Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes

Biodiesel is an interesting alternative energy source and is used as substitute for petroleum-based diesel. Microorganisms have been used for biodiesel production due to their significant environmental and economic benefits. However, few researches have investigated the regulation of fatty acid composition of these microbial diesels. Fatty acid biosynthesis in Escherichia coli has provided a paradigm for other bacteria and plants. By overexpressing two genes (fabA and fabB) associated with unsaturated fatty acid (UFA) synthesis in E. coli, we have engineered an efficient producer of UFAs. Saturated fatty acid (SFA) contents decreased from 50.2% (the control strain) to 34.6% (the recombinant strain overexpressing fabA and fabB simultaneously) and the ratio of cis-vaccenate (18:1Δ11), a major UFA in E. coli, reached 51.1% in this recombinant strain. When an Arabidopsis thaliana thioesterase (AtFatA) was coexpressed with these two genes, 0.19 mmol l⁻¹ fatty acids was produced by this E. coli strain after 18-h culture under shake-flask conditions. Free fatty acids made up about 37.5% of total fatty acid concentration in this final engineered strain carrying fabA, fabB, and AtFatA, and the ratio of UFA/SFA reached 2.3:1. This approach offers a means to improve the fatty acid composition of microdiesel and might pave the way for production of biodiesel equivalents using engineered microorganisms in the near future.     

Isotopic evidence for methane-based chemosynthesis in the Upper Floridan aquifer food web

Anecdotal observations of the Dougherty plain cave crayfish (Cambarus cryptodytes), the Georgia blind cave salamander (Haideotriton wallacei), and albinistic isopods (Caecidotea sp.) at great depths below the land surface and distant from river corridors suggest that obligate aquifer-dwelling (troglobitic) organisms are widely distributed throughout the limestone Upper Floridan aquifer (UFA). One mechanism by which subterranean life can proliferate in an environment void of plant productivity is through a microbial food web that includes chemosynthesis. We examined this possibility in the UFA by measuring the isotopic composition (¹³C, ¹⁴C, and ¹⁵N) of tissues from troglobitic macrofauna. Organisms that were captured by cave divers entering into spring conduits had δ¹³C values that suggested plant matter as a primary food resource (cave crayfish, –24.6 ± 2.7‰, n = 9). In contrast, δ¹³C values were significantly depleted in organisms retrieved from wells drilled into areas of the UFA remote from spring and sinkhole conduits (cave crayfish −34.7 ± 9.8‰, n = 10). Depleted ¹³C values in crayfish were correlated with radiocarbon (Δ¹⁴C) depletion relative to modern values. The results suggest that methane-based microbial chemosynthetic pathways support organisms living in the remote interior of the aquifer, at least in part.     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

Modulation of Bacillus pasteurii cytochrome c 553 reduction potential by structural and solution parameters

 Direct cyclic voltammetry and ¹H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c ₅₅₃ isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c ₅₅₃ at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell, revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275–308 K;ΔS°′=–162.7±1.2 J mol^–1 K^–1, ΔH°′=–53.0±0.5 kJ mol^–1, ΔG°′=–4.5±0.1 kJ mol^–1, E°′=+47.0±0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c ₅₅₃. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol^–1 K^–1, ΔH°′=–93.4±3.1 kJ mol^–1, ΔG°′=–5.8±0.6 kJ mol^–1, E°′=+60.3±5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-T conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Hückel theory, yields values of E ^°′ _(I=0) =–25.4±1.4 mV, z _red=–11.3, and z _ox=–10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7–10 and an increase at more acidic pH, allowing the calculation of pK _O=5.5 and pK _R=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c ₅₅₃. ¹H NMR spectra of the oxidized paramagnetic cytochrome c ₅₅₃ indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288–340 K at pH 7 and in the pH range 4.8–10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0–0.2 M causes a slight change of the ¹H NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5.2±1.1 M^–1. The pH dependence of the isotropically shifted ¹H NMR signals of the oxidized cytochrome displays at least one ionization step with pK _O=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c ₅₅₃. Received: 9 January 1998 / Accepted: 8 April 1998     

