Arabidopsis thaliana overexpressing glycolate oxidase in chloroplasts: H2O2-induced changes in primary metabolic pathways

Reactive oxygen species (ROS) represent both toxic by-products of aerobic metabolism as well as signaling molecules in processes like growth regulation and defense pathways. The study of signaling and oxidative-damage effects can be separated in plants expressing glycolate oxidase in the plastids (GO plants), where the production of H₂O₂ in the chloroplasts is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions. Thus, GO plants represent an ideal non-invasive model to study events related to the perception and responses to H₂O₂ accumulation. Metabolic profiling of GO plants indicated that under high light a sustained production of H₂O₂ imposes coordinate changes on central metabolic pathways. The overall metabolic scenario is consistent with decreased carbon assimilation, which results in lower abundance of glycolytic and tricarboxylic acid cycle intermediates, while simultaneously amino acid metabolism routes are specifically modulated. The GO plants, although retarded in growth and flowering, can complete their life cycle indicating that the reconfiguration of the central metabolic pathways is part of a response to survive and thus, to adapt to stress conditions imposed by the accumulation of H₂O₂ during the light period.Key words: Arabidopsis thaliana, H₂O₂, oxidative stress, reactive oxygen species, signalingReactive oxygen species (ROS) are key molecules in the regulation of plant development, stress responses and programmed cell death. Depending on the identity of ROS species or its subcellular production site, different cellular responses are provoked.¹ To assess the effects of metabolically generated H₂O₂ in chloroplasts, we have recently generated Arabidopsis plants in which the peroxisomal GO was targeted to chloroplasts.² The GO overexpressing plants (GO plants) show retardation in growth and flowering time, features also observed in catalase, ascorbate peroxidase and MnSOD deficient mutants.^3–5 The analysis of GO plants indicated that H₂O₂ is responsible for the observed phenotype. GO plants represent an ideal non-invasive model system to study the effects of H₂O₂ directly in the chloroplasts because H₂O₂ accumulation can be modulated by growing the plants under different ambient conditions. By this, growth under low light or high CO₂ concentrations minimizes the oxygenase activity of RubisCO and thus the flux through GO whereas the exposition to high light intensities enhances photorespiration and thus the flux through GO.Here, we explored the impact of H₂O₂ production on the primary metabolism of GO plants by assessing the relative levels of various metabolites by gas chromatography coupled to mass spectrometry (GC-MS)⁶ in rosettes of plants grown at low light (30 µmol quanta m⁻² s⁻¹) and after exposing the plants for 7 h to high light (600 µmol quanta m⁻² s⁻¹). The results obtained for the GO5 line are shown in

Indirect effects of tending ants on holm oak volatiles and acorn quality

The indirect effect of ants on plants through their mutualism with honeydew-producing insects has been extensively investigated. Honeydew-producing insects that are tended by ants impose a cost on plant fitness and health by reducing seed production and/or plant growth. This cost is associated with sap intake and virus transmissions but may be overcompesated by tending ants if they deter or prey on hebivorous insects. The balance between cost and benefits depends on the tending ant species. In this study we report other indirect effects on plants of the mutualism between aphids and ants. We have found that two Lasius ant species, one native and the other invasive, may change the composition of volatile organic compounds (VOCs) of the holm oak (Quercus ilex) blend when they tend the aphid Lachnus roboris. The aphid regulation of its feeding and honeydew production according to the ant demands was proposed as a plausible mechanism that triggers changes in VOCs. Additionally, we now report here that aphid feeding, which is located most of the time on acorns cap or petiole, significantly increased the relative content of linolenic acid in acorns from holm oak colonized by the invasive ant. This acid is involved in the response of plants to insect herbivory as a precursor or jasmonic acid. No effect was found on acorn production, germination or seedlings quality. These results suggest that tending-ants may trigger the physiological response of holm oaks involved in plant resistance toward aphid herbivory and this response is ant species-dependent.Key words: tended aphid, invasive ants, linolenic acid, jasmonic acid, monoterpene emissionsTo achieve an indirect effect it is necessary to have a minimun of three species, two focal species that interact directly and an associate species whose presence promotes an indirect effect on one or both focal species. In general, indirect effects of a third species are defined by how and to what degree a pairwise species interaction is influenced by the presence and density of this third species.¹ There are several examples of interactions presenting indirect effects: apparent competition,¹ facilitation,² tri-trophic level interactions,³ cascading effects⁴ and exploitative competition. 5 But, indirect effects have been studied most extensively in the context of trophic cascades when top predators are removed⁶ or added⁷ and in the context of mutualisms.^8–10 Usually, indirect effects are investigated as changes in abundance of the focal species occur. However, indirect effects may result in biologically significant changes in a species that are not reflected only to its abundance.¹¹ There are many examples of changes in physiology, behavior, morphology and/or genotypic composition of the focal species.^11,12 These changes on density and/or morphological, physiological and behavioral traits of the focal species are not mutually exclusive, and all can act at the same time.¹³ The magnitude and direction of both direct and indirect effects should influence the relative resilience of communities to perturbation, which in turn will affect species coexistence and community evolution.¹⁴ In this regard, indirect effects had been postulated as one of the main forces structuring communities² and shaping the evolution of communities.¹⁴In terrestrial communities ants interact with plants both directly and indirectly. They can disperse or consume seeds, feed from specialized plant structures such as food bodies and extrafloral nectaries, act as or deter pollinitators, prey on herbivorous insects and/or develop mutualisms with honeydew-producing insects indirectly modifying plant fitness.^15–17 Additionally, through their nesting activities in soil, ants increase soil nutrient content available to plants, may change water infiltration and soil holding-capacity and modify biodiversity and abundance of soil organisms related to the decomposition process.^18,19 As a consequence of their activities, ants may thus change behavior, density, physiology or fitness of other species.^12,22,23 In the case of ants that tend honeydew-producing insects, evidence shows that their attention may change some traits of insect life history, 22 their abundance or physiology.¹⁸ For the plant, the net outcome of the mutualism between ants and honeydew-producing insects will depend on the balance between the costs for plant fitness via consumption of plant sap and transmission of plant pathogens and the benefit of ants deterring herbivorous insects.^18,23 As a consequence, plant seed production, pod production or even plant growth may decrease when the cost of honeydew-producing insects exceed the benefit provided by tending ants.^18,23Recently, we have described the changes that two tending ant species may exert indirectly on monoterpene emissions of holm oak (Quercus ilex) saplings through its mutualism with Lachnus roboris aphids.²⁴ One of these tending ant species was Lasius neglectus, an invasive ant species that displaces the local ant Lasius grandis. We found that aphids feeding on holm oak increased the emission of total volatile organic carbon (VOCs) by 31%. In particular, aphids feeding elicited the emission of a new monoterpene, Δ³-carene, and increased the emission of myrcene (mean ± SE; sapling alone: 0.105 ± 0.011 µg g⁻¹ h⁻¹; sapling plus not tended aphid: 0.443 ± 0.057 µg g⁻1 h⁻1) and γ-terpinene (sapling alone: 0.0013 ± 0.0001; sapling plus not tended aphid: 0.0122 ± 0.0022 µg g⁻1 h⁻1) (Mann-Whitney, sapling alone vs. sapling plus not tended aphids, U_4,4 = 0, p < 0.05 for both compounds). Changes of VOC emission in response to aphid infestation were noticed also in boreal trees.²⁴ When the aphids became tended by the invasive ant, L. neglectus, VOCs emissions increased only 19% because myrcene, the main compound of the blend, decreased significantly (25 When our data was recalculated on leaf area basis (nmol m⁻² s⁻¹), the general pattern was the same independently of the units, but the differences among treatments were not statistically significant (26 These slight differences in the statitiscal significance of the differences of VOC emissions depending on the reference unit may be due to differences in leaf morphology, i.e., changes of leaf area and mass. However, in our study, all holm oaks showed a similar leaf morphology among treatments (Kruskal-Wallis, leaf mass: H_3,20 = 2.16, p = 0.53; leaf area: H_3,20 = 2.64, p = 0.45) (24^,27 This lack of consistence of aphid effect on leaf area and mass limits the development of a clear pattern linking aphids feeding, leaf area or mass and VOC emissions. On the other hand, to achieve statistical significance of emitted VOCs among treatments, values should differ strongly given the high variability of VOC emission within treatments.²⁶ Under this scenario, we recommend giving the values of leaf morphology and to give VOC emissions on both unit bases to facilite comparisons among different studies.

