and Bacillus(25%) prevailed and showed high biological activity. Bacteria of Pseudoalteromonaswere capable of extracellular hydrolases production and exhibited high antibiotic and antimicrobial properties. Five Pseudoalteromonasstrains were capable of restriction enzyme production. A significant proportion of the pseudoalteromonads inhibited growth ofProteus vulgarisand Candida albicans(81 and 84%, respectively). Spore-forming bacteria of Bacilluswere capable of chitin hydrolysis and extracellular inducible RNAses production (23%). They were active against Staphylococcus aureusand Bacillus subtilis(39 and 23%, respectively). Bacillus pumilusstrain KMM 1364 showed hemolytic activity and antagonistic properties against S. aureusand was found to produce surface active glycolipids and surfactinlike cyclic depsipeptides.     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.     

A New Family,Alteromonadaceae fam. nov., Including Marine Proteobacteria of the Genera Alteromonas,Pseudoalteromonas, Idiomarina,and Colwellia

The taxonomic positions of the marine genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia within the gamma subclass of the class Proteobacteria were specified on the basis of their phenotypic, genotypic, and phylogenetic characteristics. Gram-negative aerobic bacteria of the genera Alteromonas, Pseudoalteromonas, and Idiomarina and facultatively anaerobic bacteria of the genus Colwellia were found to form a phylogenetic cluster with a 16S rRNA sequence homology of 90% or higher. The characteristics of these genera presented in this paper allow their reliable taxonomic identification. Based on the analysis of our experimental data and analyses available in the literature, we propose to combine the genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia into a new family, Alteromonadaceae fam. nov., with the type genus Alteromonas.     

Changes of phospholipid fatty acid composition in the digestive gland of the mollusc <Emphasis Type="Italic">Littorina saxatilis</Emphasis>, caused by trematode larvae

Lipids in the digestive gland of the mollusc Littorina saxatilis from the White and Barents Seas were studied. Changes of their biochemical composition are discussed in the connection with different temperature of the habitat and with infestation with trematode larvae. Comparative analysis of the fatty acid (FA) composition of each of the phospholipids in intact molluscs has revealed essential differences. Phosphatidylcholine and monophosphatidylinositol (MPI) FA did not differ in the ω3/ω6 ratio, which is due to their tolerance to the temperature factor, whereas more unsaturated phospholipids—phosphatidylethanolamine (PEA), its plasmalogen form (pPEA), and phosphatidylserine—differed 1.5–2 times in the studied molluscs. Prevalence of ω3-acids in Barents Sea mollusc is associated undoubtedly with lower habitat temperatures, as this provides a higher fluidity of the membrane phospholipids. Infestation affected to the greatest degree the quantitative FA composition in pPEA and MPI. At infestation, there was revealed a threefold decrease of the content of eicosenoic FA, C20:1, only in MPI among all considered phospholipids, whereas it was increased, in the contrary, in all other phospholipids. Monophosphatidylinositols differed essentially from other phospholipids by the saturated FA amount as well, which changed the unsaturation index of these phospholipids. Since the functional significance of this minor phospholipid is determined by its participation in the so-called phosphatidylinositol system of the hormonal signal transduction, it seems interesting to elucidate whether the increase of this membrane phospholipid saturation at invasion affects the reflex connection between signals from receptors located in the parasite and enzymatic processes.     

Resistance of the fish-killing dinoflagellate Cochlodinium polykrikoides against algicidal bacteria isolated from the coastal sea of Japan

Noxious red tides of the dinoflagellate Cochlodinium polykrikoides tend to be long lasting and cause mass mortalities of cultured and natural fish and invertebrates along the western coast of Japan and the southern coast of Korea. In order to assess the tolerance of C. polykrikoides to attack by algicidal bacteria, the effects of algicidal bacteria strains on the growth of three C. polykrikoides strains were examined in laboratory culture experiments. Algicidal bacteria used were two strains of Cytophaga (J18/M01 and AA8-2, direct attack type and wide prey range), three strains of Alteromonas (S, K, D) and one strain of Pseudoalteromonas (R, indirect attack type), which were all isolated by using Chattonella antiqua as a prey organism. Neither Cytophaga strain showed any algicidal activity. In the cases of Alteromonas and Pseudoalteromonas, some cultures of C. polykrikoides were killed, but at least 10 days or more were required for the death of this dinoflagellate. C. polykrikoides survived in the presence of algicidal bacteria in concentrations up to 10⁶–10⁷ cells ml⁻¹, which is enough for other red tide microalgae to be killed. On the contrary, the algicidal effects of bacteria on C. antiqua were detected clearly within a few days. These results imply that C. polykrikoides is resistant to the six algicidal bacteria examined, which may reflect the capacity for mixotrophy. This resistance of C. polykrikoides to algicidal bacteria could provide a selective advantage for survival compared to other microalgae susceptible to attack by algicidal bacteria and hence prolong red tides caused by this harmful dinoflagellate.     

