Serological Specificity of the Lipopolysaccharides, the Major Antigens of Pseudomonas syringae

The nature of major antigens of Pseudomonas syringae was studied on one strain of four pathovars (pvs aptata, mors-prunorum, phaseolicola and tabaci) belonging to four separate serogroups. Bacterial antigens were prepared by 4 procedures: extraction by phenol-water (PW), by citrate-NaCl (CN), by trichloracetic acid (TCA), and precipitation of a glycoproteic extracellular complex (GP). 3-Deoxy-2-octulosonic acid (KDO) revelation in all the extracts showed that the four procedures led to antigens containing similar amounts of lipopolysaccharide (LPS). Twenty polyclonal antisera were raised in rabbits against whole bacteria and the different extracts. Serological reactions were tested by gel double diffusion (DD) and indirect immunofluorescent staining (IF). The anti-whole cell sera were shown to contain mostly anti-LPS antibodies. For each pathovar, whole bacteria used as antigens in DD gave precipitation bands identical to the bands given by the LPS extracts (PW, CN or TCA), identical to the heated bacteria (HB), and identical to LPS sidechain preparations. The GP extract itself was shown to be rich in LPS. To serotype P. syringae, it is advised to raise antisera against either whole bacteria or GP extracts; whereas the reacting antigens for DD would be heated bacteria.     

Degradation of bacterial lipopolysaccharide by the slime mould Physarum polycephalum.

A strain of the acellular slime mould Physarum polycephalum degraded lipopolysaccharides (LPS) from a variety of bacteria. The anticomplementary (AC) activity of LPS was greatly reduced, as was the content of lauric, myristic, and palmitic acids, and the ability to sensitize erythrocytes to agglutination by antibody. These results indicate that Physarum has enzymes which reduce the lipid A moiety of LPS. In contrast, 2-keto-3-deoxy-D-manno-actanoic acid (KDO), immunodominant sugars, and beta-hydroxymyristic acid were scarcely affected. Both supernates and plasmodial extracts of Physarum had LPS-degradative activity and were able to attack both purified LPS and LPS in killed bacteria.     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

The effect of tubificid oligochaetes on the uptake of zinc by Lake Erie sediments

A laboratory experiment was conducted to determine the effect of tubificid worms on the flux of zinc into lake sediments. Forty-six cores of Lake Erie sediment, with and without (control) tubificid worm populations, were exposed to aquarium water with a zinc concentration of about 5 mg 1^–1 for 139 days. Pore water and exchangeable particulate zinc concentrations in the top 12 cm of sediment were periodically determined in pairs of cores — one with worms and one without worms — at 1 cm depth increments. After 139 days, pore water zinc concentrations in sediments with and without worms were nearly identical in the 0–1 cm interval (4.1 and 4.3 mg 1^–1 respectively), but were significantly greater in the sediments with worms in the 1–2 cm (4.4 vs. 0.3 mg^1–1) and the 2–3 cm (1.3 vs. 0.3 mg 1^–1) intervals. Exchangeable particulate zinc concentrations in the 0–1, 1–2, and 2–3 cm intervals in sediments with worms were 612.3, 750.7, and 191.5 µg g^–1 dry sediment respectively, whereas in sediments without worms, concentrations were 375.4, 5.9, and 3.2 µg g^–1 dry sediment. The increased flux of zinc into tubificid-inhabited sediments was caused by the conveyor belt feeding activity of the worms, which continuously exposed sedimentary particles to the overlying water. Movement of zinc into sediments with worms was dominated by adsorption and by particle movement, whereas movement of zinc into control sediments was by adsorption at the sediment-water interface and diffusion. The increased concentration of zinc in tubificid-inhabited sediments has important implications with respect to the trophic transfer of zinc through the aquatic food chain.     

