Search

Ferrous slag produced by a historic smelter is washed from a slagheap and transported by a creek through a cave system. Slag filling cave spaces, abrasion of cave walls / calcite speleothems, and contamination of the aquatic environment with heavy metals and other toxic components are concerns. We characterize the slag in its deposition site, map its transport through the cave system, characterize the effect of slag transport, and evaluate the risks to both cave and aqueous environments. The study was based on chemical and phase analysis supported laboratory experiments and geochemical modeling. The slag in the slagheap was dominated by amorphous glass phase (66 to 99 wt%) with mean composition of 49.8 ± 2.8 wt% SiO

This article brings new information about phenomenon of the growth of floodplain forest without influence of floods over 50 years. Floodplain forest and its hydrogeological regime are under detail research for 10 years and detail 10 years data from hydrogeological well is used in this study. The area of interest belongs to nature reserve Plackuv les what together with brook Satava represents a unique relic of a floodplain forest in the confluence area of the rivers Svratka and Jihlava in southern Moravia (South-Moravian Region). The damming of the channel of the river Svratka has caused the end of floods. Despite the anthropogenic regulations performed, the hydrogeological conditions have remained preserved at the site. Stream channel was rised due to the regulation at the above the level of the existing ground; this eliminated its drainage functions for groundwater. The contact between the base of floodplain loams and the groundwater Tab. is not lost and the capillary rise in them remains preserved. Analysis of groundwater level confirmed multi-component refilling of the hydrogeological structure, which is drained in Plackuv les and the river Satava. All last 10 years, the maximum of the groundwater level measured is from July to September. It means, that groundwater reaches the highest level in the period, when the minimum flow rates are recorded on the surface flows. In this period, the locality displays the maximal infiltration of the precipitation from the last hydrological year from distant infiltration area. Because of the absence of floods, groundwater at this site is the most significant resource of water, and it represents a key-factor which enables the growth and thriving of the floodplain forest ecosystem. info:eu-repo/semantics/openAccess

The objective of this study is to investigate the potential causes of widespread Larix sibirica Ledeb. mortality observed in the Khentii massif of northern Mongolia. The ratio of deadwood to living trees in affected stands in the Goricho region, the southernmost study site situated close to the Gobi Desert, was as high as 3.6:1. Moisture fluctuations monitored over 2 years using electrical impedance spectrometry revealed that the Goricho study site had higher soil moisture levels than the two less affected sites Barun Bayan and Dzun Bayan. High soil moisture was recorded in an area characterized by highly skeletal soils, ones with more than 35% by volume of rock fragments, and comparatively shallow soil horizons, from valley to mountains. The layer of permafrost influencing hydrogeological processes is much deeper in the Goricho region compared to the undisturbed study sites. Redundancy analysis confirmed a significant number of dead L. sibirica on sites with developed soils. Live forest stands, however damaged, grow in this region on well-drained scree slopes or on rocky bastions. The mass mortality observed for L. sibirica may be directly linked to accelerated permafrost thaw in the area bordered by the Tuul and the Terelj Rivers. Our assumption is that L. sibirica root system necrosis occurred as a result of long-term waterlogging of developed soils with high spatial heterogeneity, normally able to absorb high quantities of groundwater. The areas unaffected were scree fields and rocky bastions characterized by adequate drainage. All of our findings support the primary stages of large-scale permafrost thaw, i.e., correlating increases in soil moisture with increasing permafrost active layer thickness.

Anthropogenic modifications to water regimes are one of the main factors threatening the stability and existence of floodplain forests. This study presents an analysis of topsoil biogeochemistry within three floodplain forest stands with different levels of human alteration. Decreasing contents of soil organic carbon (OC) and microbial biomass were observed along the gradient from natural to plantation forest. High annual variations were observed in soil N contents and in microbial biomass, while comparable spatial variations were observed within the natural forest. High ground-water levels resulted in increased accumulation of available Na+ and SO42− in the natural forest soil, yet the concentrations of ions were at sub-saline levels. The increasing contents of available Mn, SO42− or Cl− had mostly positive effects on soil microbial activity across the sites, though the results indicate the existence of a certain ecological limit for soil microorganisms. Reintroduction of surface-water flooding should be considered in future forest and water management to promote the dilution of ions accumulated in soils and natural deposition of sediments rich in organic matter (OM) at the sites.