Plant Uncoupling Mitochondrial Protein Activity in Mitochondria Isolated from Tomatoes at Different Stages of Ripening

In the present study we have observed a higher state of coupling in respiring mitochondriaisolated from green as compared to red tomatoes (Lycopersicon esculentum, Mill.). Greentomato mitochondria produced a membrane potential () high enough to phosphorylate ADP,whereas in red tomato mitochondria, BSA and ATP were required to restore to the levelof that obtained with green tomato mitochondria. This supports the notion that such uncouplingin red tomato mitochondria is mediated by a plant uncoupling mitochondrial protein (PUMP;cf. Vercesi et al., 1995). Nevertheless, mitochondria from both green and red tomatoes exhibitedan ATP-sensitive linoleic acid (LA)-induced decrease providing evidence that PUMP isalso present in green tomatoes. Indeed, proteoliposomes containing reconstituted green or redtomato PUMP showed LA uniport and LA-induced H⁺ transport. It is suggested that the higherconcentration of free fatty acids (PUMP substrates) in red tomatoes could explain the lowercoupling state in mitochondria isolated from these fruits.     

Changes in the Fatty Acid Composition of Hepatopancreas of the Mollusk Mytilus trossulus Fed on Microalgae

The influence of diet on the fatty acid composition of the hepatopancreas of Mytilus trossulus was studied. Three groups of mollusks were fed monocultures of the microalgae Phaeodactylum tricornutum, Chaetoceros muelleri (Bacillariophyceae), and Nannochloropsis sp. (Eustigmatophyceae) for 10 days. After 10 days, the proportion of polyunsaturated fatty acids, mainly eicosapentaenoic and docosahexaenoic, increased in the total lipids of the hepatopancreas in all mollusk groups. The content of saturated fatty acids in the mussel tissues decreased and was not dependent on the amount in the algal diet. Toward the end of the experiment, the fatty acid composition of the hepatopancreas of mussels was similar irrespective of the fatty acid composition of their food. The fatty acid analysis of M. trossulus feces suggests a selective assimilation by mussels of predominantly the n-3 polyunsaturated fatty acids. The role of fatty acid metabolism in M. trossulus is discussed.     

Marine fungus <Emphasis Type="Italic">Stilbella aciculosa</Emphasis> as a potential producer of prostaglandins

The amount and composition of fatty acids in the fungus Stilbella aciculosa associated with the marine macroorganism Apostichopus japonica (trepang) were determined by gas-liquid chromatography and gas chromatography-mass spectrometry. In the culture liquid of S. aciculosa, prostaglandins (PG) of groups E and F were revealed by UV spectroscopy. This finding was confirmed by the presence of direct precursors of PG, polyunsaturated eicosapentaenoic and docosahexaenoic acids, in the culture liquid. The biomass of this fungus contained PG of group B.     

Dispersal of Two Species of Trichoptera from Desert Springs: Conservation Implications for Isolated vs Connected Populations

During the Pleistocene, when the climate was wetter and cooler, aquatic habitats in the Great Basin of western North America were much more extensive and connected. As the climate warmed over the last 10000 years, many of these habitats dried but others remained as isolated springs and inland lakes. The isolation of desert springs and lack of dispersal between populations of non-vagile species (e.g. fish, spring snails) has led to genetic differentiation and speciation. However, the extent to which vagile species of aquatic insects disperse from spring to spring is unknown. We examined the population genetics of two caddisflies, Hesperophylax designatus (Limnephilidae) and Lepidostoma ojanum (Lepidostomatidae) that occur in isolated springs in Nevada and eastern California to determine the extent of their dispersal from spring to spring. Mitochondrial DNA sequences indicate that the populations of L. ojanum are isolated and that the populations represent management units. In contrast, H. designatus individuals are flying from spring to spring and their populations are connected by dispersal. Disturbance impacts (e.g. grazing by ungulates, water extraction) that eliminate poor dispersers (e.g. L. ojanum) locally may result in permanent losses of genetic diversity; this is less likely with broader dispersers such as H. designatus.     

Effect of altered sterol levels on the transport of amino acids and membrane structure ofMicrosporum gypseum

Ergosterol and cholesterol supplementation resulted in a significant increase (1·5-fold) in the sterol content while phospholipid remained unaffected inMicrosporum gypseum. The levels of phosphatidylethanolamine and phosphatidylcholine increased in ergosterol supplemented cells. However, a decrease in phosphatidylcholine and an increase in phosphatidylethanolamine was observed in cholesterol grown cells. The ratio of unsaturated to saturated fatty acids decreased on ergosterol/cholesterol supplementation. The uptake of amino acids (lysine, glycine and aspartic acid) decreased in sterol supplemented cells. Studies with fluorescent probe l-anilinonaphthalene-8-sulfonate showed structural changes in membrane organisation as evident by increased number of binding sites in such cells.     

Elucidation of Structure of Lipid A from the Marine Gram-Negative Bacterium Pseudoalteromonas haloplanktis ATCC 14393T

The chemical structure of lipid A, from the marine -proteobacterium Pseudoalteromonas haloplanktis 14393, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be -1,6-glucosaminobiose 1,4-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3 and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2, respectively.     

Effects of copper and cadmium ions on the physicochemical properties of lipids of the marine bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IB28 at different growth temperatures

The phospholipid and fatty acid composition and thermotropic behavior of total lipids were studied in the metal-accumulating marine strain Pseudomonas putida IB28 grown in the presence of Cu²⁺ and Cd²⁺ at 4 and 24°C. Despite the changes in acidic lipid content, unsaturated/saturated fatty acid ratio, and cyclopropane fatty acid level, the temperature range of calorimetric phase transitions of bacterial total lipids was slightly altered under these factors. The suppressive action of heavy metals on bacterial growth is attributable to the phase separation of lipids and, as a consequence, to a sharp increase in the ion permeability of the lipid bilayer. The increase in acidic phospholipid level under the influence of Cu²⁺ and Cd²⁺, especially at 24°C, is likely to be indicative of their complexation with heavy metal ions.