Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: Redirecting carbon flow

Abstract Threonine and lysine are two of the economically most important essential amino acids. They are produced industrially by species of the genera Corynebacterium and Brevibacterium . The branched biosynthetic pathway of these amino acids in corynebacteria is unusual in gene organization and in the control of key enzymatic steps with respect to other microorganisms. This article reviews the molecular control mechanisms of the biosynthetic pathways leading to threonine and lysine in corynebacteria, and their implications in the production of these amino acids. Carbon flux can be redirected at branch points by gene disruption of the competing pathways for lysine or threonine. Removal of bottlenecks has been achieved by amplification of genes which encode feedback resistant aspartokinase and homoserine dehydrogenase (obtained by in vitro directed mutagenesis).     

Sap flow in wheat under free-air CO2 enrichment

The effects of elevated carbon dioxide (CO₂) concentration on plant water use are best evaluated on plants grown under field conditions and with measurement techniques that do not disturb the natural function of the plant. Heat balance sap flow gauges were used on individual main stems of wheat (Triticum aestivum L. cv Yecora rojo) grown under normal ambient conditions (control) and in a free-air CO₂ enrichment (FACE) system in Arizona with either high (control + high H₂O = CW; FACE + high H₂O = FW) or low (control + low H₂O = CD; FACE + low H₂O = FD) irrigation regimens. Over a 30d period (stem elongation to anthesis), combinations of treatments were monitored with,10–40 gauges per treatment. The effects of increased CO₂ on tiller water use were inconsistent in both the diurnal patterns of sap flow and the statistical analyses of daily sap flow (F_tot). Initial results suggested that the reductions in F_tot, from CO₂ enrichment were small (,0–10%) in relation to the H₂O treatment effect (,20–30%). For a 3d period, F_tot of FW was,19–26% less than that of CW (P = 0.10). Examination of the different sources of variation in the study revealed that the location of gauges within the experimental plots influenced the variance of the sap flow measurements. This variation was probably related to positional variation in subsurface drip lines used to irrigate plots. A sampling design was proposed for use of sap flow gauges in FACE systems with subsurface irrigation that takes into account the main treatment effects of CO₂ enrichment and the other sources of variation identified in this study. Despite the small and often statistically non-significant differences in F_tot between the CW and FW treatments, cumulative water use of the FW treatment at the end of the first three test periods ranged from 7 to 23% lower than that of the CW treatment. Differences in sap flow between FW and CW compared well with treatment differences in evapotranspiration. The results of the study, based on the first reported sap flow measurements of wheat, suggest that irrigation requirements for wheat production, in the present climatic regimen of the south-western US, may be predicted to decrease slightly because of increasing atmospheric CO₂.     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Association of specific leaf weight, an estimate of chlorophyll, and chlorophyll concentration with apparent photosynthesis in soybean

Increasing specific leaf weight (SLW) may improve leaf apparent photosynthesis (AP) in soybean [Glycine max (L.) Merr.] but screening for SLW and AP is laborious. The Objectives of this study were (i) to determine the time course of SLW and chlorophyll concentration in experimental lines selected for differences in SLW and (ii) to evaluate the potential use of the Minolta 502 SPAD meter as a rapid estimator of SLW, AP and chlorophyll concentration in leaves of soybean. In 1991 and 1992, sixteen experimental lines representing extremes in SLW were grown at Urbana, IL, and West Lafayette, IN, with three replications at each location. SPAD values, SLW and AP were measured at the R2 (full flower), R4 (full pod) and R5 (beginning seed) growth stages. In 1992 SLW, SPAD values and chlorophyll concentration were measured weekly. Seasonal patterns of SPAD values, SLW, and chlorophyll concentration were very similar through R5. After R5, SLW continued to increase but SPAD values and chlorophyll concentration declined. SPAD values and SLW were highly correlated at the R2, R4 and R5 stages at both locations and in both years. Environmental conditions during this research were not suitable for maximum AP expression, which is likely why AP and SPAD values were correlated only at the R4 growth stage at Urbana in 1992. SPAD measurements were consistent across diverse environments and effectively separated the high SLW lines from the low SLW lines. Measuring with the Minolta 502 SPAD meter is rapid, simple and non-destructive and could be an alternative method for direct selection for SLW.Abbreviations AP- leaf apparent photosynthesis - CV- coefficient of variation - Rug- leaf rugosity - SLW- specific leaf weight - SPAD-L- SPAD value of the most recently expanded terminal leaflet - SPAD-P- SPAD value of leaflet used to measure AP - SPAD-S- SPAD value of leaflets used to measure SLW - SPAD-U- SPAD value of the terminal leaflet one node above the most recently expanded terminal leaflet The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged     