Table 1

Means and standard error of the emission rates of the main compounds emitted by Quercus ilex saplings (n = 4 for T1 and T2 and n = 8 for T3) infested with untended aphids (T1) or infested with aphids tended by the native ant Lasius grandis (T2) or by the invasive ant Lasius neglectus (T3)

Decorin regulates endothelial cell-matrix interactions during angiogenesis

Interactions between endothelial cells and the surrounding extracellular matrix are continuously adapted during angiogenesis, from early sprouting through to lumen formation and vessel maturation. Regulated control of these interactions is crucial to sustain normal responses in this rapidly changing environment, and dysfunctional endothelial cell behaviour results in angiogenic disorders. The proteoglycan decorin, an extracellular matrix component, is upregulated during angiogenesis. While it was shown previously that the absence of decorin leads to dysregulated angiogenesis in vivo, the molecular mechanisms were not clear. These abnormal endothelial cell responses have been attributed to indirect effects of decorin; however, our recent data provides evidence that decorin directly regulates endothelial cell-matrix interactions. This data will be discussed in conjunction with findings from previous studies, to better understand the role of this proteoglycan in angiogenesis.Key words: decorin, angiogenesis, motility, α2β1 integrin, insulin-like growth factor I receptor, Rac GTPaseLed by appropriate cues, the vascular system undergoes postnatal remodelling (angiogenesis), to maintain tissue homeostasis. Thus while much of the mature endothelium is quiescent, locally activated endothelial cells re-enter the cell cycle, and assume a motile phenotype essential for sprouting and neo-vessel formation. Concomitantly, the surrounding extracellular matrix (ECM) is significantly altered through de novo protein expression, deposition of plasma components and protease-mediated degradation. The latter liberates cryptic binding sites and sequestered growth factors in addition to intact and degraded ECM components, which themselves possess pro- and anti-angiogenic signalling properties. For supported blood flow, endothelium quiescence and integrity is re-established, and the ECM is organized into mature, cross-linked networks. In short, endothelial cells regulate ECM synthesis, assembly and turnover while the structure and composition of ECM in turn influences cellular phenotype. The ECM therefore, plays a critical role in control of endothelial cell behaviour during angiogenesis.Decorin is a member of the small leucine-rich repeat proteoglycan (SLRP) family, which was first discovered ‘decorating’ collagen I fibrils and was subsequently shown to regulate fibrillogenesis.^1,2 Both the protein core and the single, covalently attached glycosaminoglycan (GAG) moieties of decorin are involved in this function, the relevance of which is demonstrated by the phenotype of the decorin null mouse, which exhibits loose, fragile skin due to dysregulated fibrillogenesis.² Interestingly, a role for decorin in postnatal angiogenesis was also revealed by studies in the decorin null background. Corneal neoangiogenesis was reduced.³ Conversely, neo-angiogenesis was enhanced during dermal wound healing, although surprisingly this led to delayed wound closure.⁴ In this case, skin fragility due to the absence of decorin may have hindered wound closure, despite an increased blood supply. It is apparent however, that decorin plays a role in inflammation-associated angiogenesis. Indeed, endothelial cells undergoing angiogenic morphogenesis in this environment express decorin, while quiescent endothelial cells do not,^3–6 indicating that decorin modulates endothelial cell behaviour specifically during inflammatory-associated remodelling of the vascular system.To understand decorin effects on angiogenic morphogenesis within a minimalist environment, various in vitro models of angiogenesis have been employed (6 Similarly, decorin expression enhanced tube formation on matrigel,⁸ but in other studies utilising this substrate was found to either have no influence⁹ or to inhibit tubulogenesis induced by growth factors.¹⁰ In yet another study, decorin inhibited tube formation when presented as a substrate prior to addition of collagen I.⁷ These contrasting observations may reflect the importance of the micro-environment within which decorin is presented. Alternatively, controversial results could result from different sources of decorin since cell types differ in their post-translational modifications of the GAG moiety. Hence, varying length or sulfation patterns of GAG chains may account for different biological activities of decorin. Discrepancies can also be explained as artefacts due to different purification protocols, such as when denaturing conditions are used to extract decorin from tissue. Taken together however, these observations suggest that decorin is neither a pro- nor an anti-angiogenic factor per se, but rather a regulator of angiogenesis, dependent on local cues for different activities. Further, that decorin is capable of both enhancing and inhibiting tubulogenesis may suggest a role in balancing vessel regression versus persistence. Immature vessels have a period of plasticity prior to maturation, during which they can be remodelled, and either regress, or given the appropriate signals, proceed to maturity.¹¹ As a modulator of tube formation, it is tempting to speculate that decorin could influence the switch from immature to mature vessels, favouring one or the other in conjunction with signals from the local environment.

