Fatty Acid Distribution in Mesophilic and Thermophilic Strains of the Genus Bacillus

The fatty acid distribution of three mesophilic and three thermophilic strains of the genus Bacillus was determined by gas chromatography of the fatty acid methyl esters. Fatty acid i-15:0 was the most abundant in both the mesophiles (51%) and the thermophiles (41%). The second most abundant fatty acid was a-15:0 in the mesophiles (22%), and i-17:0 in the thermophiles (27%). The fatty acid pair i-15:0, i-17:0 was the most predominant pair in both the mesophiles (61%) and the thermophiles (66%). The fatty acid pair a-15:0, a-17:0 was the second most predominant pair and was much higher in the mesophiles (30%) than in the thermophiles (15%). The average fatty acid chain length was 15.5 for the mesophiles and 16.0 for the thermophiles. The significance of these results for the lipid theory of thermophily is discussed.     

Effect of growth phase on the fatty acid composition of Thermus spp.

Two yellow and two red pigmented strains of Thermus were monitored for changes in fatty acid content and composition with reference to growth phase at the optimum temperature. Fatty acid content per mg of dry weight increased as the cultures aged. In addition the quantities of iso C 15:0, iso C 17:0 and iso C 16:0 increased in yellow pigmented strains, but in red pigmented strains, an increase was seen in iso C 15:0, but C 16:0 and iso C 16:0 levels decreased. Thus the fatty acid composition of these organisms varies with growth phase, and shows also strain specific variability.     

Chemotaxonomic survey of sterols and fatty acids in six marine raphidophyte algae

Fatty acid and sterol profiles allowed for clear discrimination betweentheraphidophyte genera Chattonella,Heterosigma, Fibrocapsa andOlisthodiscus, but exhibited little differentiation forindividual Chattonella species(C.marina, C. antiqua and C.subsalsa). Sterol and fatty acid profiles do not support theseparation of Chattonella antiqua and C.marina as distinct species. Ecophenotypic variations in lipidprofiles were also observed between Chattonella strainsfromdifferent geographic locations. Sterol signatures which may be useful aschemotaxonomic markers were: the absence of C₂₇ sterols (cholesteroland 24-dihydrozymosterol) in Heterosigma akashiwo; thepresence of isofucosterol in Chattonella; and theoccurrence of brassicasterol, poriferasterol and fucosterol inOlisthodiscus luteus. High levels of eicosapentaenoic acid(EPA; 17-27% of fatty acids) were present in all raphidophyte species. Lipidcomposition correlated more closely to recent molecular classification ofraphidophytes than carotenoid pigments.     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Structure and composition of intracytoplasmic membranes of Ectothiorhodospira mobilis

The lamellar membrane stacks of Ectothiorhodospira mobilis were isolated and purified by a combination of lysozyme and osmotic shock treatment, followed by differential and density gradient centrifugation. Preparations of lamellar membranes were enriched at least 2.4-fold in the ratio of bacteriochlorophyll a to protein.Thin-sectioning, negative staining, platinumcarbon shadowing and freeze-etching were used to study the architecture of the membrane units. Both platinum-carbon shadowing and freeze-etching showed the outer surfaces of the isolated lamellar membrane stacks to be relatively smooth. Particles averaging 7 nm in diameter were seen on several faces following freeze-ctching.Non-polar amino acids amounted to 60% of the total amino acid composition. Lipids constituted 32% of the membrane dry weight. Phosphatidyl ethanolamine and diphosphatidyl glycerol were the major phospholipids. Fatty acids of 10–15 carbons represented a small fraction of both membrane and whole cell fatty acids. Monoenes constituted 36% of the total membrane fatty acids and 38.4% of the total whole cell fatty acids. The major fatty acids of both whole cells and purified membranes were C16:0, C18:1 and cyclopropane C19:0.     

