To gain initial structure-activity relationships regarding the highly functionalized pentyl side chain attached at C-3 of mithramycin (MTM), we focused on a post-polyketide synthase (post-PKS) tailoring step of the MTM biosynthesis by Streptomyces argillaceus ATCC 12956, which was proposed to be catalyzed by ketoreductase (KR) MtmW. In this last step of the MTM biosynthesis, a keto group of the pentyl side chain is reduced to a secondary alcohol, and we anticipated the generation of an MTM derivative with an additional keto group in the 3-side chain. Insertional inactivation of mtmW, a gene located ca. 8 kb downstream of the mithramycin-PKS genes, yielded an S. argillaceus mutant, which accumulated three new mithramycin analogues, namely mithramycin SA, demycarosyl-mithramycin SK, and mithramycin SK (MTM-SK). The structures of these three compounds confirmed indirectly the proposed role of MtmW in MTM biosynthesis. However, the new mithramycin derivatives bear unexpectedly shorter 3-side chains (ethyl or butyl) than MTM, presumably caused by nonenzymatic rearrangement or cleavage reactions of the initially formed pentyl side chain with a reactive beta-dicarbonyl functional group. The major product, MTM-SK, was tested in vitro against a variety of human cancer cell lines, as well as in an in vitro toxicity assay, and showed an improved therapeutic index, in comparison to the parent drug, MTM.

Mithramycin is an aureolic acid-type antimicrobial and antitumor agent produced by Streptomyces argillaceus. Modifying post-polyketide synthase (PKS) tailoring enzymes involved in the production of mithramycin is an effective way of gaining further information regarding the late steps of its biosynthetic pathway. In addition, new "unnatural" natural products of the aureolic acid-type class are likely to be produced. The role of two such post-PKS tailoring enzymes, encoded by mtmC and mtmTIII, was investigated, and four novel aureolic acid class drugs, two premithramycin-type molecules and two mithramycin derivatives, were isolated from mutant strains constructed by insertional gene inactivation of either of these two genes. From data bank comparisons, the corresponding proteins MtmC and MtmTIII were believed to act as a C-methyltransferase involved in the production of the D-mycarose (sugar E) of mithramycin and as a ketoreductase seemingly involved in the biosynthesis of the mithramycin aglycon, respectively. However, gene inactivation and analysis of the accumulated products revealed that both genes encode enzymes participating in the biosynthesis of the D-mycarose building block. Furthermore, the inactivation of MtmC seems to affect the ketoreductase responsible for 4-ketoreduction of sugar C, a D-olivose. Instead of obtaining premithramycin and mithramycin derivatives with a modified E-sugar upon inactivation of mtmC, compounds were obtained that completely lack the E-sugar moiety and that possess an unexpected 4-ketosugar moiety instead of the D-olivose at the beginning of the lower deoxysaccharide chain. The inactivation of mtmTIII led to the accumulation of 4E-ketomithramycin, showing that this ketoreductase is responsible for the 4-ketoreduction of the D-mycarose moiety. The new compounds of the mutant strains, 4A-ketopremithramycin A2, 4A-keto-9-demethylpremithramycin A2, 4C-keto-demycarosylmithramycin, and 4E-ketomithramycin, indicate surprising substrate flexibility of post-PKS enzymes of the mithramycin biosynthetic pathway. Although the glycosyltransferase responsible for the attachment of D-mycarose cannot transfer the unmethylated sugar to the existing lower disaccharide chain, it can transfer the 4-ketoform of sugar E. In addition, the glycosyltransferase MtmGIV, which is responsible for the linkage of sugar C, is also able to transfer an activated 4-ketosugar. The oxygenase MtmOIV, normally responsible for the oxidative cleavage of the tetracyclic premithramycin B into the tricyclic immediate precursor of mithramycin, can act on a substrate analogue with a modified or even incomplete trisaccharide chain. The same is true for glycosyltransferases MtmGI and MtmGII, both of which partake in the formation and attachment of the A-B disaccharide in mithramycin.

Mithramycin is a glycosylated aromatic polyketide produced by Streptomyces argillaceus, and is used as an antitumor drug. Three genes (mtmV, mtmU and mtmC) from the mithramycin gene cluster have been cloned, and characterized by DNA sequencing and by analysis of the products that accumulate in nonproducing mutants, which were generated by insertional inactivation of these genes. The mtm V gene codes for a 2,3-dehydratase that catalyzes early and common steps in the biosynthesis of the three sugars found in mithramycin (D-olivose, D-oliose and D-mycarose); its inactivation caused the accumulation of the nonglycosylated intermediate premithramycinone. The mtmU gene codes for a 4-ketoreductase involved in D-oliose biosynthesis, and its inactivation resulted in the accumulation of premithramycinone and premithramycin A , the first glycosylated intermediate which contains a D-olivose unit. The third gene, mtmC, is involved in D-mycarose biosynthesis and codes for a C-methyltransferase. Two mutants with lesions in the mtmC gene accumulated mithramycin intermediates lacking the D-mycarose moiety but containing D-olivose units attached to C-12a in which the 4-keto group is unreduced. This suggests that mtmC could code for a second enzyme activity, probably a D-olivose 4-ketoreductase, and that the glycosyltransferase responsible for the incorporation of D-olivose (MtmGIV) shows some degree of flexibility with respect to its sugar co-substrate, since the 4-ketoanalog is also transferred. A pathway is proposed for the biosynthesis of the three sugar moieties in mithramycin.