Table 1

Summary of the key functions of decorin in controlling cell behaviour

Stress-induced flowering

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescensFlowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.^1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.¹⁰ Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.¹¹ Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in 12^–14

Table 1

Some cases of stress-induced flowering

The speed of intracellular signal transfer for chloroplast movement

The photoreceptors for chloroplast photorelocation movement have been known, but the signal(s) raised by photoreceptors remains unknown. To know the properties of the signal(s) for chloroplast accumulation movement, we examined the speed of signal transferred from light-irradiated area to chloroplasts in gametophytes of Adiantum capillus-veneris. When dark-adapted gametophyte cells were irradiated with a microbeam of various light intensities of red or blue light for 1 min or continuously, the chloroplasts started to move towards the irradiated area. The speed of signal transfer was calculated from the relationship between the timing of start moving and the distance of chloroplasts from the microbeam and was found to be constant at any light conditions. In prothallial cells, the speed was about 1.0 µm min⁻¹ and in protonemal cells about 0.7 µm min⁻¹ towards base and about 2.3 µm min⁻¹ towards the apex. We confirmed the speed of signal transfer in Arabidopsis thaliana mesophyll cells under continuous irradiation of blue light, as was about 0.8 µm min⁻¹. Possible candidates of the signal are discussed depending on the speed of signal transfer.Key words: Adiantum capillus-veneris, Arabidopsis thaliana, blue light, chloroplast movement, microbeam, red light, signalOrganelle movement is essential for plant growth and development and tightly regulated by environmental conditions.¹ It is well known that light regulates chloroplast movement in various plant species. Chloroplast movement can be separated into three categories, (1) photoperception by photoreceptors, (2) signal transduction from photoreceptor to chloroplasts and (3) movement of chloroplasts and has been analyzed from a physiological point of view.² We recently identified the photoreceptors in Arabidopsis thaliana, fern Adiantum capillus-veneris, and moss Physcomitrella patens. In A. thaliana, phototropin 2 (phot2) mediates the avoidance movement,^3,4 whereas both phototropin 1 (phot1) and phot2 mediate the accumulation response.⁵ A chimeric photoreceptor neochrome 1 (neo1)⁶ was identified as a red/far-red and blue light receptor that mediates red as well as blue light-induced chloroplast movement in A. capillusveneris.⁷ Interestingly, neo1 mediated red and blue light-induced nuclear movement and negative phototropic response of A. capillus-veneris rhizoid cells.^8,9 On the mechanism of chloroplast movement, we also found a novel structure of actin filaments that appeared between chloroplast and the plasma membrane at the front side of moving chloroplast.¹⁰ Recent studies using the technique of microbeam irradiation have revealed that chloroplasts do not have a polarity for light-induced accumulation movement and can move freely in any direction both in A. capillus-veneris prothallial cells and in A. thaliana mesophyll cells.¹¹ However, the signal that may be released from photoreceptors and transferred to chloroplasts remains unknown.To understand the properties of the signal for the chloroplast accumulation response, we examined the speed of signal transfer in dark-adapted A. capillus-veneris gametophyte cells and A. thaliana mesophyll cells by partial cell irradiation with a red and/or blue microbeam of various light intensities for 1 min and the following continuous irradiation, respectively.¹²As shown in Figure 1, the relation between the distance of chloroplasts from the microbeam and the timing when each chloroplast started moving toward the microbeam irradiated area (shown as black dots in Fig. 1) was obtained and plotted. The lag time between the onset of microbeam irradiation and the timing of start moving of chloroplasts is the time period needed for a signal to reach each chloroplast. To obtain more accurate data many chloroplasts at various positions were used. The slope of the approximate line indicates the average speed of the signal transfer. Shown with a protonemal cell at the left side of this figure is an instance where the speed of signal transfer from basal-to-apical (acropetal) direction is obtained.Open in a separate windowFigure 1How to calculate the speed of signal transfer in the basal cell of two-celled protonema of Adiantum capillus-veneris. The relationship between the distance of chloroplast position from the edge of the microbeam to the center of each chloroplast as shown in left side of figure and the timing of chloroplast movement initiated shown as the black dots was obtained. Inclination of the approximate lines connecting dots indicates the speeds of the signal transfer.In protonemal cells, which are tip-growing linear cells, the average speed of signal transfer was about 2.3 µm min⁻¹ from basal-to-apical (acropetal) and about 0.7 µm min⁻¹ from apical-to-basal (basipetal) directions. These values were almost constant irrespective of light intensity, wavelength, irradiation period, and the region of the cell irradiated.¹² The difference of speed between basipetal and acropetal directions may be depending on cell polarity. The signal transfer in prothallial cells of A. capillus-veneris and mesophyll cells of A. thaliana was about 1.0 µm min⁻¹ to any direction, probably because they may not have a polarity comparing to protonemal cells or have a weak polarity if any. Thus, the speed of signal transfer must be conserved in most land plants,¹² if not influenced by strong polarity. R1W m⁻²R1W m⁻²B1W m⁻²R0.1W m⁻²R10W m⁻²B10W m⁻²1 mincountinuouscountinuouscountinuouscountinuouscountinuousProtonemal cell (towards apical region)2.322.372.282.412.39Protonemal cell (towards basal region)0.580.730.800.740.86Prothallial cell1.130.921.101.080.95Arabidopsis thaliana0.70Open in a separate windowThe speeds of signal transfer under different light intensities and wave length in Adiantum capillus-veneris gametophyte cells and Arabidopsis thaliana mesophyll cells are summarized. When dark-adapted cells were irradiated with various light intensities (red light: 10, 1, 0.1 W m⁻²) of a microbeam of red or blue light for 1 min or continuously, the chloroplasts moved towards the irradiated area. The speed of signal transfer was measured from the relationship between the timing of onset of moving and the distance of chloroplalsts from the microbeam irradiated area.Calcium ions have been proposed as one of the candidates of the signal. Calcium is reported to be necessary for chloroplast movement in some plants.^13,14 Chloroplast movement under polarized light could not be induced in the existence of EGTA in protonemal cells of A. capillus-veneris, although chloroplasts show slight movement in random direction.¹³ In Lemna trisulca, chloroplast movement correlates with an increase of cytoplasmic calcium levels and is inhibited by antagonists of calcium homeostasis.¹⁴ The speed of intracellular transfer of calcium ions in plant cells was measured only in moss Physcomitrella patens by microinjection of a calcium indicator into protonemal cells.¹⁵ The speed of calcium waves in the cytoplasm of protonemal cell was about 3.4 µm sec⁻¹. The speed of substance transfer as signals is not known in plant cells except for the above instance, as far as we know, but in animal cells various experimental data has been accumulated.^16–21The transfer speed of calcium waves visualizing cytoplasmic free calcium by microinjection of aequorin was about 8 µm sec⁻¹ in Xenopus eggs.¹⁶ Calcium ion expands as a spherical wave and the wave speed in plane is 50 µm sec⁻¹ in rat cardiac myocytes when measured by loading a membrane-permeable indicator of calcium into the cell. The maximum velocity was 112 µm sec⁻¹.¹⁷ Calcium waves could also be observed in the SR-free single isolated rabbit cardiac myofibrils with a propagation velocity of 15.5 µm sec⁻¹.¹⁸ The propagation velocity of the calcium wave was about 65–100 µm sec⁻¹ by calciuminduced calcium release (CICR) in pig heart muscle cells.^19–21 Comparing these values to our data in A. capillus-veneris, the speed of signal transfer in chloroplast movement in fern gametophytes was 100–200 times slower than those measured for calcium ion transfers in animal cells, suggesting that the calcium might not be the signal involved in chloroplast movement.Intracellular transport is depended on the cytoskeleton systems in many cases. So the speed of movement of the cytoskeleton itself has been examined. When motor-proteins (such as 22s dynein, 14s dynein, kinesin) were anchored on a slide glass microtubules overlaid moved with a speed of about 4.52, 4.29, 0.422 µm sec⁻¹, respectively. In similar ways, actin filaments placed over myosin-coated glass moved at about 5.21 µm sec-1.²² On the other hand, the motor domain of the Centromere Binding Factor (CBF) protein complex moves at 4.04 µm min⁻¹ on microtubules.²³ In A. capillus-veneris protonemal cells, the speed of cytoplasmic streaming depending on the actomyosin system was calculated from the speed of oil drop movement.²⁴ The speed was dependent upon the position of long protonemal cells and was about 2 µm min⁻¹ in the apical region and gradually increased to 10 µm min⁻¹ in the basal region. In comparison to the data cited here, the speed of signal transfer involved in chloroplast accumulation was 30–120 times slower than the speed of the actomyosin system or the microtubule-kinesin/dynein system, but it is similar to the moving speed of a protein complex on a microtubule²³ and oil droplets in a protonemal cell.²⁴Polymerization rates of cytoskeletal proteins have been measured using in vitro systems. For instance, the plus end of microtubules from bovine brains grew at 1.04–1.88 µm min⁻¹.^25,26 Polymerization rate of actin filaments from rabbit muscle was about 0.13–0.49 µm min⁻¹ and depended on the G-actin concentration.²⁷ Live BHK21 fibroblasts, mouse melanoma cells and Dictyostelium amoebae expressing GFP-actin fusion proteins move on glass by using three-dimensional F-actin bands. These structures propagate throughout the cytoplasm at rates ranging between 2–5 µm min⁻¹ in each cell type and produce lamellipodia or pseudopodia at the cell boundary.²⁸ The extending speed of these cytoskeletons is roughly equal to the speed of signal transfer for the chloroplast accumulation response. We therefore aim to measure the speed of extension of these filaments when a method of gene transformation has been established for A. capillus-veneris.     