Changing fatty acid composition during somatic embryogenesis in cultures of Daucus carota

The fatty acid composition of five embryo stages following the induction of embryogenesis in cultures of Daucus carota have been studied. Quantitative rather than qualitative changes in fatty acid content were observed, although globular embryos do possess some fatty acids of long (C 20 and above) chain length not observed in earlier or later stages in development. The major fatty acids present were C 16:0, 18:0, 18:1, 18:2 and 18:3.Abbreviations SM small meristematic - LV large vacuolated - GLC gas-liquid chromatography - TLC thin lager chromatography     

Differences in the envelope proteins ofChlamydia pneumoniae,Chlamydia trachomatis,andChlamydia psittaci shown by two-dimensional gel electrophoresis

Analysis by two-dimensional gel electrophoresis of theN-laurylsarkosinate(Sarkosyl)-insoluble envelope complexes ofl-^[35]S-cysteine-iabeled elementary bodies ofChlamydia pneumoniae strain IOL-207,Chlamydia trachomatis serovar LGV2, D, and F, andChlamydia psittaci strain 6BC showed differences in the molecular charges of chlamydial outer membrane proteins. The apparent isoelectric point (pI) of the major outer membrane protein ofC. pneumoniae strain IOL-207 was 6.4, whereas the pI of the major outer membrane protein of theC. trachomatis andC. psittaci strains differed little from one another, ranging from 5.3 to 5.5. The 60-kDa cysteinerich protein ofC. pneumoniae was the only 60-kDa chlamydial protein with a pI value (5.9) more acidic than that of the corresponding major outer membrane protein. As a general rule, the charges of both the 60-kDa and the lowmolecular-mass (12–15 kDa) cysteine-rich proteins were widely variable, depending on the strain. However, in cach individual strain, the variation of the charge of the 60-kDa protein had a compensatory change in the lowmolecular-mass cysteine-rich protein.     

Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Arsukibacterium ikkense gen. nov., sp. nov, a novel alkaliphilic, enzyme-producing gamma-Proteobacterium isolated from a cold and alkaline environment in Greenland

A novel aerobic, Gram-negative, non-pigmented bacterium, GCM72(T), was isolated from the alkaline, low-saline ikaite columns in the Ikka Fjord, SW Greenland. Strain GCM72(T) is a motile, non-pigmented, amylase- and protease-producing, oxidase-positive, and catalase-negative bacterium, showing optimal growth at pH 9.2-10.0, at 15 degrees C, and at 3% (w/v) NaCl. Major fatty acids were C(12:0) 3-OH (12.2+/-0.1%), C(16:00) (18.0+/-0.1%), C(18:1)omega7c (10.7+/-0.5%), and summed feature 3 comprising C(16:1)omega7c and/or iso-C(15:0) 2-OH (36.3+/-0.7%). Phylogenetic analysis based on 16S rRNA gene sequences showed that isolate GCM72(T) was most closely related to Rheinheimera baltica and Alishewanella fetalis of the gamma-Proteobacteria with a 93% sequence similarity to both. The G+C content of DNA isolated from GCM72(T) was 49.9mol% and DNA-DNA hybridization between GCM72T and R. baltica was 9.5%. Fatty acid analysis and G+C content supports a relationship primarily to R. baltica, but several different features, such as a negative catalase-response and optimal growth at low temperature and high pH, together with the large phylogenetic distance and low DNA similarity to its closest relatives, lead us to propose a new genus, Arsukibacterium, gen. nov., with the new species Arsukibacterium ikkense sp. nov. (type strain is GCM72(T)).     

Alterations in growth temperature range and fatty acid composition of Thermus as a result of plasmid elimination

Elimination of plasmids from Thermus flavus, T. thermophilus and three wild Thermus strains caused alterations in growth temperature range, pigmentation and membrane fatty acids without affecting viability. Following plasmid elimination all Thermus strains lost their ability to grow above 70°C. In addition, the minimum growth temperature was lowered by 5–10°C. Fatty acids were reduced by an average of approximately 35%. In addition, the contribution of iso- and anteisobranched fatty acids were altered in four of the five strains. The iso C_15:0/iso C_17:0 ratio approached 1.0 in all strains, whereas the anteiso C_15:0/anteiso C_17:0 was reduced to 0.2. The iso C_16:0/normal-C_16:0 ratio increased in all strains due to an increase in iso C_16:0 in four strains and a reduction in normal-C_16:0 relative to iso C_16:0 in one strain. However, it was evident that the plasmid-free strains were able to compensate for these alterations in membrane fluidity to a certain extent by reducing the average chain length of isobranched acids. Altered fatty acid metabolism at the level of precursors may have influenced membrane composition and consequently growth temperature range.     