Diatom bloom in the tidal freshwater zone of a turbid and shallow estuary,Rupert Bay (James Bay,Canada)

Phytoplankton biomass and species composition were studied from June to September 1991 at the mouth of four major rivers and in the freshwater (sal. 0 %), the estuarine (sal. 2–10%) and the coastal (sal. 10–12%) zones of Rupert Bay, located at the southeast tip of James Bay, Canada.A chlorophyll a maximum (5–14 µg 1^–1) was observed in the freshwater zone from July to September. Chlorophyll values were low at the mouth of the rivers and in the estuarine and coastal zones (chl a < 1.00 µg 1^–1). Diatoms were dominant in the freshwater zone (30–80 % abundance), with flagellates dominating in the estuarine and coastal zones (60–95% abundance). Diversity was low (H: 1.5–2.5) in the freshwater zone and decreased seaward (H: 0.5–1.5).The diatom bloom was composed almost exclusively of the autochthonous planktonic diatom Cyclotella meneghiniana Kütz., which contributed 25–85% of the species composition, and of the subdominant benthic species Diploneis smithii, Navicula lanceolata and Surirella robusta. Peak abundance occurred upstream of the turbidity maximum, in the tidal freshwater zone. In this zone the mean photic depth was 1 m and residence time was from 7 to 8 days during the bloom. Residence time is considered to be the dominant factor controlling the phytoplankton bloom, with light not acting as a limiting factor. The high turbidity due to resuspension and shallow depth of the bay controlled the species composition.     

Pyrite accumulation in salt marshes in the Eastern Scheldt,southwest Netherlands

Pore water composition, pyrite distribution and pyrite crystal morphology of sediments from salt marshes in the Eastern Scheldt, southwestern Netherlands, were examined from July 1984 to October 1986.Hydrology and marsh vegetation were the chief determinants of pyrite accumulation. In the bare sediments of pans in the low marsh, highly reducing conditions prevailed just below the surface. At these sites, practically all the incoming detrital pyrite (0.5–1% FeS₂) was preserved. The in-situ formation of pyrites was negligible in these anoxic sediments.All incoming detrital pyrite was oxidized in the surface layers (0–10 cm) of the medium-high marsh overgrown withSpartina anglica. Pyrite was formed at a rate of 2.6–3.8 mol S-FeS₂m^–2yr^–1 in a narrow range of depths (15–20cm), at the interface of the oxidizing and underlying reducing sediment. At this interface the concentration profiles of Fe²⁺ and dissolved S intersected. The role of the rhizosphere is discussed in connection with pyrite formation. No further pyrite formation occurred deeper in the sediment. This resulted in the build up of high concentrations of dissolved S and acid volatile sulfides (AVS). The decrease with depth in oxalate-extractable Fe indicated that most of the iron oxyhydroxides (70–80%) had been transformed to pyrite. Another 10–20% of oxalate-extractable Fe was present as AVS. The abundance of framboidal pyrite particles and the high concentrations of AVS and dissolved S indicated that the formation of pyrite occurred via iron monosulfide intermediatesThere was a linear relationship between the organic carbon and the S-FeS₂ content in theSpartina overgrown reducing sediment. The mean C/S ratio was 4.2.     

Is bacterioplankton production in the Ria de Aveiro influenced by salt marshes and bed sediment?

The contribution of potential export of materials from bottom sedimentsand salt marshes into the water column of a shallow estuarine system of Ria deAveiro to the observed high bacterial productivity in the mid section of thisestuary was evaluated. Vertical profiles of physical, chemical and bacterialvariables were studied in the marine and brackish water zones, and oftransversal profiles in the brackish zone only. Although the concentrations ofseston (17–241 mg l^–1), particulate organiccarbon (3–15.5 mg l^–1) and chlorophyll(1.2–7.0 g l^–1) varied widely, thevertical and transversal profiles were without much variation. Total bacterialnumber (0.2–8.5 × 10⁹ cellsl^–1) and the number of particle-attached bacteria(0.02–2.50 × 10⁹ cellsl^–1)along vertical and transversal profiles did not differ much, but the rate ofbacterial production (0.05–14.2 g C l^–1h^–1) and dissolved organic carbon concentration(6.0–69.2 mg l^–1) were frequently highernear the salt marsh margin at the brackish water transect. The increase inproductivity could not be associated with runoff of particulate matter butcoincided with the inputs of dissolved organic carbon. The results of verticaland transversal profiles point to a minor role of particulate matter additionsfrom the salt marsh area or from bed sediments.     