The in vivo rituximab effects in B cell malignancies are only partially understood. Here we analyzed in a large chronic lymphocytic leukemia (CLL) cohort (n = 80) the inter-patient variability in CLL cell count reduction within the first 24 h of rituximab administration in vivo, and a phenomenon of blood repopulation by malignant cells after anti-CD20 antibody therapy. Larger CLL cell elimination after rituximab infusion was associated with lower pre-therapy CLL cell counts, higher CD20 levels, and the non-exhausted capacity of complement-dependent cytotoxicity (CDC). The absolute amount of cell-surface CD20 molecules (CD20 density x CLL lymphocytosis) was a predictor for complement exhaustion during therapy. We also describe that a highly variable decrease in CLL cell counts at 5 h (88 %–2%) following rituximab infusion is accompanied in most patients by peripheral blood repopulation with CLL cells at 24 h, and in ∼20 % of patients, this resulted in CLL counts higher than before therapy. We provide evidence that CLL cells recrudescence is linked with i) CDC exhaustion, which leads to the formation of an insufficient amount of membrane attack complexes, likely resulting in temporary retention of surviving rituximab-opsonized cells by the mononuclear-phagocyte system (followed by their release back to blood), and ii) CLL cells regression from immune niches (CXCR4dimCD5bright intraclonal subpopulation). Patients with major peripheral blood CLL cell repopulation exhibited a longer time-to-progression after chemoimmunotherapy compared to patients with lower or no repopulation, suggesting chemotherapy vulnerability of CLL cells that repopulate the blood.

The use of the anti-CD20 antibody rituximab has improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab was shown to act via various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct apoptosis, or sensitization to chemotherapy (reviewed in ref. [1]). However, the biological function of CD20 and the reasons for the impressive activity of rituximab and other anti-CD20 antibodies remain elusive. It has been suggested, but remains controversial, whether CD20 functions as a calcium channel, couples with CD40 and MHCII, or B-cell receptor (BCR), and whether it has a role in T cell-dependent and -independent immunity [1,2,3,4]. This is also of great clinical interest for the design of combinatorial treatment of rituximab with BCR inhibitors, DNA damaging or immunomodulatory drugs, or CAR T cells. We have previously shown that microenvironmental interactions upregulate the CD20 levels on CLL cells through the CXCR4/SDF-1 axis [5]. Here we describe that higher CD20 expression has a direct role in the BCR signaling in CLL cells, and a BCR-proficient intra-clonal CLL cell subpopulation is more efficiently eliminated by rituximab in vivo due to higher CD20 levels.

Histone modifications have a profound impact on the chromatin structure and gene expression and their correct establishment and recognition is essential for correct cell functioning. Malfunction of histone modifying proteins is associated with developmental defects and diseases and detailed characterization of these proteins is therefore very important. The lysine specific demethylase KDM2A is a CpG island binding protein that has been studied predominantly for its ability to regulate CpG island-associated gene promoters by demethylating their H3K36me2. However, very little attention has been paid to the alternative KDM2A isoform that lacks the N-terminal demethylation domain, KDM2A-SF. Here we characterized KDM2A-SF more in detail and we found that, unlike the canonical full length KDM2A-LF isoform, KDM2A-SF forms distinct nuclear heterochromatic bodies in an HP1a dependent manner. Our chromatin immunoprecipitation experiments further showed that KDM2A binds to transcriptionally silent pericentro...