Prion interference with multiple prion isolates

Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.Key words: prion diseases, prion interference, prion strainsPrion diseases are fatal neurodegenerative diseases that are caused by an abnormal isoform of the prion protein, PrP^Sc.¹ Prion strains are hypothesized to be encoded by strain-specific conformations of PrP^Sc resulting in strain-specific differences in clinical signs, incubation periods and neuropathology.^2–7 However, a universally agreed upon definition of prion strains does not exist. Interspecies transmission and adaptation of prions to a new host species leads to the emergence of a dominant prion strain, which can be due to selection of strains from a mixture present in the inoculum, or produced upon interspecies transmission.^8,9 Prion strains, when present in the same host, can interfere with each other.Prion interference was first described in mice where a long incubation period strain 22C extended the incubation period of a short incubation period strain 22A following intracerebral inoculation.¹⁰ Interference between other prion strains has been described in mice and hamsters using rodent-adapted strains of scrapie, TME, Creutzfeldt-Jacob disease and Gerstmannn-Sträussler-Scheinker syndrome following intracerebral, intraperitoneal, intravenous and sciatic nerve routes of inoculation.^10–15 We previously demonstrated the detection of PrP^Sc from the long incubation period DY TME agent correlated with its ability to extend the incubation period or completely block the superinfecting short incubation period HY TME agent from causing disease and results in a reduction of HY PrP^Sc levels following sciatic nerve inoculation.¹² However, it is not known if a single long incubation period agent (e.g., DY TME) can interfere with more than one short incubation period agent or if interference can occur by a natural route of infection.To examine the question if one long incubation period agent can extend the incubation period of additional short incubation period agents, hamsters were first inoculated in the sciatic nerve with the DY TME agent 120 days prior to superinfection with the short-incubation period agents HY TME, 263K scrapie and HaCWD.^16–18 The HY TME and 263K scrapie agents have been biologically cloned and have distinct PrP^Sc properties.^19,20 The HaCWD agent used in this study is seventh hamster passage that has not been biologically cloned and therefore will be referred to as a prion isolate. Sciatic nerve inoculations were performed as previously described.^11,12 Briefly, hamsters were inoculated with 103.0 i.c. LD₅₀ of the DY TME agent or equal volume (2 µl of a 1% w/v brain homogenate) of uninfected brain homogenate 120 days prior to superinfection of the same sciatic nerve with either 10^4.6 i.c. LD₅₀ of the HY TME agent, 10^5.2 i.c. LD₅₀ of the HaCWD agent or 10^4.6 i.c. LD₅₀/g 263K scrapie agent (Bartz J, unpublished data).^16,18,21 Animals were observed three times per week for the onset of clinical signs of HY TME, 263K and HaCWD based on the presence of ataxia and hyperexcitability, while the clinical diagnosis of DY TME was based on the appearance of progressive lethargy.^16–18 The incubation period was calculated as the number of days between the onset of clinical signs of the agent strain that caused disease and the inoculation of that strain. The Student''s t-test was used to compare incubation periods.¹² We found that sciatic nerve inoculation of both the HaCWD agent and 263K scrapie agent caused disease with a similar incubation period to animals infected with the HY TME agent (12 In hamsters inoculated with the DY TME agent 120 days prior to superinfection with the HaCWD or 263K agents, the animals developed clinical signs of DY TME with an incubation period that was not different from the DY TME agent control group (12 The PrP^Sc migration properties were consistent with the clinical diagnosis and all co-infected animals had PrP^Sc that migrated similar to PrP^Sc from the DY TME agent infected control animal (Fig. 1, lanes 1–10). This data indicates that the DY TME agent can interfere with more than one isolate and that interference in the CNS may be a more generalized phenomenon of prion strains.Open in a separate windowFigure 1The strain-specific properties of PrP^Sc correspond to the clinical diagnosis of disease. Western blot analysis of 250 µg brain equivalents of proteinase K digested brain homogenate from prion-infected hamsters following intracerebral (i.c.), sciatic nerve (i.sc.) or per os inoculation with either the HY TME (HY), DY TME (DY), 263K scrapie (263K), hamster-adapted CWD (CWD) agents or mock-infected (UN). The unglycoyslated PrP^Sc glycoform of HY TME, 263K scrapie and hamster-adapted CWD migrates at 21 kDa. The unglycosylated PrP^Sc glycoform of DY PrP^Sc migrates at 19 kDa. Migration of 19 and 21 kDa PrP^Sc are indicated by the arrows on the left of the figure. n.a., not applicable.