Distribution of zinc-tolerant bacteria in stream sediments

The distribution of zinc-tolerant bacteria in sediments from three stream sites containing high (3125 g g^–1), medium (291 g g^–1), and low (109 g g^–1) concentrations of Zn was determined. Zinc tolerance was estimated by the ability of bacteria to grow on media amended with Zn concentrations ranging from 4 to 512 mg 1^–1. The presence of Zn-tolerant bacteria was correlated with the degree of heavy metal contamination; this correlation was more closely associated with readily extractable heavy metal concentrations than with the more rigorously extracted heavy metals. Low concentrations of Zn in media (4 to 16 mg 1^–1) were stimulatory to growth of bacteria from contaminated sites while concentrations as low as 4 mg 1^–1 were inhibitory to bacteria from the control site.     

The effects of sediment slurrying on microbial processes,and the role of amino acids as substrates for sulfate reduction in anoxic marine sediments

In sediment slurry experiments with anoxic marine sediments collected in Cape Lookout Bight, NC, and a site in mid-Chesapeake Bay, the rates of sulfate reduction and ammonium production decrease with increasing dilution of sediment with oxygen-free sea-water. The effect of sediment dilution on the rates of these processes can be described by a simple mathematical relationship, and when these rates are corrected for sediment dilution they yield values which agree well with direct measurements of these processes.In sediment slurry studies of amino acid utilization in Cape Lookout Bight sediments, the fermentative decarboxylation of glutamic acid (to -aminobutyric acid) or aspartic acid (to alanine or -alanine) did not occur when either of these amino acids were added to Cape Lookout Bight slurries. The addition of glutamic acid did however lead to a small (1) transient build-up of -aminoglutaric acid. Measured rates of glutamic acid uptake in these slurries also decreased with increasing sediment dilution.Molybdate inhibition experiments demonstrated that dissolved free amino acids represent 1–3% of the carbon sources/electron donors used for sulfate reduction in Cape Lookout Bight sediments. The direct oxidation of amino acids by sulfate reducing bacteria also accounts for 13–20% of the total ammonium produced. Glutamic acid, alanine, -aminoglutaric acid, aspartic acid and asparagine are the major amino acids oxidized by sulfate reducing bacteria in Cape Lookout Bight sediments.     

Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-octulosonate for synthesis of lipopolysaccharide.

Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42 degrees C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grow at 42 degrees C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10(-6), required exogenous KDO for growth at 42 degrees C. The growth rate at 42 degrees C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent Km for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of KDO per min per 10(8) cells. Bacteria incorporated exogenous [3H]KDO exclusively into LPS, with less than 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (3H, 13C, and 14C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.     

Phosphate in the sediments of the Gulf of Lions (NW Mediterranean Sea), relationship with input by the river Rhone

We investigated P-input by the Rhone river into the Mediterranean Sea taking into account P trapped in the surface sediment of the Gulf of Lions. Total phosphate concentration was determined every cm in the upper 10 cm-layer of sediments sampled at 11 stations in the Gulf of Lions during two cruises (March 1998 and January 1999). Two low downward gradients, one East–West and another North–South, with distance to the Rhone river mouth were found. Except at one station, total phosphate concentration in surface sediments was found to be constant with depth down to 10 cm. Values for individual stations ranged between 400 and 700 g g^–1 with an average value of 547 g g^–1 (st. dev. = 63 g g^–1) for the whole gulf. The low variability in total-P concentration in sediments is in contrast to the large variability in suspended matter load of the river Rhone and suggests the dominance of authigenic P removal mechanisms in P burial. The total P-pool in the upper 10 cm-layer of the sediments in the gulf was estimated at 562 kt, with about 80% trapped into the shelf and 20% into the slope. Annual P-deposition was estimated as 7.2–12.4 kt y^–1, from the P-pool in the sediment and the sedimentation rates. This is equivalent to a previous estimation of the river Rhone input, estimated to be about 6.5–12.2 kt y^–1. As the Rhone is the major river flowing into the Mediterranean Sea, total P in surface sediments of the Gulf of Lions should be taken into account in P-budgets at the scale of the Mediterranean Sea.     