International audience; Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria-BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed -propeller fold. Functional studies conducted on a series of oligo-and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for β-L-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

Waste rock dumps in the aftermath of pegmatite mining enable the growth of healthy populations of Larix sibirica in, for the forest unsuitable semi-arid steppe habitat, the Khentii Mountains, Mongolia. Trees can thrive in such extreme habitats due to water vapour condensation and adsorption in the topsoil of a dump. During the growing season 2015–2017, continual measurement of thermal regime (hourly) was carried out at the depths of 10, 30 and 60 cm in the dumps and surrounding steppes. At the depth of 10 cm, temperatures (up to 40% of cases) enabling dew formation in dumps, in the time period of the highest possible air humidity (2 a.m. to 8 a.m.) were confirmed there. The suitable temperature conditions to form dew were found primarily at the beginning of the growing season characterized by a low precipitation. Later in the growing season with the following increasing precipitation, the temperature potential of dumps for dew formation decreased continuously. The cross-correlation indicated active exchange of heat between the atmosphere and the dump up to the depth of 30 cm. The outcomes further indicate that water vapour adsorption shall contribute significantly to the moisture regime of dumps and the effective porosity of sandy soils of dumps is likely to form ideal conditions for it. The construction thickness of the cover soil material between the depths of 30 and 60 cm with the predefined physico-chemical properties can be recommended to augment the potential of the dew yield in future projects. Effective water vapour condensation and adsorption may support success of landscape afforestation or reclamation of mining areas in arid or semi-arid conditions.

Chemical diversity: Two SAM-dependent N-methyltransferases-LmbJ from the biosynthesis of the antibiotic lincomycin and CcbJ from celesticetin biosynthesis-have been characterized and compared. Both tested enzymes form multimers and are able to utilize N-demethyllincomycin, the natural substrate of LmbJ, with comparable efficiency.

Members of the Actinobacteria are among the most important litter decomposers in soil. The site of a waterlogged deciduous forest with acidic soil was explored for actinobacteria because seasonality of litter inputs, temperature, and precipitation provided contrasting environmental conditions, particularly variation of organic matter quantity and quality. We hypothesized that these factors, which are known to influence decomposition, were also likely to affect actinobacterial community composition. The relationship between the actinobacterial community, soil moisture and organic matter content was assessed in two soil horizons in the summer and winter seasons using a 16S rRNA taxonomic microarray and cloning-sequencing of 16S rRNA genes. Both approaches showed that the community differed significantly between horizons and seasons, paralleling the changes in soil moisture and organic matter content. The microarray analysis further indicated that the actinobacterial community of the upper horizon was characterized by high incidence of the genus Mycobacterium. In both horizons and seasons, the actinobacterial clone libraries were dominated (by 80%) by sequences of a separate clade sharing an ancestral node with Streptosporangineae. This relatedness is supported also by some common adaptations, for example, to soil acidity and periodic oxygen deprivation or dryness.

The diversity of environmental mycobacteria was studied in water-logged acidic forest soil. Mycobacteria were assessed in upper and lower soil horizons and summer and winter seasons using T-RFLP and sequencing of 16S rRNA gene produced with Mycobacterium-specific primers. Mycobacteria diversity differed between both the two seasons and soil horizons. Cloning revealed the presence of mycobacteria belonging to three major clusters recognized within the genus, i.e. fast-growing, intermediate, and slow-growing species, with unprecedented abundance and diversity of the latter. Two novel clusters of sequences unrelated to the known mycobacteria were identified. This study raises the possibility that forest wetlands could serve as environmental reservoirs for an unexplored diversity of mycobacteria including those related to pathogenic species.