The effect of low temperature on fatty acid composition and tocopherols of the red microalga, <Emphasis Type="Italic">Porphyridium cruentum</Emphasis>

Porphyridium cruentum was grown in 10 L batch culture at 18°C, pH 8.0 and 28‰ salinity. The cells were harvested in the stationary phase and the fatty acid composition analysed by GC and tocopherol content by HPLC. A total of 14 fatty acids were identified including saturated fatty acids (13:0, 14:0, 14:0 iso, 15:0, 16:0, 16:0iso) and monounsaturated fatty acids (MUFAs; 16:1(n-7), 18:1(n-7), 18:1(n-9). Polyunsaturated fatty acids (PUFAs) were the predominant fatty acids detected, reaching 43.7% of total fatty acids in the stationary phase of culture. Among the PUFAs, eicosapentaenoic acid (EPA, 20:5(n-3)) was dominant (25.4%), followed by 12.8% arachidonic acid (AA, 20:4(n-6)). α-Tocopherol and γ-tocopherol contents were 55.2 μg g⁻¹ dry weight and 51.3 μg g⁻¹ dry weight respectively.     

Enzymatic characterisation of high-palmitic acid sunflower (Helianthus annuus L.) mutants

Two high-palmitic acid sunflower (Helianthus annuus L.) mutants, CAS-5 and CAS-12, have been biochemically characterised. The enzymatic activities found to be responsible for the mutant characteristics are β-keto-acyl-acyl carrier protein synthetase II (KASII; EC 2.3.1.41) and acyl-acyl carrier protein thioesterase (EC 3.1.2.14). Our data suggest that the high-palmitic acid phenotype observed in both mutant lines is due to the combined effect of a lower KASII activity and a higher thioesterase activity with respect to palmitoyl-acyl carrier protein (16:0-ACP). The level of the latter enzyme appeared to be insufficient to hydrolyse the produced 16:0-ACP completely. As a consequence of this, three new fatty acids appear: palmitoleic acid (16:1 Δ9), asclepic acid (18:1 Δ11), and palmitolinoleic acid (16:2 Δ9 Δ12). These fatty acids should be synthesised from palmitoyl-ACP or a derivative by the action of the stearoyl-ACP desaturase, fatty acid synthetase II and oleoyl-phosphatidylcholine desaturase, respectively. Received: 11 July 1998 / Accepted: 10 October 1998     

Abscisic acid metabolism and lipid accumulation of a cell suspension culture of <Emphasis Type="Italic">Lesquerella fendleri</Emphasis>

Lesquerella fendleri (commonly known as “Fendler’s bladderpod” or “yellowtop”) is a member of the Brassicaceae and is an important seed oil-producing plant. The lipid profile of L. fendleri seed indicates potential for producing a high quality replacement for castor oil. In this work, characterization of the lipid content of a suspension cell culture, derived from seedlings of L. fendleri, is provided. Under the described suspension cell culture conditions, 16:0, 18:1Δ9, 18:2 Δ9, Δ12 and 18:3 Δ9, Δ12, Δ15 fatty acids were found to accumulate in the cells, while 16:0, 26:0 and 28:0 fatty acids were predominant in the culture medium. Subsequently, the effect of application of abscisic acid (ABA), which modulates lipid accumulation, was assessed. Exogenously applied ABA was taken up by the cells and metabolized via the conjugation pathway, resulting in the accumulation of ABA-glucose ester. Preliminary tests demonstrate the cell line is responsive to exogenous ABA, resulting in increased cellular lipid content and increased accumulation of lipids in the culture medium. This novel L. fendleri suspension culture presents a valuable model system for efficient characterization of mechanisms associated with ABA-induced accumulation of lipids.     

