The plant thioredoxin system.

Thioredoxins are small proteins catalyzing thiol-disulfide interchange and are involved in the regulation of the redox environment of the cell. In plants, the thioredoxin system is particularly complex since at least 20 thioredoxin isoforms are found in the plant model Arabidopsis thaliana. Based upon primary sequence analysis and subcellular localization, thioredoxins can be classified into different groups and subgroups. Different pathways allowing thioredoxin reduction also coexist in the plant involving ferredoxin-thioredoxin reductase, thioredoxin reductases and the glutathione/glutaredoxin system. This review discusses the literature of plant thioredoxins with emphasis on recent findings in the field.

Plant glutaredoxins: still mysterious reducing systems.

Glutaredoxins are ubiquitous oxidoreductases which are similar to thioredoxins and possess a typical glutathione-reducible CxxC or CxxS active site. We present here the current knowledge about these proteins in plants. At least 31 glutaredoxin genes are present in Arabidopsis thaliana, a value close to the thioredoxin gene number. Based essentially on active site sequences, a classification of these multiple genes is proposed. The specificity of the various apparently redundant forms within the glutaredoxin group or between glutaredoxin and thioredoxin can be analysed in terms of differential spatiotemporal expression of the genes, specificity vs. target proteins and mode of catalysis (glutathiolation/ deglutathiolation processes appear to be a specific function of glutaredoxin). Additional putative functions are proposed for plant glutaredoxins based on their targets in other organisms and in the light of the existence of hybrid proteins containing glutaredoxin modules in their N- or C-terminal part.

Influence of tropolone on Poria placenta wood degradation.

Fenton reactions are believed to play important roles in wood degradation by brown rot fungi. In this context, the effect of tropolone (2-hydroxycyclohepta-2,4,6-trienone), a metal chelator, on wood degradation by Poria placenta was investigated. Tropolone (50 micro M) strongly inhibits fungal growth on malt agar, but this inhibition could be relieved by adding iron salts. With an experimental system containing two separate parts, one supplemented with tropolone (100 micro M) and the other not, it was shown that the fungus is able to reallocate essential minerals from the area where they are available and also to grow in these conditions on malt-agar in the presence of tropolone. Nevertheless, even in the presence of an external source of metals, P. placenta is not able to attack pine blocks impregnated with tropolone (5 mM). This wood degradation inhibition is related to the presence of the tropolone hydroxyl group, as shown by the use of analogs (cyclohepta-2,4,6-trienone and 2-methoxycyclohepta-2,4,6-trienone). Furthermore, tropolone possesses both weak antioxidative and weak radical-scavenging properties and a strong affinity for ferric ion and is able to inhibit ferric iron reduction by catecholates, lowering the redox potential of the iron couple. These data are consistent with the hypothesis that tropolone inhibits wood degradation by P. placenta by chelating iron present in wood, thus avoiding initiation of the Fenton reaction. This study demonstrates that iron chelators such as tropolone could be also involved in novel and more environmentally benign preservative systems.

Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as a proton donor.

A sequence coding for a peroxiredoxin (Prx) was isolated from a xylem/phloem cDNA library from Populus trichocarpa and subsequently inserted into an expression plasmid yielding the construction pET-Prx. The recombinant protein was produced in Escherichia coli cells and purified to homogeneity with a high yield. The poplar Prx is composed of 162 residues, a property that makes it the shortest plant Prx sequence isolated so far. It was shown that the protein is monomeric and possesses two conserved cysteines (Cys). The Prx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of an exogenous proton donor that can be either thioredoxin or glutaredoxin (Grx). Based on this finding, we propose that the poplar protein represents a new type of Prx that differs from the so-called 2-Cys and 1-Cys Prx, a suggestion supported by the existence of natural fusion sequences constituted of a Prx motif coupled to a Grx motif. The protein was shown to be highly expressed in sieve tubes where thioredoxin h and Grx are also major proteins.

Fungicidal activity of beta-thujaplicin analogues.