The hallmark of chronic lymphocytic leukemia (CLL) cells is their re-circulation between peripheral blood and immune niches to obtain pro-proliferative and pro-survival signals. CLL cells that have recently exited the immune niches to the peripheral blood are characterized by low cell-surface levels of chemokine receptor CXCR4 and high levels of activation molecule CD5. These CXCR4dimCD5bright CLL cells have a ~2-fold higher CD20 expression due to the activation of the CXCR4/SDF-1 axis (Pavlasova et al., Blood, 2016). We hypothesized that CD20 up-regulation in the context of a microenvironment is required for some functional regulation. We hypothesized that CD20 expression is of importance for B-cell receptor (BCR) signaling as we observed that CXCR4dimCD5brightCD20bright CLL cells have also ~2-fold higher surface IgM levels (P Citation Format: Gabriela Pavlasova, Marek Borsky, Veronika Svobodova, Jan Oppelt, Katerina Cerna, Jitka Novotna, Katerina Musilova, Vaclav Seda, Eva Vojackova, Yvona Brychtova, Michael Doubek, Sarka Pospisilova, Jiri Mayer, Marek Mraz. CD20 supports BCR signaling in an intra-clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR-proficient B cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1012.

The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl- l -proline. Feeding this mutant strain with precursor derivatives resulted in production of 4′-butyl-4′-depropyllincomycin and 4′-pentyl-4′-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4′-pentyl-4′-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.

The first insight into celesticetin biosynthetic gene cluster of S. caelestis is presented. The genomic DNA of producing strain was digested, digoxigenin-labeled and hybridized with a set of probes designed according to S. lincolnensis gene sequences. Genes with high homology to the lincomycin biosynthetic genes coding for the predicted common parts of the pathway were identified in S. caelestis. Then, genomic DNA of S. caelestis treated by a multiple digestion was hybridized with five digoxigenin-labeled probes to construct a rough restriction map. Two consecutive islands formed by the genes with a putative function in biosynthesis of the shared saccharide moiety revealed an organization similar to the lincomycin biosynthetic gene cluster. The celesticetin cluster was mapped and essential information was obtained for subsequent steps, i.e. isolation and sequence analysis of the cluster.

An assay of L-tyrosine (Tyr) hydroxylating activity operating in lincomycin biosynthesis is described. The assay development consisted of HPLC procedure development, assessing the effect of reaction mixture components on non-enzymatic Dopa and Tyr oxidation, and sample stability evaluation. The HPLC procedure with isocratic elution and fluorescence detection was developed and validated. The method showed a wide linear range of Dopa determination of 0.125-25 micromol/L with lower limit of quantification (LLOQ) of 0.125 micromol/L, RSD of 7.2% and accuracy of 101.7%. The studied linear range of Tyr was 15.625 mmol/L to 500 mmol/L with LLOQ of 15.625 mmol/L, RSD of 1.1%, and accuracy of 98.1%. Recoveries for Dopa and Tyr were 100.66 +/- 0.89% and 94.76 +/- 0.94%, respectively. The inter- and intra-day accuracies and precisions were all within 10%. Samples of the reaction mixture were stable for at least 24 h at room temperature (RT) and 28 days at -20 degrees C. The method was tested for the enzyme activity monitoring in purified as well as crude preparations and enabled micro preparation of the enzyme product during confirmation of its identity. The influence of pH and ascorbic acid content in reaction mixture was studied with respect to non-enzymatic Tyr oxidation.

Structurally different and functionally diverse natural compounds – antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin – share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C–C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C–C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agreement with its proposed structure, 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3-extradiol cleavage of the l-3,4-dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3-secodopa. A possible role of LmbB1 in 2,3-secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.

The lincomycin-production gene cluster of the industrial overproduction strain Streptomyces lincolnensis 78-11 has been sequenced (Peschke et al. 1995) and twenty-seven putative open reading frames with biosynthetic or regulatory functions (lmb genes) identified. Two distinct hypothetical genes, lmbI and lmbH, were found downstream of the lmbJ gene, coding for LmbJ protein, which is believed to participate in the last lincomycin biosynthetic step, i.e. conversion of N-demethyllincomycin (NDL) to lincomycin. In the present study, we demonstrate the presence of a single larger open reading frame, called lmbIH, in the lincomycin low-production type strain Streptomyces lincolnensis ATCC 25466, instead of two smaller lmbI and lmbH genes. The product, LmbIH, is a protein of an unknown function and is homologous with the TldD protein family. Escherichia coli TldD protein was previously shown to be involved in the control of DNA gyrase by LetD protein. Moreover, our experiments indicate co-regulation of lmbJ and lmbIH expression. This translation coupling probably reflects an eight nucleotide overlap between the lmbJ and lmbIH genes, as well as the lack of a Shine-Dalgarno sequence upstream of the lmbIH gene.