<Emphasis Type="Italic">Halomonas qijiaojingensis</Emphasis> sp. nov. and <Emphasis Type="Italic">Halomonas flava</Emphasis> sp. nov., two moderately halophilic bacteria isolated from a salt lake

Two moderately halophilic, Gram-negative, rod-shaped bacteria, designated YIM 93003^T and YIM 94343^T, were isolated from a salt lake in Xinjiang province, north-west China. The two strains YIM 93003^T and YIM 94343^T grew at 20–40°C, pH 6–9, 0.5–24% (w/v) NaCl and at 20–40°C, pH 6–9, 0.5–23% (w/v) NaCl, respectively. No growth occurred in absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains YIM 93003^T and YIM 94343^T were phylogenetically affiliated to the genus Halomonas and exhibited sequence similarity of 97.5% and 97.4% to the type strain Halomonas anticariensis DSM 16096^T, respectively. The strains possessed chemotaxonomic markers that were consistent with their classification in the genus Halomonas (Q-9 as predominant respiratory quinine; C18:1ω7c, C16:0 and C16:1 ω7c/iso-C15:02-OH as the major fatty acids). The DNA–DNA hybridization values for strains YIM 93003^T and YIM 94343^T, YIM 93003^T and DSM 16096^T, YIM 94343^T and DSM 16096^T were 38.1 ± 3.0, 18.3 ± 4.7, and 20.8 ± 4.6%, respectively. The G+C contents of the strains YIM 93003^T and YIM 94343^T were 63.4 and 64.0 mol%, respectively. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA–DNA hybridization results, two novel species, Halomonas qijiaojingensis sp. nov., and Halomonas flava sp. nov., are proposed. The type strains are YIM 93003^T (=CCTCC AB 208133^T =KCTC 22228^T) and YIM 94343^T (=CCTCC AB 2010382^T =KCTC 23356^T), respectively.     

Type I blue copper proteins as enantioselective quenchers of the photoluminescence of Δ,Λ-Eu(pyridine-2,6-dicarboxylate)3 3–: azurin from Pseudomonas aeruginosa and its Met44→Lys mutant, amicyanin from Paracoccus versutus and parsley plastocyanin

 The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-dicarboxylate=dpa)₃ ^3– by the title proteins is investigated and the enantioselectivity of the proteins in the quenching of the Δ and Λ enantiomers of Eu(dpa)₃ ^3– is determined. The two diastereomeric quenching rate constants pertaining to azurin (k _q ^Δ=3.3×10⁶, k _q ^Λ=2.7×10⁶ M^–1s^–1, pH 7.2, ionic strength I=22 mM) are lower than for its Met→44Lys mutant (k _q ^Δ=1.9×10⁷, k _q ^Λ=1.4×10⁷ M^–1 s^–1, same pH and I), indicating that energy transfer occurs from Eu(dpa)₃ ^3– to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity remains unaltered by the mutation: k _q ^Δ/k _q ^Λ=1.27±0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and pH dependence of k _q indicate that the lysine residue interacts electrostatically with Eu(dpa)₃ ^3–. For plastocyanin the quenching rates are of the order of 10⁶ M^–1 s^–1; for amicyanin they are two orders of magnitude larger (k _q ^Δ=12×10⁷, k _q ^Λ=11×10⁷ M^–1 s^–1, pH 7.2, I=22 mM). The variation of k _q is attributed to differences in the charge distribution on the proteins, which influences the binding of the luminophore to the protein surface. For amicyanin the anion binding site near Lys59 and Lys60 may be involved in the energy transfer. Received: 16 June 1998 / Accepted: 18 September 1998     

Stereoselective and driving-force-dependent photoinduced electron-transfer reactions of zinc myoglobin with optically active N,N′-dimethylcinchoninium and N,N′-dimethylcinchonidinium ions