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The aim of this study was to investigate the potential of indigenous arsenic-tolerant bacteria to enhance arsenic phytoremediation by the autochthonous pseudometallophyte The first goal was to perform an initial analysis of the entire rhizosphere and endophytic bacterial communities of the above-named accumulator plant, including the cultivable bacterial species.'s microbiome was dominated by taxa related to ,, and , especially the and genera. A total of 54 cultivable rhizobacteria and 41 root endophytes, mainly affiliated with the phyla, ,, and , were isolated and characterized with respect to several potentially useful features for metal plant accumulation, such as the ability to promote plant growth, metal chelation, and/or mitigation of heavy-metal stress. Seven bacterial isolates were further selected and tested for accumulation of arsenic in plants; four of them were finally assayed in field-scale bioaugmentation experiments. The exposure to arsenic caused an increase in the total nonprotein thiol compound content in roots, suggesting a detoxification mechanism through phytochelatin complexation. In the contaminated field, the siderophore and indole-3-acetic acid producers of the endophytic bacterial consortium enhanced arsenic accumulation in the leaves and roots of , whereas the rhizosphere isolate strain 91R mainly promoted plant growth. Field experimentation showed that additional factors, such as soil arsenic content and pH, influenced arsenic uptake in the plant, attesting to the relevance of field conditions in the success of phytoextraction strategies. Microorganisms and plants have developed several ways of dealing with arsenic, allowing them to resist and metabolize this metalloid. These properties form the basis of phytoremediation treatments and the understanding that the interactions of plants with soil bacteria are crucial for the optimization of arsenic uptake. To address this in our work, we initially performed a microbiome analysis of the autochthonous plants growing in arsenic-contaminated soils, including endosphere and rhizosphere bacterial communities. We then proceeded to isolate and characterize the cultivable bacteria that were potentially better suited to enhance phytoextraction efficiency. Eventually, we went to the field application stage. Our results corroborated the idea that recovery of pseudometallophyte-associated bacteria adapted to a large historically contaminated site and their use in bioaugmentation technologies are affordable experimental approaches and potentially very useful for implementing effective phytoremediation strategies with plants and their indigenous bacteria.

Explants of Actinidia deliciosa Chev. Liang and Ferguson var. Hayward were cultured in controlled CO2 atmospheres in the presence of different sucrose concentrations. Organogenesis was measured after 45 days in explants from the different assays, and quantification of photosynthesis, transpiration, chlorophylls, RUBISCO and total soluble protein content was performed in leaves from the different treatments. The best results were those of explants cultured at 600 &mgr;mol CO2 mol-1 on 20 g l-1 of sucrose for the first 20 days and then transferred to sucrose-free medium until the end of the culture period. Increasing CO2 to 2000 &mgr;mol CO2 mol-1 in the atmosphere of the culture vessel reduced all the parameters studied. Photosynthesis of autotrophically developed explants trebled that of the reference heterotrophic explants, as there was an apparent inverse relationship between photosynthesis and transpiration. Photosynthesis was saturated at 300 &mgr;mol m-2 s-1 PPFD and 600 &mgr;mol CO2 mol-1. Chlorophylls and RUBISCO presented differences between treatments, mainly between different CO2 concentrations, with the highest values in autotrophically cultured explants. Explants grown at 2000 &mgr;mol CO2 mol-1 showed the lowest RUBISCO/Prots ratio, probably due to negative adaptation of RUBISCO to long-term high CO2. In short, explants grown in a controlled microenvironment, with increased CO2 and under autotrophic conditions, developed wholly functional photosynthetic apparatus well prepared to be transferred to ex vitro conditions, which has many advantages in micropropagation.

In recent years, the use of woody plants in phytoremediation has gained popularity due to their high biomass production and their association with mycorrhizal fungi, which can improve their survival and development rates under stress conditions. In this study, mycorrhized and non-mycorrhized white birch plants (Betula pubescens Ehr.) were grown in control and a metal-polluted industrial soil. After 60days of culture, plant growth and metal accumulation, the content of photosynthetic pigments and oxidative-stress markers, as well as the enzymatic activities and gene expressions of antioxidant enzymes were measured. According to our results, mycorrhized birch plants grown in control soil showed an increased activity and gene expression of catalase and ascorbate peroxidase, along with hydrogen peroxide overproduction, which could support the importance of the reactive oxygen species as signaling molecules in the regulation of plant-fungus interactions. Additionally, in polluted soil mycorrhized plants had higher biomass but lower metal accumulation, probably because the symbiotic fungus acted as a barrier to the entrance of metals into the host plants. This behavior led to mitigation in the oxidative challenge, reduced hydrogen peroxide content and diminished activities of the antioxidant enzymes in comparison to non-mycorrhized plants.