A method for the analysis of acetate turnover in a coastal marine sediment

The concentrations of volatile fatty acids were measured in the pore water of sediment from the Limfjorden, Denmark. The pore water was freeze-dried and the acids, which were redissolved in formic acid, were analyzed by gas chromatography on a Carbopack column. The limit of detection was 0.1 mol l^–1 pore water. The concentration ranges (mol l^–1 pore water) were as follows: 0.1 to 6.0 for acetate; <0.1 to 0.6 for propionate, and <0.1 to 0.5 for butyrate. The rate constants for the disappearance of injected tracer concentrations of U-¹⁴C-acetate were measured at 2 cm depth intervals in sediment strata (0 to 10 cm). The rate constant for acetate turnover at 4 to 6 cm depth did not vary greatly with season, 2.1 h^–1, SD 0.6 for 7 values. In spring, the rate constants were highest in the 0 to 2 cm stratum and decreased with sediment depth. The calculated rates for acetate turnover of 7.2 mmol m^–2 day^–1 for early spring (2°C) and of 19.6 mmol m^–2 day^–1 for late autumn (7°C) were higher than would be expected from published values for carbon oxidation by sulfate in these sediments.     

Peptides as requirement for immunotherapy of the guinea-pig line-10 tumor with endotoxins

Summary The transplantable line-10 hepatocellular carcinoma of guinea-pigs has been used as a model for the study of immunotherapy of malignant tumors. Cure rates of up to 100% have been obtained with ReGl-CM from 0 antigen-deficient (Re) mutant strains of Enterobacteriaceae, provided they were combined with mycobacterial trehalose dimycolate (cord factor, P3). Whereas highly endotoxic LPS extracts from all wild-type strains so far tested have failed to cause tumor regression, acid hydrolysis of such LPS samples led to residual fractions (RESI) that cross-reacted serologically with ReGl-CM samples (Chang and Nowotny, 1975) and provided cure rates up to 100%. RESI from Serratia marcescens was essentially nonpyrogenic and 100 times less lethal for chick embryos than potent endotoxins. Antigenic material associated with endotoxic extracts appears to be cryptic or sterically hindered from being effective in wild-type LPS but is exposed in ReGl and RESI samples.Reduction of the aminoacid content of ReGl-CM by microparticulate silica gel chromatography or by treatment with Triton X-100 significantly lowered the ability to bring about tumor regression without affecting endotoxicity. Antitumor activity could be restored by the addition of synthetic N-acetyl-muramyl-l-seryl-d-isoglutamine (MDP) or a nontoxic lipoid side fraction recovered during the isolation of ReGl-CM, which contained a small amount of peptidic substances. It is concluded that the addition of peptidic material, which may act as an adjuvant, to endotoxins is required to make them useful for immunotherapy of the weakly immunogenic line-10 tumor.Chemical procedures known to detoxify endotoxins while retaining adjuvanticity, such as succinylation and phthalylation, resulted in complete loss of endotoxicity and tumor-regressive potency of ReGl-CM. Transesterification with sodium methoxide led to a water-soluble phase, which cured 50% of tumor-bearing animals even though lethality and pyrogenicity were reduced by 100 times and 50 times, respectively. Thus there was no direct correlation between endotoxic potency and tumor-regressive activity. In addition, our findings indicate that a low level of toxicity may be required to obtain optimal levels of tumoricidal action.Abbreviations P3 trehalose dimycolate isolated by microparticulate silica gel chromatography (Ribi et al., 1974) - LPS lipopolysaccharide from wild-type gram-negative bacteria - ReGl endotoxic glycolipids from Re mutant gram-negative bacteria - CM chloroformmethanol - PW phenol-water - PCP phenol-chloroform-petroleum ether - ReGl-CM ReGl-PW, ReGl-PCP refer to ReGl extracted with CM, PW, or PCP, respectively - ACP acetone-precipitated by-product of ReGl-CM - B1, B2, B4 chromatographic fractions of ReGl-CM - lipid A hydrochloric acid hydrolyzate of LPS or ReGl - RESI organic solvent-insoluble fraction of lipid A (Chang and Nowotny, 1975) - KDO keto-3-deoxyoctonate - BSA bovine serum albumin - CWS cell wall skeleton of BCG (Bacillus Calmette-Guérin) - PPD purified protein derivative (tuberculoprotein) - TAP tuberculin-active peptide - MDP N-acetyl-muramyl-l-seryl-d-isoglutamine     