In order to understand the detailed mechanism of the stereoselective photoinduced electron-transfer (ET) reactions of zinc-substituted myoglobin (ZnMb) with optically active molecules by flash photolysis, we designed and prepared new optically active agents, such as N,N′-dimethylcinchoninium diiodide ([MCN]I₂) and N,N′-dimethylcinchonidinium diiodide ([MCD]I₂). The photoexcited triplet state of ZnMb, ³(ZnMb)*, was successfully quenched by [MCN]²⁺ and [MCD]²⁺ ions to form the radical pair of ZnMb cation (ZnMb^·+) and reduced [MCN]^·+ and [MCD]^·+, followed by a thermal back ET reaction to the ground state. The rate constants (k _q) for the ET quenching at 25 °C were obtained as k _q(MCN)=(1.9±0.1)×10⁶ M⁻¹ s⁻¹ and k _q(MCD)=(3.0±0.2)×10⁶ M⁻¹ s⁻¹, respectively. The ratio of k _q(MCD)/k _q(MCN)=1.6 indicates that the [MCD]²⁺ preferentially quenches ³(ZnMb)*. The second-order rate constants (k _b) for the thermal back ET reaction from [MCN]^·+ and [MCD]^·+ to ZnMb^·+ at 25 °C were k _b(MCN)=(0.79±0.04)×10⁸ M⁻¹ s⁻¹ and k _b(MCD)=(1.0±0.1)×10⁸ M⁻¹ s⁻¹, respectively, and the selectivity was k _q(MCD)/k _q(MCN)=1.3. Both quenching and thermal back ET reactions are controlled by the ET step. In the quenching reaction, the energy differences of ΔΔH ^≠(MCD–MCN) and ΔΔS ^≠(MCD–MCN) at 25 °C were obtained as −1.1 and 0 kJ mol⁻¹, respectively. On the other hand, ΔΔH ^≠(MCD–MCN)=11±2 kJ mol⁻¹ and TΔΔS ^≠(MCD–MCN)=−10±2 kJ mol⁻¹ were given in the thermal back ET reaction. The highest stereoselectivity of 1.7 for [MCD]^·+ found at low temperature (10 °C) was due to the ΔΔS ^≠ value obtained in the thermal back ET reaction. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

Fatty acid composition and ultrastructure of photoreceptive membranes in the crayfish Procambarus clarkii under conditions of thermal and photic stress

The ultrastructural state of the crayfish visual membrane is correlated with its fatty acid composition during times of photic and thermal stress and the period over which the dynamic events occur is investigated. Crayfish kept at 4 °C under constant darkness contain in their rhabdoms significantly increased amounts of unsaturated fatty acids such as 16:1, 18:1, 20:5, and 22:6 compared with individuals kept at 25 °C. The ratio of unsaturated/saturated fatty acids (UFA/SFA-ratio) amounts to 2.17 in the cold-water- and 1.46 in the warm water-acclimated animals. The visual membranes of crayfish suddenly transferred from 4 °C to 25 °C exhibited ultrastructural modifications such as membrane collapse and disappearance of microvillar dense␣core-filaments most clearly 3 h post-transfer. Parallel to the structural changes a significant increase in fatty acid 18:0 was observed, while the amounts of 16:1 and 20:1 decreased. When 4 °C, dark-adapted crayfish were exposed to light alone and not a temperature increase, only fatty acid 22:6 showed a significant reduction to 10% of its pre-experimental level within 2 h of exposure. Thereafter, it slowly increased again. In cold water-acclimated crayfish that had been exposed to light of 5000 lx for 3␣weeks no significant change of the UFA/SFA ratio was observed, although fatty acid species 18:0, 20:4, and 20:5 had increased at the expense of fatty acids 14:0, 16:0, 16:1, 18:1, 20:1, and 22:6. The total amount of fatty acids, however, had become significantly smaller (from 0.058 ng g⁻¹ body weight in the dark-adapted to 0.048 ng g⁻¹ in the light-adapted crayfish). Morphologically the rhabdom volume had decreased by approx. 20%, but ultrastructurally rhabdom microvilli remained almost unchanged. The amount of peroxidized lipids in the retina following irradiation with bright white light in the cold-adapted crayfish fell during the first 2 h of exposure from 0.4 nmol g⁻¹ to 0.32 nmol g⁻¹, but after 12 h of exposure had reached a level of 0.48 nmol g⁻¹. Greatest structural abnormalities to the visual membranes occurred when dark-adapted, cold-acclimated crayfish were suddenly subjected to bright light and an increase in water temperature. Under such conditions the microvillar arrangement was disrupted and membrane collapse and disappearance of core-filaments were apparent. Our results provide evidence that the fatty acid composition of the membranes determines to a considerable extent the structural integrity of the photoreceptor, but that it is too simplistic a model to think that peroxidation of membrane lipids alone is responsible for the disintegration of the photoreceptive membranes in the crayfish eye following exposure to bright light. Accepted: 4 July 1996     