Modelling crop growth and biomass partitioning to shoots and roots in relation to nitrogen and water availability,using a maximization principle

Many crop models relate the allocation of dry matter between shoots and roots exclusively to the crop development stage. Such models may not take into account the effects of changes in environment on allocation, unless the allocation parameters are altered. In this paper a crop model with a dynamic allocation parameter for dry matter between shoots and roots is described. The basis of the model is that a plant allocates dry matter such that its growth is maximized. Consequently, the demand and supply of carbon, nitrogen, and water is maintained in balance. This model supports the hypothesis that a functional equilibrium exists between shoots and roots.This paper explains the mathematical computation procedure of the crop model. Moreover, an analysis was made of the ability of a crop model to simulate plant dry matter production and allocation of dry matter between plant organs. The model was tested using data from a greenhouse experiment in which spring wheat (Triticum aestivum L.) was grown under different soil moisture and nitrogen (N) levels.Generally, the model simulations agreed well with data recorded for total plant dry matter. For validation data the coefficient of determination (r²) between simulated and measured shoot dry weight was 0.96. For the validation treatments r² was slightly lower, 0.94. In addition to dry matter production the model succeeded satisfactorily in simulating the dry weight of different plant organs. The response of simulated root to shoot ratio to the level of soil moisture was mainly in accordance with the measured data. In contrast, the simulated ratio seemed to be insensitive to the changes in the levels soil N concentration used in the experiment.The data used in the present study were not extensive, and more data are needed to validate the model. However, the results showed that the model responses to the changes in soil N and water level were realistic and mostly agreed with the data. Thus, we suggest that the model and the method employed to allocate dry matter between roots and shoots are useful when modelling the growth of crops under N and water limited conditions.     

Three-year measurement of methane emission from an Indonesian paddy field

Yearly and seasonal (rainy and dry seasons) variations of CH₄ emission from a Sumatra paddy field were measured for 3 years. The mean CH₄ emission rates during the growth period were in the range of 16.0–26.1 mg CH₄ m^-2 h^-1 for the chemical fertilizer plots and 23.3–34.9 mg CH₄ m^-2 h^-1 for the plots with rice straw application, respectively. The increase in the amounts of CH₄ emission by rice straw application were from 1.3 to 1.6 times. There was no significant difference in the mean CH₄ emission rates between rainy and dry seasons.Total amounts of CH₄ emitted during the period of rice growth were in the ranges of 29.5–48.2 and 43.0–64.6 g CH₄ m^-2 for the plots applied with chemical fertilizer and those with rice straw application, respectively. Nearly the same amounts of CH₄ were emitted in the first and second half of the growth period, irrespective of rice straw application.     

芽孢杆菌冷冻真空干燥法和砂管法保藏过程中存活率的变化

以芽孢杆菌(Bacillus)为材料,用冷冻真空干燥法和砂管法,在4℃条件下进行了六年的菌种保藏对比试验.结果表明,冷冻真空干燥法比砂管法保藏的菌种存活率高,存活率下降速率小;建立了菌种保藏过程中菌种相对存活率随时间变化规律的数学模型L′_f(t)=e~(-0.1323t)和L′s(t)=e~(-0.2900t).该模型的模拟结果能与试验结果较好地吻合.在室温条件下,砂管法的菌种存活率下降速率比4℃条件下大,三个月时存活率为零或接近零;而冷冻真空干燥法保藏菌种存活率与4℃的效果接近.因此冷冻真空干燥法是芽孢杆菌的比较有效的保藏方法.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Sedimentology of the Upper Triassic reef complex at the Hochkönig Massif (Northern Calcareous Alps,Austria)

