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Suicide gene therapy with the herpes simplex virus type-1 thymidine kinase gene (HSV-1 tk) is considered to be a promising approach to the treatment of cancer. Making use of the lower specificity of the viral enzyme compared to human thymidine kinase, the therapy involves the administration of antiviral agents (e.g., ganciclovir) as prodrugs to induce enzymatic cell death in those cells that express the transferred gene. 18F-labelled derivatives have been described for monitoring location, duration, and magnitude of the viral kinase enzyme activity by positron emission tomography (PET). Since an optimal radiotracer has not been developed, novel substances were synthesized for monitoring gene expression. A group of 13 nucleoside analogues were synthesized, among them N1-methyl-9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (5) and N1-methyl-9-[(4-hydroxy)-3-hydroxymethylbutyl]guanine (7) as methyl analogues of ganciclovir and penciclovir and their related fluoro compounds (6, 8). Further novel derivatives include N6-methyl-9-[(1,3-dihydroxy-2-propoxy)methyl]-, N6-methyl-9-[(4-hydroxy)-3-hydroxymethylbutyl]adenine (9, 10), as well as the uracil derivatives 5-hydroxy-1-[(1,3-dihydroxy-2-propoxy)methyl]uracil (11), 6-methyl-1-[(1,3-dihydroxy-2-propoxy)-methyl]uracil (12), and its 3-fluoro-derivative (13).
Copper-based fungicides are routinely used for wood and plant protection, which can lead to an enrichment of copper-tolerant microbial communities in soil. To investigate the effect of such wood preservatives on the soil fungal and bacterial community compositions, five different vineyard and fruit-growing soil environments were evaluated using incubation studies over time. Pine sapwood specimens were impregnated with either water or different biocide treatment solutions containing a mixture of copper, triazoles, and quaternary ammonium compounds (CuTriQAC), a mixture of triazoles and quaternary ammonium compounds (TriQAC), or copper alone (Cu). Specimens were incubated in soil from each sample site for 8, 16, 24, and 32 weeks. The effects of preservative treatment on the modulus of elasticity (MOE) of the wood specimens and on the soil fungal as well as bacterial community composition at the soil-wood interface were assessed by quantitative PCR and amplicon sequencing of the fungal internal transcribed spacer (ITS) region and bacterial 16S rRNA gene. Specimens impregnated with CuTriQAC and Cu showed decreased MOE and reduced fungal and bacterial copy numbers over time compared to those impregnated with water and TriQAC. Fungal but not bacterial community composition was significantly affected by wood preservative treatment. The relative abundance of members of the family Trichocomaceae compared to other genera increased in the presence of the Cu and CuTriQAC treatments at three sites, suggesting these to be Cu-tolerant fungi. In conclusion, the copper-containing treatments resulted in marginally increased MOE, lowered microbial gene copy numbers compared to those in the TriQAC and water treatments, and thus enhanced wood protection against soil microbial wood degradation
Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH4 into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH4 flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using 13C-labelled CH4 directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to 13CH4 for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.
As a glacier retreats, it leaves behind it a forefield that has a natural gradient of soil formation age. We systematically sampled the Damma glacier forefield (Switzerland) along a temporal gradient of soils deglaciated between 1956 and 2002. A significant change in organic carbon content, sulfate concentration, pH, water and nucleic acid content was observed along the forefield chronosequence. Based on 16S rRNA gene based fingerprinting, the structure of the bacterial community also shifted along the forefield. Shifts in the structure of the bacterial community were significantly correlated to changes of pH, soil water content and soil age. To test the impacts of an array of environmental variables including soil age, soil water content, and different anions on the structure of the bacterial community we incubated different glacier forefield soils under specific treatment conditions for 30 days at 25 °C. The incubation of recently deglaciated soils resulted into an increase of bacteria from the family Methylocystaceae and from the class Betaproteobacteria while the community composition from matured soil changed to a lesser extent. The total nitrogen concentration in matured soil doubled during incubation, whereas the nitrogen concentrations in recently deglaciated soil stayed constant. This suggested that the microbial ecosystem functioned differently in the mature versus the recently deglaciated soils. Only soil age and soil water content could be singled out as having significant effects on the structure and composition of the bacterial community, despite the fact that bacterial communities in glacier forefields are exposed to other steep environmental gradients.
Most of the methane (CH4) emission from rice fields is derived from plant photosynthates,
which are converted to CH4. Rice cluster I (RC-1) archaea colonizing the
rhizosphere were found to be the methanogens responsible for this process. Hence, RC-1
methanogens seem to play a crucial role in emission of the greenhouse gas CH4. We
determined the community composition and activity of methanogens colonizing the
roots of eight different rice cultivars after growth on both Italian rice soil and river bank
soil, which contained different communities of methanogenic archaea. The community
composition was analyzed by terminal restriction fragment length polymorphism and
cloning/sequencing of the archaeal 16S rRNA gene and the mcrA gene coding for a
subunit of the methyl coenzyme M reductase. When grown on rice field soil, the
methanogenic community of the different rice cultivars was always dominated by RC-1
methanogens. In contrast, roots were colonized by Methanomicrobiales when grown on
river bank soil, in which RC-1 methanogens were initially not detectable. Roots
colonized with Methanomicrobiales compared with RC-1 exhibited lower CH4 production
and CH4 emission rates. The results show that the type of methanogens colonizing
rice roots has a potentially important impact on the global CH4 cycle.