Table 1

Clinical signs and incubation periods of hamsters inoculated in the sciatic nerve with either the HY TME, HaCWD or 263K scrapie agents, or co-infected with the DY TME agent 120 days prior to superinfection of hamsters with the HY TME, HaCWD or 263K agents

Antimicrobial Activity of Simulated Solar Disinfection against Bacterial,Fungal, and Protozoan Pathogens and Its Enhancement by Riboflavin

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m⁻²) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log₁₀ after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log₁₀ after 6 h) was obtained only in the presence of riboflavin and 250 W m⁻² irradiance.Solar disinfection (SODIS) is an established and proven technique for the generation of safer drinking water (11). Water is collected into transparent plastic polyethylene terephthalate (PET) bottles and placed in direct sunlight for 6 to 8 h prior to consumption (14). The application of SODIS has been shown to be a simple and cost-effective method for reducing the incidence of gastrointestinal infection in communities where potable water is not available (2-4). Under laboratory conditions using simulated sunlight, SODIS has been shown to inactivate pathogenic bacteria, fungi, viruses, and protozoa (6, 12, 15). Although SODIS is not fully understood, it is believed to achieve microbial killing through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating (21).The combination of UVA radiation and riboflavin (vitamin B₂) has recently been reported to have therapeutic application in the treatment of bacterial and fungal ocular pathogens (13, 17) and has also been proposed as a method for decontaminating donor blood products prior to transfusion (1). In the present study, we report that the addition of riboflavin significantly enhances the disinfectant efficacy of simulated SODIS against bacterial, fungal, and protozoan pathogens.Chemicals and media were obtained from Sigma (Dorset, United Kingdom), Oxoid (Basingstoke, United Kingdom), and BD (Oxford, United Kingdom). Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), and Fusarium solani (ATCC 36031) were obtained from ATCC (through LGC Standards, United Kingdom). Escherichia coli (JM101) was obtained in house, and the Legionella pneumophila strain used was a recent environmental isolate.B. subtilis spores were produced from culture on a previously published defined sporulation medium (19). L. pneumophila was grown on buffered charcoal-yeast extract agar (5). All other bacteria were cultured on tryptone soy agar, and C. albicans was cultured on Sabouraud dextrose agar as described previously (9). Fusarium solani was cultured on potato dextrose agar, and conidia were prepared as reported previously (7). Acanthamoeba polyphaga (Ros) was isolated from an unpublished keratitis case at Moorfields Eye Hospital, London, United Kingdom, in 1991. Trophozoites were maintained and cysts prepared as described previously (8, 18).Assays were conducted in transparent 12-well tissue culture microtiter plates with UV-transparent lids (Helena Biosciences, United Kingdom). Test organisms (1 × 10⁶/ml) were suspended in 3 ml of one-quarter-strength Ringer''s solution or natural freshwater (as pretreated water from a reservoir in United Kingdom) with or without riboflavin (250 μM). The plates were exposed to simulated sunlight at an optical output irradiance of 150 watts per square meter (W m⁻²) delivered from an HPR125 W quartz mercury arc lamp (Philips, Guildford, United Kingdom). Optical irradiances were measured using a calibrated broadband optical power meter (Melles Griot, Netherlands). Test plates were maintained at 30°C by partial submersion in a water bath.At timed intervals for bacteria and fungi, the aliquots were plated out by using a WASP spiral plater and colonies subsequently counted by using a ProtoCOL automated colony counter (Don Whitley, West Yorkshire, United Kingdom). Acanthamoeba trophozoite and cyst viabilities were determined as described previously (6). Statistical analysis was performed using a one-way analysis of variance (ANOVA) of data from triplicate experiments via the InStat statistical software package (GraphPad, La Jolla, CA).The efficacies of simulated sunlight at an optical output irradiance of 150 W m⁻² alone (SODIS) and in the presence of 250 μM riboflavin (SODIS-R) against the test organisms are shown in Table ​Table1.1. With the exception of B. subtilis spores and A. polyphaga cysts, SODIS-R resulted in a significant increase in microbial killing compared to SODIS alone (P < 0.001). In most instances, SODIS-R achieved total inactivation by 2 h, compared to 6 h for SODIS alone (Table ​(Table1).1). For F. solani, C. albicans, ands A. polyphaga trophozoites, only SODIS-R achieved a complete organism kill after 4 to 6 h (P < 0.001). All control experiments in which the experiments were protected from the light source showed no reduction in organism viability over the time course (results not shown).

TABLE 1.

Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)