The genes lmbA,B1,B2 in the lincomycin A production gene cluster of Streptomyces lincolnensis were shown to form a common transcription unit with the promoter located directly upstream of lmbA. The proteins LmbB1 (mol. mass, 18 kDa) and LmbB2 (mol. mass 34 kDa), when over-produced together in Escherichia coli, brought about enzyme activities for the specific conversion of both l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA) to a yellow-colored product. The LmbB1 protein alone catalyzed the conversion of l-DOPA, but not of l-tyrosine. The purified LmbB1 protein showed a K m for l-DOPA of 258.3 μM. The l-tyrosine converting activity could not been demonstrated in vitro. The preliminary interpretation of these data suggests that the protein LmbB1 is an l-DOPA extradiol-cleaving 2,3-dioxygenase and that the protein LmbB2, either alone or in accord with LmbB1, represents an l-tyrosine 3-hydroxylase. This sequence of putative oxidation reactions on l-tyrosine seems to represent a new pathway different from the ones catalyzed by mammalian l-tyrosine hydroxylases or the wide-spread tyrosinases. The protein LmbA seemed not to be involved in this process. The labile, yellow-colored product from l-DOPA could not be converted to a picolinic acid derivative [3-(2-carboxy-5-pyridyl)alanine] in the presence of ammonia. Therefore, it probably is not a derivative of a cis,cis-3-hydroxymuconic acid semialdehyde; instead, its speculative structure represents a heterocyclic precursor of the propylhygric acid moiety of lincomycin A.

Determination of polynuclear aromatic hydrocarbons in air was carried out in two monitoring sites of ‘Program Teplice’ throughout the year 1993. The town of Teplice is located in an industrialized region of the Northern Bohemia, Prachatice, a small town in Southern Bohemia, was chosen as reference. Measurement for PAHs was done in air at both locations and in bulk deposition at Teplice. Analysis of PAHs was done in air by HPLC with fluorescence monitor and in bulk deposition by CGC‐MS. Difference in the mean PAHs levels in air is not significant; particularly when taking benzo(a)pyrene as an indicator compound. Higher concentrations of PAHs were observed during the winter heating period at both locations.

Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 microM benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.

Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO 3 or purification of extracted DNA by CaCl 2 . The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl 2 purification and in 93% of cases by the method based on CaCO 3 pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agreement with its proposed structure, 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3-extradiol cleavage of the l-3,4-dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3-secodopa. A possible role of LmbB1 in 2,3-secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.

The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.

Publisher Summary Natural lincosamides are produced by several Streptomyces species, mainly by Streptomyces lincolnensis, S. roseolus, and S. caelestis and by Micromonospora halophytica. Lincosamides constitute a relatively small group of antibiotics with a chemical structure consisting of amino acid and sugar moieties. Their mechanism of action is via inhibition of protein synthesis in sensitive micro-organisms. Lincosamides have an unusual antimicrobial spectrum, being active against only Gram-positive and not Gram-negative aerobic bacteria. They exhibit a significant antibiotic activity against some anaerobic bacteria. They are used therapeutically, especially in cases where synergistic effects of a mixed anaerobic and aerobic microflora are anticipated. Lincomycin and clindamycin are also useful alternatives to penicillin and its derivatives in the treatment of upper respiratory tract infections in patients with allergy to penicillin. The chapter also discusses the major route of resistance to lincosamides. Furthermore, with the current knowledge of gene clusters specifying biosynthesis of lincomycin and the related antibiotic celesticetin, production of new derivatives of lincosamides by genetic engineering can be easily imagined, resulting in production of new derivatives of lincosamides.