The microbial community structure was investigated together with the path of methane production in Italian rice field soil incubated at moderate (35 °C) and high (45 °C) temperature using terminal restriction fragment length polymorphism and stable isotope fractionation. The structure of both the archaeal and bacterial communities differed at 35 °C compared with 45 °C, and acetoclastic and hydrogenotrophic methanogenesis dominated, respectively. Changing the incubation of the 45 °C soil to different temperatures (25, 30, 35, 40, 45, 50 °C) resulted in a dynamic change of both microbial community structure and stable isotope fractionation. In all treatments, acetate first accumulated and then decreased. Propionate was also transiently produced and consumed. It is noteworthy that acetate was also consumed at thermophilic conditions, although archaeal community composition and stable isotope fractionation indicated that acetoclastic methanogenesis did not operate. Instead, acetate must have been consumed by syntrophic acetate oxidizers. The transient accumulation and subsequent consumption of acetate at thermophilic conditions was specifically paralleled by terminal restriction fragments characteristic for clostridial cluster I, whereas those of clostridial clusters I and III, Acidaminococcaceae and Heliobacteraceae, paralleled the thermophilic turnover of both acetate and propionate.
The microbial community in anoxic rice field soil produces CH4 over a wide temperature range up to 55°C. However, at temperatures higher than about 40°C, the methanogenic path changes from CH4 production by hydrogenotrophic plus acetoclastic methanogenesis to exclusively hydrogenotrophic methanogenesis and simultaneously, the methanogenic community consisting of Methanosarcinaceae, Methanoseataceae, Methanomicrobiales, Methanobacteriales and Rice Cluster I (RC-1) changes to almost complete dominance of RC-1. We studied changes in structure and function of the methanogenic community with temperature to see whether microbial members of the community were lost or their function impaired by exposure to high temperature. We characterized the function of the community by the path of CH4 production measuring δ13C in CH4 and CO2 and calculating the apparent fractionation factor (αapp) and the structure of the community by analysis of the terminal restriction fragment length polymorphism (T-RFLP) of the microbial 16S rRNA genes. Shift of the temperature from 45°C to 35°C resulted in a corresponding shift of function and structure, especially when some 35°C soil was added to the 45°C soil. The bacterial community (T-RFLP patterns), which was much more diverse than the archaeal community, changed in a similar manner upon temperature shift. Incubation of a mixture of 35°C and 50°C pre-incubated methanogenic rice field soil at different temperatures resulted in functionally and structurally well-defined communities. Although function changed from a mixture of acetoclastic and hydrogenotrophic methanogenesis to exclusively hydrogenotrophic methanogenesis over a rather narrow temperature range of 42-46°C, each of these temperatures also resulted in only one characteristic function and structure. Our study showed that temperature conditions defined structure and function of the methanogenic microbial community.
Four azulene dyads have been synthesized and studied by spectroscopic and electrochemical methods. A triarylamine, a boron-dipyrromethene (BDP or BODIPY), a porphyrin, and an isoalloxazine moiety have been linked to an extended π electron system at the 2-position of azulene, leading to the dyads 1–4, respectively. For the synthesis of 1–4, first 2-(4-ethynyl-phenyl)azulene (EPA) was prepared, which was further reacted with the halogenated chromophores by Pd-catalyzed cross-coupling reactions. The dyads 1–4 exhibit strong absorption bands in the visible range, which are dominated by the absorption spectra of the individual subchromophores. Fluorometric studies of 2–4 revealed that after excitation of the subchromophoric unit attached to the parent azulene moiety, quenching mainly through energy transfer to azulene is effective, whereas possible charge-transfer interactions play only a minor role. Potentiodynamic oxidation of the dyads 1–4 leads to the formation of polymer films, which are deposited at the electrode. The polymer film derived from 1 was further characterized by spectroelectrochemistry. During positive doping of poly-1, a strong absorption band appears at 13,200 cm1, which is typical for triarylamine radical cations. This band is overlapping with a broad absorption band in the low-energy region that might be caused by charge-transfer interactions within the polymer.
Bacterial community succession corresponds to changes in the phylogenetic identity, growth-response time and rRNA operon (rrn) copy number of culturable populations. To test this hypothesis, we compared the bacterial fractions culturable from the oxic zone of flooded, unplanted paddy soil microcosms after 1-day (early succession) and 70-day (late succession) incubation periods. The proportion of bacteria that was cultivable on solid media corresponded for early and late succession to 3740% and 3135% of total DAPI cell counts, which were 7.40 (±0.36) × 108 and 5.54 (±0.28) × 108 cells per gram of dry soil, respectively. In colony-forming curve analysis, late successional bacteria showed a significant delay in their growth response compared with those from early succession. A total of 59 early successional isolates grouped into 16 species-level clusters (SLC) plus three Bacilli-like SLC, while 66 late successional isolates formed 25 SLC plus five Bacilli-like SLC. Except Bacilli-like spp., isolates from early succession always belonged to different SLC than those from late succession. Betaproteobacteria and Gammaproteobacteria were typical of the early stage, while Alphaproteobacteria and Actinobacteria prevailed in late succession. Considering all SLC except those assigned to Bacilli, growth-response time and rrn copy number were significantly correlated with successional stage. Isolates of most early successional SLC (14 of 16) formed visible colonies within 1 (11 SLC) or 2 days (three SLC) and contained = 4 rrn copies. In contrast, isolates of late successional SLC (23 of 25) formed visible colonies within 2 days (four SLC) or, in most cases, only within 315 days (19 SLC) and contained = 2 rrn copies. Regardless of whether isolated from early or late succession, Bacilli-like isolates always showed a colony-forming time of 2 days and had 911 rrn copies.