Stretching the timescale of intravital imaging in tumors

Since the time it was pioneered in 1992, intravital imaging of tumors at cellular resolution has offered us the extremely important opportunity of “seeing biology.” However, until now, most studies were monitoring tumor cell behavior in the same animal over short times, requiring the combining of acquired data into a hypothesis via statistical analysis. In the last year, different groups have independently developed techniques to extend the time scale of intravital imaging to several days. This improvement allows one to address the connection between tumor cell behavior and the microenvironment which surrounds them. We can now assess dynamics of the cell-cell interactions in tumors, analyze tumor cell fate and changes in the tumor extracellular matrix which accompany tumor progression.Key words: intravital, multiphoton, spinning disc, microenvironment, second harmonic generation, mammary imaging window, dorsal skinfold chamber, photoswitchingIntravital imaging of tumors at cellular resolution offers insight into the physiology of cells in vivo in real time. The first published study which included injectable dyes to monitor tumor metastasis inside the embryo was done by the group of Groom.¹ Some years later, Farina,² and then Naumov,³ and co-workers, used GFP-labeled tumor cells to study tumors by confocal scanning microscopy. Soon after, Brown,⁴ and Wang,⁵ and co-workers, introduced two-photon microscopes into their studies.Until recently, single cell-resolved intravital imaging in tumors commonly involved recording movies 4D (3D through time) with one or two channels, collecting data via multiphoton microscopy from one region at a time.^6–8 The inner side of the orthotopic tumor is exposed by making a small incision in the skin and skin folding. This technique, termed ‘skin-flap’, allows for several hours of imaging in one animal. Data from several animals are combined into the final result averaging measurements as well as differences in tumor preparation, animal condition and genotype. Some low-resolution studies have proposed a reversible flap⁹ on the tumor tissue implanted several days earlier. However, visualized areas were not the same at each of the timepoints. Also, as skin flaps were opened repeatedly, they were potentially influencing the microenvironment by surgery-related immune/inflamatory-responses. In addition, several groups have been using a dorsal skinfold chamber¹⁰ in which the tumor is grown ectopically, in the space between the skin and glass coverslip on the back of the mouse. This preparation could be used for either low resolution measurements over several days, or short-term measurements at cellular resolution.In the last few months, several studies have included techniques which extend the time-scale of intravital imaging in tumors from hours to days (TechniqueMIW + photoswitching12Dorsal skinfold + SHG recognition¹³Extended skinflap¹⁵Orthotopic tumorsYesNoYesLong-term anesthesia neededNoNoYesMultiple imaging sessions availableYesYesNoMicroscopyConfocal and multiphotonMultiphotonSpinning disc confocalDepth of imaging∼120 µm¹²∼100 µm¹³<70 µm¹⁵DetectorsPMT (1 for each channel)PMT (1 for each channel)CameraNumber of channels424Open in a separate windowSegall-Condeelis groups^11,12 have developed a technique to visualize and quantify invasion and intravasation of single tumor cells in orthotopic mammary tumors. They designed a mammary imaging window (MIW), which enables imaging the tumor in serial imaging sessions. Moreover, to properly position the animal on the microscope and keep the animal orientation the same over several sessions, they use a stereotactic imaging box.¹¹ Due to cell replication and motility, angiogenesis and consequent changes in tissue shape, a registration landmark is essential in order to recognize the region of interest in each of the imaging sessions. In Kedrin et al.¹² a photoswitchable protein Dendra2 was used as a tumor cell marker, making it possible to differentiate between total tumor cells (green) and chosen cells of interest (red). By photomarking and visualizing selected populations of cells within the tumor, team quantified and compared the metastatic behavior of cells in different tumor microenvironments within the same tumor. The number of imaging sessions which visualize a specific group of cells in areas surrounding major blood vessels is limited by high cell motility and intravasation. However, in areas where only microvessels are present (Fig. 1), this technique can monitor cell invasion of the surrounding environment for up to seven days.Open in a separate windowFigure 1Photoconverted regions which are not in the vicinity of major blood vessels show a relatively slow dispersion of cells throughout a seven day period. Images are the result of serial intravital imaging sessions (0–168 h after photoswitching) of mammary tumor cells which express cytoplasmic Dendra2. Fluorescence intensity at each time point was normalized to 0 h level. Photo converted region (red) is 150× 150µm at 0h.Similarly, in Perentes et al.,¹³ Boucher-Jain groups use serial imaging sessions made possible via dorsal chamber implantation and intravital multiphoton microscopy to study the mechanism of collagen fiber remodeling by tumor-associated fibroblasts. The internal landmark used in order to recognize and image the same microenvironment in several imaging sessions is collagen itself. Fibers are visualized by second harmonic generation (SHG), without any additional labeling. Since the resulting images are misaligned due to different animal orientations and tissue changes over serial imaging sessions, additional registration approach based on fluorescence intensity (Turboreg¹⁴) was applied during data post-processing. Images taken over nine-day periods were aligned based on similar bulk distribution of collagen fibers. Further, individual fibers were analyzed for a decrease in length and an increase in area overlap with surrounding GFP-fibroblasts.Werb and co-workers have used a different method when comparing the dynamics of stromal cells in different microenvironments of breast carcinoma, as presented in Egeblad et al.¹⁵ In order to optically access the tumor, they used an improved version of the ‘skin-flap’ technique. This allows work on transgenic mouse models, such as MMTV-PyMT,¹⁶ which can have several tumor stages present in one animal. Imaging was done over a single session that extends up to 27 h by carefully controlling temperature, anesthesia and animal hydration. While the use of spinning disc confocal microscopy limits imaging depth to ∼2 cell diameters deep into the tissue, large areas of the tumor can be imaged and with high speed.¹⁵ The high speed of acquisition results from simultaneous illumination of ∼1,000 rotating pinholes at a time¹⁷ and using cameras as detectors. This means that the limiting factor in the speed of data acquisition is the brightness of cells inside the tumor. As the excitation is achieved via single photon events, the implementation of additional laser lines is much cheaper and fairly straightforward. Moreover, by using a motorized stage which is controlled by software, several fields of view can be combined into mosaic images of a larger area. The final output of this set up is a 4D movie which contains up to four-channels, three z-sections, 45 timepoints per hour and compiles five fields into a mosaic view.Finally, Dunphy et al.¹⁸ recently proposed an interesting microcartography approach in which fluorescent beads are inserted inside the dorsal skinfold chamber as reference points. Based on the visualization of beads, coordinates of the region of interest are recalculated in each of the series of imaging sessions.We can now map the fate of tumor cells over days or monitor changes in the extracellular matrix inside the tumor as the tumor grows and progresses. These improvements allow assessment of the dynamics of cell-cell and cell-matrix interactions inside the tumor. Visualization and quantification of these interactions, the more precise definitions of microenvironments and the identification of stromal cells essential to tumor progression are all within reach. In addition, the analysis of mechanisms of drug action on single cells in real time in vivo, is now an achievable goal.     

Growth of Arthrobacter sp. Strain JBH1 on Nitroglycerin as the Sole Source of Carbon and Nitrogen