Cloning,expression, and biochemical characterization of 3-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-<Emphasis Type="Italic">manno</Emphasis>-2-octulosonate-8-phosphate (KDO8P) synthase from the hyperthermophilic bacterium<Emphasis Type="Italic"> Aquifex pyrophilus</Emphasis>

3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase, catalyzes the aldol-type condensation between phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P) to produce the unusual 8-carbon sugar KDO8P, and inorganic phosphate. A 15.5-kb segment containing the kdsA gene from the hyperthermophilic bacterium Aquifex pyrophilus was cloned from a genomic library and sequenced. The native kdsA gene lacks a typical ribosome binding site, but contains a conserved U,A-rich sequence upstream to the start codon. The purified kdsA gene product catalyzes the formation of KDO8P from its natural substrates, PEP and A5P, as determined by ¹H NMR analysis. KDO8P synthase showed maximum activity at 80 °C and pH 5.5–6.0 at 10-min reaction assay. At temperatures of 70, 80, and 90 °C, the enzyme exhibited half-lives of 8.0, 2.25, and 0.5 h, respectively. The kinetic constants at 60 °C were K_m^A5P=70 M, K_m^PEP=290 M, and k_cat=4 s^–1. The isolated enzyme contained 0.19 and 0.26 mol iron and zinc, respectively, per mole of enzyme subunit. Treatment with metal chelators eliminated enzyme activity, and by the addition of several divalent metal ions, the activity was restored and even exceeded the original activity. These results indicate that A. pyrophilus KDO8P synthase is a metal-dependent enzyme. A C11A mutant of KDO8P synthase from A. pyrophulis retained less than 1% of the wild-type activity and was shown to be incapable of metal binding.Communicated by G. Antranikian     

Integrated cultivation of salmonids and seaweeds in open systems

Bacterial abundance and production in a vertical profile in Lake Kariba (17dgS), Zimbabwe, were affected by solar irradiance. At the surface, 1.87 × 10⁹ bacteria 1^–1 were found and abundance peaked at 10 m (2.5 × 10⁹ bacteria l^-1), then decreasing with depth. Bacterial reproduction at the surface(0.145 µg C1^–1 h^–1) was nearly four times less than the production at 10 m although bacterial numbers were only 26% less. Thus, bacterial production per cell was lower at the surface than deeper down, suggesting that bacterial production is inhibited at the surface.Bacterial production in GF/F filtered lake water in Whirl Pack bags showed an exponential decrease down to 3 m depth. The inhibition was well in accordance with light extinction in the UV region. Phosphatase activity was low in light exposed bags compared to dark, indicating photolysis of extracellular enzymes, or phototransformation of recalcitrant DOM, which substitutes enzyme activity. Hypolimnetic enzyme activity was less affected by solar light than epilimnetic.     

Isolation of lipopolysaccharides and the effect of Polymyxin B on the outer membrane of Corynebacterium autotrophicum

Lipopolysaccharides (LPS) from Corynebacterium autotrophicum were isolated and analyzed. Autotrophically grown cells contained 2–5 mg of partly purified LPS per g dry weight of lyophilized cells. Serological cross reaction with Lipid A antigen of Salmonella minnesota confirmed the presence of LPS in C. autotrophicum. Electron microscopy of negatively stained Polymyxin B-treated cells showed formation of blebs on the Outer Membrane indicating an interaction of Polymyxin B specifically with LPS. Up to now, no Gram-positive organisms are known which contain any LPS. Thus, C. autotrophicum, though giving opposite results when the Gram-staining reaction was applied by several authors, has to be classified into the group of Gram-negative bacteria.Non-Common Abbreviations LPS lipopolysaccharide - KDO 2-keto-3-deoxyoctonate     

Distribution and speciation of phosphorus along a salinity gradient in intertidal marsh sediments