Effect of temperature on survival, development and reproduction of the predatory lacewing Dichochrysa prasina (Neuroptera: Chrysopidae) reared on Ephestia kuehniella eggs (Lepidoptera: Pyralidae)

Preimaginal development and adult longevity and reproduction of Dichochrysa prasina Burmeister were studied at six constant temperatures (15, 20, 25, 27, 30 and 33 °C) and a photoperiod of 16:8 (L:D). Eggs of the flour moth Ephestia kuehniella (Zeller) were used as food throughout preimaginal development, whereas the adults of D. prasina fed on a liquid diet of water, yeast hydrolysate, sugar and honey. At the highest tested temperature of 33 °C no larvae completed their development. At the rest of the tested temperatures the egg to adult developmental period ranged from approximately 92 days at 15 °C to 25 days at 30 °C. Percentages of adult emergence ranged from 36% at 15 °C to 84% at 30 °C. Both adult longevity and fecundity were significantly affected by temperature and the intrinsic rate of increase (r_m) reached its maximum value at 27 °C. These results could be useful for the establishment of a small scale rearing and mass production of D. prasina.     

Fatty acids, unusual glycophospholipids and DNA analyses of thermophilic bacteria isolated from hot springs

The composition of fatty acids in 12 strains of the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus was analyzed by gas chromatography–mass spectrometry. Major FAs found in the profiles included i-15:0, i-17:0, ai-15:0, i-16:0, 16:0, ai-17:0, together with some minor components. Branched FAs were predominant, forming more than 80% of all FAs measured. Fast atom bombardment-mass spectrometry was used for analysis of unusual glycophospholipids, i.e., acylglycosylcardiolipins from genera Geobacillus and Alicyclobacillus and 1-(hydroxy(2-(O-acylglycosyl-oxy)hexadecyloxy)phosphoryloxy) hexadecan-2-yl esters of C15–C17 acids from genera Thermus and Meiothermus. Cloning and preliminary sequence analysis of 16S rDNA showed that these isolates belong to the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus.     

Seed germination and dormancy in the medicinal woodland herbs Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae)

We used a double germination phenology or “move-along” experiment (sensu Baskin and Baskin, 2003) to characterize seed dormancy in two medicinal woodland herbs, Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae). Imbibed seeds of both species were moved through the following two sequences of simulated thermoperiods: (a) 30/15 °C→20/10 °C→15/6 °C→5 °C→15/6 °C→20/10 °C→30/15 °C, and (b) 5 °C→15/6 °C→20/10 °C→30/15 °C→20/10 °C→15/6 °C→5 °C. In each sequence, seeds of both species germinated to high rates (>85%) at cool temperatures (15/6 and 20/10 °C) only if seeds were previously exposed to cold temperatures (5 °C). Seeds kept at four control thermoperiods (5, 15/6, 20/10, 30/15 °C) for 30 d showed little or no germination. Seeds of both species, therefore, have physiological dormancy that is broken by 12 weeks of cold (5 °C) stratification. Morphological studies indicated that embryos of C. canadensis have “investing” embryos at maturity (morphological dormancy absent), whereas embryos of D. villosa are undeveloped at maturity (morphological dormancy present). Because warm temperatures are required for embryo growth and cold stratification breaks physiological dormancy, D. villosa seeds have non-deep simple morphophysiological dormancy (MPD). Neither species afterripened in a 6-month dry storage treatment. Cold stratification treatments of 4 and 8 weeks alleviated dormancy in both species but C. canadensis seeds germinated at slower speeds and lower rates compared to seeds given 12 weeks of cold stratification. In their natural habitat, both species disperse seeds in mid- to late autumn and germinate in the spring after cold winter temperatures alleviate endogenous dormancy.     

Chromatin-bound DNA polymerase from higher plants

Chromatin-bound DNA polymerase has been extracted from pea (Pisum sativum L.) seedlings, and partially purified by solubilization from chromatin followed by chromatography on columns of either DEAE-cellulose or DEAE-Sephadex. The enzyme elutes from DEAE-cellulose as a single peak, but is fractionated into two peaks, CI and CII, by DEAE-Sephadex chromatography. If the enzyme is stored at-15°C for several days prior to chromatography, a third peak, CIII, derived from CII, is obtained. The polymerase is devoid of nuclease activity, and is relatively insensitive to N-ethyl-maleimide. These features, taken with the ion requirements and with data obtained from other plant species, lead to the suggestion that the chromatin-bound DNA polymerase of higher plants is similar to the DNA polymerase- from vertebrates.     