Arthrobacter sp. strain JBH1 was isolated from nitroglycerin-contaminated soil by selective enrichment. Detection of transient intermediates and simultaneous adaptation studies with potential intermediates indicated that the degradation pathway involves the conversion of nitroglycerin to glycerol via 1,2-dinitroglycerin and 1-mononitroglycerin, with concomitant release of nitrite. Glycerol then serves as the source of carbon and energy.Nitroglycerin (NG) is manufactured widely for use as an explosive and a pharmaceutical vasodilator. It has been found as a contaminant in soil and groundwater (7, 9). Due to NG''s health effects as well as its highly explosive nature, NG contamination in soils and groundwater poses a concern that requires remedial action (3). Natural attenuation and in situ bioremediation have been used for remediation in soils contaminated with certain other explosives (16), but the mineralization of NG in soil and groundwater has not been reported.To date, no pure cultures able to grow on NG as the sole carbon, energy, and nitrogen source have been isolated. Accashian et al. (1) observed growth associated with the degradation of NG under aerobic conditions by a mixed culture originating from activated sludge. The use of NG as a source of nitrogen has been studied in mixed and pure cultures during growth on alternative sources of carbon and energy (3, 9, 11, 20). Under such conditions, NG undergoes a sequential denitration pathway in which NG is transformed to 1,2-dinitroglycerin (1,2DNG) or 1,3DNG followed by 1-mononitroglycerin (1MNG) or 2MNG and then glycerol, under both aerobic and anaerobic conditions (3, 6, 9, 11, 20), and the enzymes involved in denitration have been characterized in some detail (4, 8, 15, 21). Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8, 9). Cultures capable of incomplete denitration to MNG in the presence of additional carbon sources were identified as Pseudomonas putida, Pseudomonas fluorescens (4), an Arthobacter species, a Klebsiella species (8, 9), and Agrobacterium radiobacter (20).Here we describe the isolation of bacteria able to degrade NG as the sole source of carbon, nitrogen, and energy. The inoculum for selective enrichment was soil historically contaminated with NG obtained at a facility that formerly manufactured explosives located in the northeastern United States. The enrichment medium consisted of minimal medium prepared as previously described (17) supplemented with NG (0.26 mM), which was synthesized as previously described (18). During enrichment, samples of the inoculum (optical density at 600 nm [OD₆₀₀] ∼ 0.03) were diluted 1/16 in fresh enrichment medium every 2 to 3 weeks. Isolates were obtained by dilution to extinction in NG-supplemented minimal medium. Cultures were grown under aerobic conditions in minimal medium at pH 7.2 and 23°C or in tryptic soy agar (TSA; 1/4 strength).Early stages of enrichment cultures required extended incubation with lag phases of over 200 h and exhibited slow degradation of NG (less than 1 μmol substrate/mg protein/h). After a number of transfers over 8 months, the degradation rates increased substantially (2.2 μmol substrate/mg protein/h). A pure culture capable of growth on NG was identified based on 16S rRNA gene analysis (504 bp) as an Arthrobacter species with 99.5% similarity to Arthrobacter pascens (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU246730","term_id":"289474572","term_text":"GU246730"}}GU246730). Purity of the cultures was confirmed microscopically and by formation of a single colony type on TSA plates. 16S gene sequencing and identification were done by MIDI Labs (Newark, DE) and SeqWright DNA Technology Services (Houston, TX). The Arthrobacter cells stained primarily as Gram-negative rods with a small number of Gram-positive cocci (data not shown); Gram variability is also a characteristic of the closely related Arthrobacter globiformis (2, 19). The optimum growth temperature is 30°C, and the optimum pH is 7.2. Higher pH values were not investigated because NG begins to undergo hydrolysis above pH 7.5 (data not shown). The isolated culture can grow on glycerol, acetate, succinate, citrate, and lactate, with nitrite as the nitrogen source. Previous authors described an Arthrobacter species able to use NG as a nitrogen source in the presence of additional sources of carbon. However, only dinitroesters were formed, and complete mineralization was not achieved (9).To determine the degradation pathway, cultures of the isolated strain (5 ml of inoculum grown on NG to an OD₆₀₀ of 0.3) were grown in minimal medium (100 ml) supplemented with NG at a final concentration of 0.27 mM. Inoculated bottles and abiotic controls were continuously mixed, and NG, 1,2DNG, 1,3DNG, 1MNG, 2MNG, nitrite, nitrate, CO₂, total protein, and optical density were measured at appropriate intervals. Nitroesters were analyzed with an Agilent high-performance liquid chromatograph (HPLC) equipped with an LC-18 column (250 by 4.6 mm, 5 μm; Supelco) and a UV detector at a wavelength of 214 nm (13). Methanol-water (50%, vol/vol) was used as the mobile phase at a flow rate of 1 ml/min. Nitrite and nitrate were analyzed with an ion chromatograph (IC) equipped with an IonPac AS14A anion-exchange column (Dionex, CA) at a flow rate of 1 ml/min. Carbon dioxide production was measured with a Micro Oxymax respirometer (Columbus Instruments, OH), and total protein was quantified using the Micro BCA protein assay kit (Pierce Biotechnology, IL) according to manufacturer''s instructions. During the degradation of NG the 1,2DNG concentration was relatively high at 46 and 72 h (Fig. ​(Fig.1).1). 1,3DNG, detected only at time zero, resulted from trace impurities in the NG stock solution. Trace amounts of 1MNG appeared transiently, and trace amounts of 2MNG accumulated and did not disappear. Traces of nitrite at time zero were from the inoculum. The concentration of NG in the abiotic control did not change during the experiment (data not shown).Open in a separate windowFIG. 1.Growth of strain JBH1 on NG. ×, NG; ▵, 1,2DNG; ⋄, 1MNG; □, 2MNG; ○, protein.Results from the experiment described above were used to calculate nitrogen and carbon mass balances (Tables ​(Tables11 and ​and2).2). Nitrogen content in protein was approximated using the formula C₅H₇O₂N (14). Because all nitrogen was accounted for throughout, we conclude that the only nitrogen-containing intermediate compounds are 1,2DNG and 1MNG, which is consistent with previous studies (6, 9, 20). The fact that most of the nitrogen was released as nitrite is consistent with previous reports of denitration catalyzed by reductase enzymes (4, 8, 21). The minor amounts of nitrate observed could be from abiotic hydrolysis (5, 12) or from oxidation of nitrite. Cultures supplemented with glycerol or other carbon sources assimilated all of the nitrite (data not shown).

TABLE 1.

Nitrogen mass balance

Peptidoglycan Fine Structure of the Radiotolerant Bacterium Deinococcus radiodurans Sark

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine–N-acetylmuramic acid–l-Ala–d-Glu-(γ)–l-Orn-[(δ)Gly-Gly]–d-Ala–d-Ala. Cross-linkage was mediated by (Gly)₂ bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.The gram-positive bacterium Deinococcus radiodurans is remarkable because of its extreme resistance to ionizing radiation (14). Phylogenetically the closest relatives of Deinococcus are the extreme thermophiles of the genus Thermus (4, 11). In 16S rRNA phylogenetic trees, the genera Thermus and Deinococcus group together as one of the older branches in bacterial evolution (11). Both microorganisms have complex cell envelopes with outer membranes, S-layers, and ornithine-Gly-containing mureins (7, 12, 19, 20, 22, 23). However, Deinococcus and Thermus differ in their response to the Gram reaction, having positive and negative reactions, respectively (4, 14). The murein structure for Thermus thermophilus HB8 has been recently elucidated (19). Here we report the murein structure of Deinococcus radiodurans with similar detail.D. radiodurans Sark (23) was used in the present study. Cultures were grown in Luria-Bertani medium (13) at 30°C with aeration. Murein was purified and subjected to amino acid and high-performance liquid chromatography (HPLC) analyses as previously described (6, 9, 10, 19). For further analysis muropeptides were purified, lyophilized, and desalted as reported elsewhere (6, 19). Purified muropeptides were subjected to plasma desorption linear time-of-flight mass spectrometry (PDMS) as described previously (1, 5, 16, 19). Positive and negative ion mass spectra were obtained on a short linear ²⁵²californium time-of-flight instrument (BioIon AB, Uppsala, Sweden). The acceleration voltage was between 17 and 19 kV, and spectra were accumulated for 1 to 10 million fission events. Calibration of the mass spectra was done in the positive ion mode with H⁺ and Na⁺ ions and in the negative ion mode with H⁻ and CN⁻ ions. Calculated m/z values are based on average masses.Amino acid analysis of muramidase (Cellosyl; Hoechst, Frankfurt am Main, Germany)-digested sacculi (50 μg) revealed Glu, Orn, Ala, and Gly as the only amino acids in the muramidase-solubilized material. Less than 3% of the total Orn remained in the muramidase-insoluble fraction, indicating an essentially complete solubilization of murein.Muramidase-digested murein samples (200 μg) were analyzed by HPLC as described in reference 19. The muropeptide pattern (Fig. ​(Fig.1)1) was relatively simple, with five dominating components (DR5 and DR10 to DR13 [Fig. 1]). The muropeptides resolved by HPLC were collected, desalted, and subjected to PDMS. The results are presented in Table ​Table11 compared with the m/z values calculated for best-matching muropeptides made up of N-acetylglucosamine (GlucNAc), N-acetylmuramic acid (MurNAc), and the amino acids detected in the murein. The more likely structures are shown in Fig. ​Fig.1.1. According to the m/z values, muropeptides DR1 to DR7 and DR9 were monomers; DR8, DR10, and DR11 were dimers; and DR12 and DR13 were trimers. The best-fitting structures for DR3 to DR8, DR11, and DR13 coincided with muropeptides previously characterized in T. thermophilus HB8 (19) and had identical retention times in comparative HPLC runs. The minor muropeptide DR7 (Fig. ​(Fig.1)1) was the only one detected with a d-Ala–d-Ala dipeptide and most likely represents the basic monomeric subunit. The composition of the major cross-linked species DR11 and DR13 confirmed that cross-linking is mediated by (Gly)₂ bridges, as proposed previously (20). Open in a separate windowFIG. 1HPLC muropeptide elution patterns of murein purified from D. radiodurans. Muramidase-digested murein samples were subjected to HPLC analysis, and the A₂₀₄ of the eluate was recorded. The most likely structures for each muroeptide as deduced by PDMS are shown. The position of residues in brackets is the most likely one as deduced from the structures of other muropeptides but could not be formally demonstrated. R = GlucNac–MurNac–l-Ala–d-Glu-(γ)→.