Summary The Upper Triassic Dachsteinkalk of the Hochk?nig Massif, situated 50 km south of Salzburg in the Northern Calcareous Alps, corresponds to a platform margin reef complex of exceptional thickness. The platform interior limestones form equally thick sequences of the well known cyclic Lofer facies. Sedimentation in the reef complex was not so strongly controlled by low-amplitude sea-level oscillations as was the Lofer facies. The westernmost of the 8 facies of the reef complex is an oncolite-dominated lagoon, in which wave-resistant stromatolite mounds with a relief of a few metres were periodically developed. The transition to the central reef area is accomplished across the back-reef facies. In the back-reef facies patch reefs and calcisponges appear. The proportion of coarse bioclastic sediment increases rapidly over a few hundred metres before the central reef area is encountered. The central reef area consists of relatively widely spaced small patch reefs that did not develop wave-resistant reef framework structures. The bulk of the sediment in the central reef area is coarse bioclastic material, provided by the dense growth of reef organisms and the wave-induced disintegration of patch reefs. Collapse of the reef margin is recorded by the supply of large blocks of patch reef material to the upper reef slope. Additionally, coarse, loose bioclastic debris was supplied to the upper reef slope and this was incorporated into debris flows on the reef slope and turbidites found at the base of the slope and in the off-reef facies. Partially lithified packstones and wackestones of the lower to middle reef slope were modified by mass movement to form breccia and rudstone sheets. The latter reach out hundreds of metres into the off-reef facies environment. A reef profile is presented which was derived by the restoration of strike and dip information. In conjunction with constraints imposed by sedimentary facies related to slope processes, the angle of slope in the reef margin area ranged from 11° to 5°, forming a concave (dished downwards) slope. Water depth estimations require that the central reef area did not develop in water of less than 10 metres depth. At the reef margin water depths were about 30 metres, at the base of the reef slope 200 metres and deepening in the off-reef facies to 250 metres. While previous work on reef complexes from this type of setting suggests growth in a heavily storm-dominated environment, the present author finds little evidence for the storm generation of the fore reef breccias, although there is good evidence for storm-influenced sedimentation and reworking in the central reef area. Post-depositional processes were characterised by continued slope processes causing brecciation and hydraulic injection of red internal sediments downwards into the reef slope and off-reef limestones. Hydrothermal circulation caused a number of phases of post-depositional (diagenetic) brecciation. There appears not to have been an important period of emergence at the Triassic/Jurassic boundary.     

Impairment of raw 264.7 macrophage function by antiarrhythmic drugs

The effect of the antiarrhythmic drugs lidocaine, quinidine and procainamide on macrophage function was investigated in RAW 264.7 mouse monocytic macrophage cell. Cells stimulated by either zymosan or phorbol ester were found to generate both superoxide (O ₂ ^– ) and H₂O₂. The production of O₂ was detected as superoxide dismutase inhibitable ferricytochrome c reduction. H₂O₂ production was monitored in both chemical and flow cytometric fluorescent assays. Although all three drugs inhibited both O₂ and H₂O₂ release in a dose dependent manner, only quinidine was found to have significant inhibitory effects. The amounts of quinidine required to cause a 50% inhibition in O₂ production in zymosan and phorbol ester stimulated cells were found to be 250 M and 300 M, respectively and the amounts required to cause one-half optimum levels of H₂O₂ production in these cells were found to be 50 M and 100 M, respectively. The effect of these drugs on O₂ producing NADPH oxidase was investigated and only procainamide was found to have a significant effect (p<0.001) in inhibiting the oxidase activity. Lidocaine and quinidine had no significant effect on the activation of the respiratory burst oxidase. A sensitive and convenient differential phagocytosis assay was devised on the basis of number of particles engulfed by individual phagocytes using flow cytometric techniques. It appears to be remarkably free of interference and was applied to investigate the role of antiarrhythmic drugs on the phagocytosis of fluorescent latex beads. All three antiarrhythmic drugs inhibited phagocytosis of latex beads in a dose dependent manner irrespective of the number of particles phagocitized by the cells. The results of these studies do not conclusively establish a mechanism of action of these drugs on the generation of O₂ and H₂O₂ by stimulated macrophages; nevertheless, it is interesting that all three drugs inhibited the phagocytic activity.