Carnitine palmitoyl transferase activity and whole muscle oxidation rates vary with fatty acid substrate in avian flight muscles

Birds primarily fuel migratory flights with fat, and the composition of that fat has the potential to affect overall lipid oxidation rates. We measured the whole muscle lipid oxidation rates in extensor digitorum communis muscles from white-throated sparrows (Zonotrichia albicollis Gmelin) incubated for 20 min at 20°C with radiolabeled stearate (18:0), oleate (18:1ω9), or linoleate (18:2ω6). Lipid oxidation rates were ~40% higher with linoleate than oleate (oleate: 36 ± 8.54 μmol CO₂ g⁻¹ h⁻¹), and ~75% lower with stearate compared with oleate, indicating that maximal lipid oxidation rates can indeed be affected by the type of fatty acid supplied to the muscle. Additionally, we investigated the activity of the mitochondrial fatty acid transport-associated enzyme carnitine palmitoyl transferase (CPT) in pectoralis muscles of 5 bird species (Zonotrichia albicollis, Philomachus pugnax, Sturnus vulgaris, Taeniopygia guttata, Passer domesticus). Activity was measured in homogenized samples using various fatty acyl-CoA substrates (16:0, 16:1, 18:0, 18:1ω9, 18:2ω6, 18:3ω3, 18:3ω6, 20:0, 20:4ω6, 22:6ω3) in a spectrophotometric assay. CPT activity increased with the degree of unsaturation and decreased with chain length. CPT activity did not differ between ω3 and ω6 isomers of 18:3, nor was the pattern of CPT substrate preference different between captive white-throated sparrows in a migratory (i.e., displaying Zugunruhe) or non-migratory state. These findings can explain previously observed differences in peak performance induced by dietary fat composition and suggest that lipid supply is limiting to maximal exercise performance in birds.     

Taxonomy of Pinus species based on the seed oil fatty acid compositions

 The fatty acid compositions of the seed oils from ten pine species have been established by capillary gas-liquid chromatography of the methyl esters. With regard to either normal fatty acids or Δ5-olefinic acids, the general pattern of fatty acids did not differ from that of other pine seed oils reported previously. The main fatty acid was linoleic (9,12–18:2) acid (44.4–57.1%), followed by either oleic (9–18:1) acid (13.4–24.5%) or pinolenic (5,9,12–18:3) acid (1.5–25.2%). When applying multivariate analyses to the chemometric data (13 variables) of 49 pine species (ca. 40% of the living pine species), it was possible to distinguish between several sections: Pinea, Longifolia, Halepensis, Ponderosa-Banksiana, Sylvestris, and Cembra. The latter section was clearly divided into two sub-groups. A few species that presented a low overall content of Δ5-olefinic acids, and that grow in warm-temperate regions, were isolated from the bulk of other pine species. It is hypothesized that Δ5-olefinic acids might be related to cold-acclimation. Received: 5 June 1997 / Accepted: 17 August 1997     

Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems

In this study, four real-time polymerase chain reaction (PCR) primer sets were developed for the 16S rRNA genes of specific ammonia-oxidizing bacteria (AOB) found in activated sludge of sewage treatment systems. The primer sets target two of several sequence types of the Nitrosomonas oligotropha cluster, members within the Nitrosomonas communis cluster, and all members of the Nitrosomonas europaea–Nitrosococcus mobilis cluster. The detection limit of each primer set was in the range of 3×10¹–6×10² genes reaction⁻¹. Reliable quantification of the target AOB DNA was obtained when the target AOB DNA comprised more than 0.1% of total AOB DNA in the sample. The application of the primer sets to samples taken from five sewage treatment systems showed that, in all systems, the majority of the AOB population was comprised of one sequence type of the N. oligotropha cluster (3.9±1.5×10⁹–1.7±0.5×10¹⁰ cell l⁻¹) and, in most systems, followed by members within the N. communis cluster (2.8±0.3×10⁹–1.0±0.1×10¹⁰ cell l⁻¹) or/and another sequence type of the N. oligotropha cluster (1.5±0.6×10⁸–5.5±0.5×10⁸ cell l⁻¹). N. europaea–N. mobilis cluster arose solely in small numbers (4.9±0.8×10⁸ cell l⁻¹) in one system. Real-time PCR-amplified products obtained from genomic DNA extracted from samples were verified using clone library, and it revealed that only the target AOB DNA were PCR amplified, without amplification of the nontarget sequences.     

<Emphasis Type="Italic">Marinimicrobium haloxylanilyticum</Emphasis> sp. nov., a new moderately halophilic,polysaccharide-degrading bacterium isolated from Great Salt Lake,Utah

A new moderately halophilic, strictly aerobic, Gram-negative bacterium, strain SX15^T, was isolated from hypersaline surface sediment of the southern arm of Great Salt Lake (Utah, USA). The strain grew on a number of carbohydrates and carbohydrate polymers such as xylan, starch, carboxymethyl cellulose and galactomannan. The strain grew at salinities ranging from 2 to 22% NaCl (w/v). Optimal growth occurred in the presence of 7–11% NaCl (w/v) at a temperature of 35°C and a pH of 6.7–8.2. Major whole-cell fatty acids were C16:0 (30.5%), C18:0 (14.8%), C18:1ω7c (13.1%) and C12:0 (7.8%). The G+C content of the DNA was 60 ± 0.5 mol%. By 16S rRNA gene sequence analysis, strain SX15^T was shown to be affiliated to members of the gammaproteobacterial genus Marinimicrobium with pair wise identity values of 92.9–94.6%. The pheno- and genotypic properties suggest that strain SX15^T represents a novel species of the genus Marinimicrobium for which the name Marinimicrobium haloxylanilyticum is proposed. The type strain is SX15^T (= DSM 23100^T = CCUG 59572^T).     

Lipid content and fatty acid consumption in zoospores/developing gametophytes of Saccharina latissima (Laminariales, Phaeophyceae) as potential precursors for secondary metabolites as phlorotannins

Phlorotannins are considered inter alia to act protective to a variety of stressors, while lipids in spores are known to fuel various metabolic processes during spore release and settlement. Here, phlorotannin production in zoospores/juvenile gametophytes in relation to lipid metabolism was investigated for the first time in order to study-related metabolic costs. The experiment was carried out in Ny-?lesund (Svalbard, Arctic) within the development from spores to juvenile gametophytes of the brown alga Saccharina latissima over 20 days. In the release stage, the total phlorotannin content of single zoospores was 1.5 × 10⁻⁷ μg and 1.9 × 10⁻⁷ μg in the surrounding medium. Upon release, the total amount of lipids was 1.76 × 10⁻⁵ μg lipid zoospore⁻¹ containing the major fatty acids 16:0 and 18:0, 18:1(n-9), 18:2 (n-6), 18:3(n-3), 20:4(n-6), and 20:5(n-3). During development from spores to gametophytes, a decrease in fatty acids was observed via electron microscopy and a decrease in the fatty acid 18:1(n-9) from 45 to 30% was measured by gas chromatography in particular. While phlorotannin content within the spores remained stable, phlorotannins accumulated in the surrounding medium of gametophytes to 4.0 × 10⁻⁷ μg phlorotannins spore⁻¹ indicating exudation processes. Results obtained support the key and multifunctional role of lipids in zoospore/gametophyte metabolism and may indicate that gametophytes of S. latissima need approximately 10 days to switch to photo-autotrophic strategies, becoming independent of storage lipids. In addition, fatty acids might represent an essential energy source to fuel adaptive responses.     