The fungicidal activity of analogues of beta-thujaplicin, a natural product responsible for the durability of heartwood of several cupressaceous trees, was investigated in vitro on the growth of different white and brown rot fungi involved in wood biodegradation, Coriolus versicolor, Phanerochaete chrysosporium, Poria placenta and Gloephyllum trabeum. The study shows that 2-hydroxycyclohepta-2,4,6-trienone (tropolone), easily prepared according to a literature procedure, possesses interesting fungicidal activity when compared to beta-thujaplicin, azaconazole, tebuconazole and copper oxine, which suggests this compound should be examined further as a potential biocide for wood preservation.

Induction of lactate production associated with a decrease in NADH cell content enables growth resumption of Clostridium cellulolyticum in batch cultures on cellobiose.

When grown in batch cultures in fermentors with 23.4 mM cellobiose, Clostridium cellulolyticum displayed biphasic growth kinetics not associated with sequential substrate consumption and which led to a twofold higher production of biomass than previously reported. In the first growth phase, acetate was the major product of cellobiose metabolism, since lactate and ethanol productions remained low. Furthermore, an accumulation of intracellular NADH was observed. The transition towards the second growth phase was accompanied by an induction of lactate production, in such a way that lactate became the major product of C. cellulolyticum metabolism. In addition, a decrease in NADH concentration was measured, concomitant with this induction of lactate production and with the growth resumption. During both growth phases, the NADH-ferredoxin reductase-hydrogenase system played a major function in NADH regeneration, since H2 production was 1.4- to 1.5-fold higher than that of CO2. Thus, we found that lactate production serves as an additional catabolic pathway enabling C. cellulolyticum to cope with excesses of carbon and NADH produced. Growth experiments on C. cellulolyticum under an atmosphere of carbon monoxide mimicked this phenomenon and confirmed that a high intracellular level of NADH can provide a barrier to bacterial growth.

Effect of dilution rate, cellobiose and ammonium availabilities on Clostridium cellulolyticum sporulation.

The nutritional and physiological factors affecting sporulation of Clostridium cellulolyticum were studied using steady-state continuous cultures grown in both complex and synthetic media. Under cellobiose limitation, the probability that cells will sporulate appears to be directly related to the growth rate. In complex medium, the highest percentage of sporulation was 20% at a dilution rate of 0.015 h-1 whereas in synthetic medium it was 10% at 0.035 h-1. In both media, when the dilution rate was either higher or lower the percentage of sporulation decreased by between 2% and 4%. At low dilution rates, endospore formation was repressed under cellobiose-sufficient concentrations, suggesting catabolite repression by cellobiose. Furthermore, the concentration of ammonium was important in determining the percentage of sporulation, as ammonium limitation induced extensive sporulation at low growth rates even in an excess of cellobiose. The sporulation process is not triggered when cells are cellobiose-exhausted both in complex and synthetic media. These data suggest that, in C. cellulolyticum, an exogenous supply of carbon is required throughout the sporulation process. In the experimental conditions used in this work, no relationship between glycogen accumulation or glycogen mobilization and endospore formation was detected in C. cellulolyticum.

The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production.

In this study, we demonstrate that the cellulosome of Clostridium cellulolyticum grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate, C. cellulolyticum is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.

Reconstitution of the B800 bacteriochlorophylls in the peripheral light harvesting complex B800-850 of rhodobacter sphaeroides 2.4.1 with BChl a and modified (bacterio-)chlorophylls

A method is described for reversibly removing bacteriochlorophyll from the B800-site of the B850-850 antenna complex from Rhodobacter sphaeroides. This method uses the oligosaccharidic detergent Triton BG-10, together with an incubation at pH 5.0. Reconstitution at the B800-site has been successfully achieved for a range of modified bacteriochlorophylls. Copyright 1998 Elsevier Science B.V. All rights reserved.

Metabolism of cellobiose by Clostridium cellulolyticum growing in continuous culture: evidence for decreased NADH reoxidation as a factor limiting growth.