We examined forms of solid phosphorus fractions in intertidal marsh sediments along a salinity (0–22%.) gradient in a river-dominated estuary and in a marine-dominated salt marsh with insignificant freshwater input. Freshwater marsh sediments had the highest ratio of organic N:P of between 28:1 and 47:1 mol:mol, compared to 211 to 311 molmol in the saltmarshes, which is consistent with a trend toward P-limitation of primary production in freshwater and N-limitation in salt marshes. However, total P concentration, 24.7±11.1mol P g dw^–1 (±1 SD) averaged over the upper meter of sediment, was greatest in the freshwater marsh where bioavailablity of P is apparently limited. In the freshwater marsh the greatest fraction of total P (24–51%.) was associated with humic acids, while the importance of humic-P decreased with increasing salinity to 1–23%. in the salt marshes. Inorganic P contributed considerably less to total sediment P in the freshwater marsh (15–40%.) than in the salt marshes (33–85%.). In reduced sediments at all sites, phosphate bound to aluminum oxides and clays was an important inorganic P pool irrespective of salinity. Inorganic P associated with ferric iron [Fe(III)] phases was most abundant in surface sediments of freshwater and brackish marshes, while Ca-bound P dominated inorganic P pools in the salt marshes. Thus, our results showed that particle-bound P in marsh sediments exhibited changes in chemical association along the salinity gradient of an estuarine system, which is a likely consequence of changes in ionic strength and the availability of iron and calcium.     

Metabolic network analysis on Phaffia rhodozyma yeast using C–labeled glucose and gas chromatography-mass spectrometry

Carotenoid production by microorganisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for ‘all natural’ products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metabolism, labeling experiments with [1-¹³C]glucose were conducted with the wildtype strain (CBS5905 T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes.Amino acid labeling patterns, as determined by GC–MS, were in accordance with a metabolic network consisting of the Embden–Meyerhof–Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway (65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower.     

Mitochondrial enzymes in aerobically and anaerobically germinated seedlings of Echinochloa and rice

Activities of tricarboxylic acid (TCA) cycle enzymes in seedlings of barnyard grass (Echinochloa phyllopogon (Stapf.) Koss) and rice (Oryza sativa L.) germinated under aerobic and anaerobic conditions were investigated. In E. phyllopogon, development of TCA-cycle enzyme activities during 10 d of anoxia generally paralleled those in air, although at lower rates. After 5 d, E. phyllopogon seedlings germinating under N₂ exhibited 50–80% of the activity of seedlings grown in air, except for 2-oxoglutarate dehydrogenase (EC 1.2.4.2) and fumarate reductase (EC 1.3.1.6) which exhibited only 25–35% of aerobic activity. In anaerobically germinated rice, development of TCA-cycle enzyme activities also paralleled those in air except for aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.41), and 2-oxoglutarate dehydrogenase. Those enzymes did not increase in activity under anoxia. Development of maximum enzyme activities generally occurred more rapidly and persisted longer in E. phyllopogon compared to rice. The data indicate that mitochondria of E. phyllopogon function better during anaerobiosis than those of rice and this factor may contribute to the successful biochemical strategy of this weed in rice paddies throughout the world.Abbreviation TCA tricarboxylic acid This work was supported by U.S. Department of Agriculture Competitive Research grant No. 87-CRCR1-2595 and a Herman Frasch Foundation grant in Agricultural Chemistry to R.A.K.     

A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gram-negative bacteria

A procedure is described to determine 2-keto-3-deoxyoctonate (KDO) present in lipopolysaccharide (LPS) of gram-negative bacteria. The method involves the treatment of LPS with 0.2 n H₂SO₄ at 100°C for 30 min to release KDO, followed by its reaction with periodic acid, sodium arsenite, and thiobarbituric acid. The red chromophore thus formed is kept in solution at room temperature by adding dimethylsulfoxide to the reaction mixture. The final color is stable for days at room temperature and facilitates accurate determination of KDO in microgram quantities. KDO contents of cell surface antigens and glycolipids from gram-negative bacteria are presented as illustrations of the accuracy and sensitivity of the assay.