Lipopolysaccharide ofRhodospirillum salinarum 40: structural studies on the core and lipid A region

The structural elucidation of lipid A of the cell wall lipopolysaccharide (LPS) ofRhodospirillum salinarum 40 by chemical methods and laser desorption mass spectrometry revealed the presence of a mixed lipid A composed of three different 1,4 bisphosphorylated β(1→6)-linked backbone hexosaminyl-hexosamine disaccharides, i.e. those composed of GlCN→GlcN, 2,3-diamino-2,3-dideoxy-d-Glc-(DAG)→DAG, and DAG→GlcN. Lipid A ofR. salinarum contained preferentially 3-OH-18:0 and 3-OH-14:0 as amide-linked andcisΔ¹¹-18:1 and c19:0 as ester-linked fatty acids. The mass spectra of the liberated acyl-oxyacyl residues proved the concomitant presence of 3-O-(cisΔ¹¹-18:1)-18:0 and 3-O-(c19:0)-14:0 as the predominating diesters in this mixed lipid A. The glycosidically linked and the ester-linked phosphate groups of the backbone disaccharide were neither substituted by ethanolamine phosphorylethanolamine, nor by 4-amino-4-deoxy-l-arabinose, in contrast to most of the enterobacterial lipid As. In the core oligosaccharide fraction, a HexA (1→4)HexA(1→5)Kdo-trisaccharide was identified by methylation analysis. The terminal HexA (hexuronic acid) is possibly 4-OMe-GalA, a component described here as an LPS constituent for the first time. LPS ofR. salinarum showed a lethality in C57BL/10 ScSN (LPS-responder)-mice) of an order of 10⁻¹–10⁻² of that reported forSalmonella abortus equi LPS, and it was also capable of inducing TNFα and IL6 in macrophages of C57BL/10ScSN mice.     

<Emphasis Type="Italic">Luteibacter jiangsuensis</Emphasis> sp. nov.: A Methamidophos-Degrading Bacterium Isolated from a Methamidophos-Manufacturing Factory

A Gram-stain-negative, non-motile, rod-shaped bacterial strain, JW-64-1^T, capable of degrading methamidophos was isolated from a methamidophos-manufacturing factory in China, and was subjected to a polyphasic taxonomic investigation. Strain JW-64-1^T produced circular, smooth, transparent, yellow-colored colonies (1.0–2.0 mm) on LB agar after 2 days incubation. It grew optimally at 25–30°C and pH 7.0 without the presence of NaCl. The G+C content of the total DNA was 63.6 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain JW-64-1^T fell within the cluster comprising Luteibacter species. The 16S rRNA gene sequence of strain JW-64-1^T was most closely related to Luteibacter rhizovicinus DSM 16549^T (98.6%), followed by Luteibacter yeojuensis DSM 17673^T (98.4%) and L. anthropi CCUG 25036^T (98.2%). The major cellular fatty acids of strain JW-64-1^T were iso-C_15:0 (24.1%), iso-C_17:0 (20.2%) and summed feature 9 comprising iso-C_17:1 ω9c and/or C_16:0 10-methyl (20.3%). The major isoprenoid quinine was Q-8 (98%), and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphoaminolipid, aminolipids-1, aminolipids-2, and phospholipids. The values for DNA–DNA relatedness between strain JW-64-1^T and the closest phylogenetic relatives of L. rhizovicinus and Luteibacter yeojuensis were 34.8 ± 2.6 and 25.6 ± 3.1%, respectively. On the basis of the phenotypic, chemotaxonomic, DNA–DNA relatedness and phylogenetic analysis based on the 16S rRNA gene sequences, strain JW-64-1^T represents a novel species of the genus Luteibacter, for which the name Luteibacter jiangsuensis sp. nov. is proposed. The type strain is JW-64-1^T (=CGMCC 1.10133^T = DSM 22396^T).