Previous results indicated that molar growth yields are reduced when Clostridium cellulolyticum is cultured in media containing cellobiose concentrations greater than 1 g I-1. Continuous cultures were examined to determine the physiological basis of these poor growth yields. Acetate was the main product of C. cellulolyticum metabolism, whereas the production of reduced compounds such as ethanol or lactate was low. Such patterns of product formation were accompanied by a 12-fold increase in intracellular NADH concentration when the cellobiose flow was increased. Catabolic enzymic activities were measured in vitro. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acetate kinase and phosphoroclastic activities were found at similar levels as in cells metabolizing higher substrate concentrations. In contrast, lactate dehydrogenase activity was low and correlated with the rate of lactate production. Furthermore, an inhibition of GAPDH activity by high NADH/NAD+ ratios was established. These results suggested that a decreased NADH reoxidation could be responsible for limiting C. cellulolyticum growth. Lactate and ethanol production were not sufficient to balance out the NADH produced in the GAPDH step of glycolysis. One consequence of poor NADH reoxidation would be an increase in intracellular concentration of NADH, which in turn could inhibit GAPDH activity.

Clostridium cellulolyticum Viability and Sporulation under Cellobiose Starvation Conditions.

Depending on the moment of cellobiose starvation, Clostridium cellulolyticum cells behave in different ways. Cells starved during the exponential phase of growth sporulate at 30%, whereas exhaustion of the carbon substrate at the beginning of growth does not provoke cell sporulation. Growth in the presence of excess cellobiose generates 3% spores. The response of C. cellulolyticum to carbon starvation involves changes in proteolytic activities; higher activities (20% protein degradation) corresponded to a higher level of sporulation; lower proteolysis (5%) was observed in cells starved during the beginning of exponential growth, when sporulation was not observed; with an excess of cellobiose, an intermediate value (10%), accompanied by a low level of sporulation, was observed in cells taken at the end of the exponential growth phase. The basal percentage of the protein breakdown in nonstarved culture was 4%. Cells lacking proteolytic activities failed to induce sporulation. High concentrations of cellobiose repressed proteolytic activities and sporulation. The onset of carbon starvation during the growth phase affected the survival response of C. cellulolyticum via the sporulation process and also via cell-cellulose interaction. Cells from the exponential growth phase were more adhesive to filter paper than cells from the stationary growth phase but less than cells from the late stationary growth phase.

Colonization of Crystalline Cellulose by Clostridium cellulolyticum ATCC 35319.

Cellulose colonization by Clostridium cellulolyticum was studied by using [methyl-H]thymidine incorporation. The colonization process indicated that a part of the bacterial population was released from cellulose to the liquid phase before binding and colonizing another adhesion site of the cellulose. We postulate that cellulose colonization occurs according to the following process: adhesion, colonization, release, and readhesion.

Adhesion and growth rate of Clostridium cellulolyticum ATCC 35319 on crystalline cellulose.

The rate of tritiated-thymidine incorporation into DNA was used to estimate Clostridium cellulolyticum H10 growth rates on Avicel cellulose, taking into consideration both the unattached cells and the cells adhered to the substrate. The generation time on cellobiose calculated from the data on cell density (4.5 h) agreed well with the generation time calculated by tritiated-thymidine incorporation (3.8 h). Growth on Avicel cellulose occurred when bacteria were adhered to their substrate; 80% of the biomass was detected on the cellulose. Taking into consideration attached and free bacteria, the generation time as determined by thymidine incorporation was about 8 h, whereas by bacterial-protein estimation it was about 13 h. In addition to the growth rate of the bacteria on the cellulose, the release of adhered cells constituted an important factor in the efficiency of the cellulolysis. The stage of growth influenced adhesion of C. cellulolyticum; maximum adhesion was found during the exponential phase. Under the conditions used, the end of growth was characterized by an acute release of biomass and cellulase activity from the cellulose. An exhaustion of the accessible cellulose could be responsible for this release.

Characteristics of cellulose colonization by a mesophilic, cellulolytic Clostridium (strain C401).

Using the technique of incorporation of 3H-thymidine into DNA, we describe colonization properties of an anaerobic, mesophilic, cellulolytic Clostridium, strain C401. This method took into account both bacteria which adhered and those which did not adhere to the substrate. The observed generation time (7.5 h) was faster than that detected (26 h) with other methods. Under the conditions used, the end of growth was characterized by a sharp release of biomass. A depletion in the supply of carbon source and therefore, of the adhesion site, was responsible for this release.