Occurrence and structure of lipoteichoic acids in the genus Staphylococcus

Lipoteichoic acids were isolated from eleven species of the genus Staphylococcus using phenol-water partition and hydrophobic chromatography on octyl-Sepharose CL-4B. The lipoteichoic acids purified could be visualized by SDS-PAGE. They were shown to be composed of a hydrophilic poly(glycerophosphate) chain covalently linked to gentiobiosyldiacylglycerol, the common lipid anchor of these molecules. Glycerophosphate units of the hydrophilic chain were found to be partly substituted with ester-linked d-alanine, except in the case of S. cohnii. The lipoteichoic acids isolated from S. cohnii, S. hominis, S. saprophyticus and S. simulans contain (1–2)-linked N-acetylglucosamine as an additional substituent of the poly(glycerophosphate) backbone.Abbreviations GLC gas-liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TLC thin-layer chromatography     

Gas chromatographic method using nitrogen–phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen–phosphorus detection (GC–NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179°C at 4°C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10 000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within ±15%.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

<Emphasis Type="Italic">Anoxybacillus rupiensis</Emphasis> sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria)

Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55–58°C, and pH at 6.0–6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270^T with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA–DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270^T, represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127^T = NBIMCC 8387^T). The 16S rRNA gene sequence data of a strain R270^T have been deposited in the EMBL databases under the accession number AJ879076.     

Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand

One hundred and eighty-seven strains of Cryptococcus neoformans isolated from patients in Thailand were charcterized by biochemical varieties relating to serogroups. Canavanine-glycine-bromothymol blue (CGB) agar was used for differentiating the varieties of C. neoformans. Slide agglutination tests were performed with Crypto Check (Iatron, Inc., Tokyo) to determine their serotypes. Fifty-five percent (10 out of 18) of the pre-AIDS isolates were serotype B, 28% were serotype A, 5% were serotype D, and an unexpected 11% (2 out of 18) were serotype C. These are the first to be recorded in Asia. In contrast, among the 169 clinical isolates obtained between January 1993 and March 1995 (AIDS epidemic), serotype A was outstandingly predominant-93% (157 out of 169), serotype B was relatively low (3.6%) and both serotypes D and AD were 1.8%. The pattern of serotypes of the 59 isolates from known HIV-positive patients was closely similar to the total isolates during the AIDS epidemic. In determining the varieties of C. neoformans by CGB, only 1 of the 187 isolates gave a false reaction. On the basis of our findings, we believe that in the pre-AIDS era either C. neoformans var. gattii serorype B or serotype C were the common causative agents of cryptococcosis in Thailand. The advent of AIDS changed the pattern of serotypes with serotype A becoming predominant as has been reported world wide.     

Effect of Temperature on the Life History of Cirrospilus sp. near lyncus (Hymenoptera: Eulophidae), a Parasitoid of Phyllocnistis citrella (Lepidoptera: Gracillariidae)

The effect of four constant temperatures on the life history of Cirrospilus sp. near lyncus was examined in the laboratory. This species is one of the most abundant generalist indigenous parasitoids of the citrus leafminer, Phyllocnistis citrella Stainton, in Spain. Adult lifespan of C. sp. near lyncus decreased from 50.2 to 9.1 days as temperatures increased from 15 to 30°C, respectively. Both gross fecundity and host-feeding were highest at 20°C (170.48 eggs and 32.33 hosts). Oviposition rates were optimal at higher temperatures (5.22 eggs per day at 25°C and 4.79 eggs per day at 30°C) and were dependent on female age. In contrast, host-feeding rates for a given temperature did not depend on age. Generation time decreased with increasing temperatures from 68.05 days at 15°C to 12.19 days at 30°C. Net reproduction peaked at 20°C (68.86 viable females per female). Intrinsic rate of increase doubled from 15°C (0.059 females per female per day) to 20°C (0.127 females per female per day) and almost doubled again from 20 to 30°C (0.210 females per female per day). Given these parameters, C. sp. near lyncus could perform optimally in the area occupied by P. citrella in the Mediterranean region.