TABLE 1

Calculated and measured m/z values for the molecular ions of the major muropeptides from D. radiodurans

Effect of Growth Phase Feeding Strategies on Succinate Production by Metabolically Engineered Escherichia coli

Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.Succinic acid is valued as one of the key basic chemicals used in the preparation of biodegradable polymers or as raw material for chemicals of the C₄ family (8, 19). The fermentative production of succinic acid from renewable resources is environmentally acceptable and sustainable (3). A breakthrough in genetically engineering Escherichia coli (6, 7, 11, 18) for succinate production was the isolation of strain AFP111 (1, 4), a mutant of NZN111 with a spontaneous ptsG mutation (pflAB ldhA double mutant). The process involves a two-stage fermentation, with aerobic cell growth followed by anaerobic conditions for succinate production (16, 21, 22). The aerobically induced enzymes can maintain their activity during the anaerobic phase and significantly affect succinate fermentation (22, 23). Using the best transition time based on the activities of the key enzymes and other physiological states, a two-stage fermentation using the recombinant AFP111 strain harboring pTrc99A-pyc achieved a final succinic acid concentration and productivity of 99.2 g·liter⁻¹ and 1.3 g·liter⁻¹·h⁻¹, respectively (21).Aerobic cell growth is essential for the subsequent anaerobic fermentation. However, few studies have focused on the regulation of aerobic cell growth. As a regulation method, gluconeogenic carbon sources were used instead of glucose for the aerobic growth of Escherichia coli NZN111 and the activities of enzymes that are favorable for the anaerobic synthesis of succinate were enhanced (23, 24). Unfortunately, a gluconeogenic carbon source (e.g., sodium acetate) might increase the osmotic pressure of culture media, which would be detrimental to succinate production (23). As another regulation method, a glucose feeding strategy controlling the glucose concentration at about 0.5 g·liter⁻¹ up to 1 g·liter⁻¹ was reported to prevent excessive formation of acetic acid (16).In this study, we investigated different glucose feeding strategies for the aerobic growth phase of the two-phase process for succinate production by E. coli AFP111. Specifically, we compared several growth rates by using glucose limitation in addition to maximum growth under conditions of excess glucose.E. coli AFP111 [F⁺ λ⁻ rpoS396(Am) rph-1 ΔpflAB::Cam ldhA::Kan ptsG] (4, 16), which was a kind gift from D. P. Clark (Southern Illinois University), was the only strain used in this study. Luria-Bertani (LB) medium (60 ml) was used for inoculum culture in 1,000-ml flasks, and 3 liters of chemically defined medium (13, 14) was used for two-stage culture in a 7-liter fermentor. Two-stage fermentations were divided into three types, based on the glucose feeding strategy used during the aerobic stage. For type I culture, the glucose concentration was maintained at about 20 g·liter⁻¹ during aerobic cell growth. Type II and III cultures comprised a batch process and subsequent glucose-limited fed-batch process (Fig. ​(Fig.1).1). The batch process initially contained 13 g/liter of glucose. The fed-batch process began when the dry cell weight (DCW) reached about 6 g/liter, with type II and type III cultures using a 600 g/liter glucose feed to achieve cell growth rates of 0.15 h⁻¹ and 0.07 h⁻¹, respectively (10). When the DCW reached 12 g·liter⁻¹, the aerobically grown cells were directly transferred to anaerobic conditions (Fig. ​(Fig.1).1). For the anaerobic process, oxygen-free CO₂ was sparged at 0.5 liter·min⁻¹, the pH was controlled between 6.4 and 6.8 with intermittent supplementation of solid magnesium carbonate hydroxide, and the glucose concentration was maintained at about 20 g·liter⁻¹ by supplying glucose in an 800-g·liter⁻¹ solution.Open in a separate windowFIG. 1.Concentrations of glucose (circles), DCW (triangles), and succinic acid (squares) in the three types of two-stage fermentation by AFP111. μ, growth rate.The optical density at 600 nm was used to monitor cell growth, and this value was correlated to DCW. The concentration of glucose was assayed with an enzyme electrode analyzer, and organic acids were quantified by high-performance liquid chromatography (HPLC). The intracellular concentrations of NADH and NAD⁺ were assayed with a cycling method (12). The activities of isocitrate lyase (ICL) (20), pyruvate kinase (PYK) (17), phosphoenolpyruvate (PEP) carboxykinase (PCK) (20, 23), PEP carboxylase (PPC) (23), and malate dehydrogenase (MDH) (23) were measured spectrophotometrically at the end of the aerobic phase and 12 h after the onset of the anaerobic phase.All three types of fermentations were terminated when the succinate concentration increased less than 1 g·liter⁻¹ in 5 h. Type III fermentation was terminated at a final succinic acid concentration of 101.2 g·liter⁻¹ and an anaerobic-phase productivity of 1.89 g·liter⁻¹·h⁻¹ (Fig. ​(Fig.1).1). Trace amounts of by-products (such as acetate, ethanol, and pyruvate) accumulated and did not follow any trend in the anaerobic phase (data not shown).At the end of the aerobic culture phase, the specific enzyme activities of PCK, PYK, and ICL in type III culture were 2.9, 2.5, and 11.4 times higher, respectively, than the activities in type I culture (Table ​(Table1)1) . This phenomenon is consistent with published reports that suggest that the expression of enzymes involved in anaplerotic metabolism and the glyoxylate shunt (5, 15) is elevated in E. coli grown under glucose-limited conditions. These enzymes maintained their activities in the subsequent anaerobic phase (Table ​(Table1)1) and would be central to succinate production (22, 23). The elevated levels of PCK and PPC would provide the reductive branch of the tricarboxylic acid (TCA) cycle with oxaloacetate (OAA) at a higher rate (9), thereby supplying both malate and citrate (Table ​(Table11).

TABLE 1.

Activities of enzymes at the end of the aerobic culture phase and 12 h after the onset of the anaerobic phase