Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs are widespread in the bacterial kingdom except for the industrially and medicinally important Actinobacteria. While Ms1 RNA was identified in Mycobacterium, it is not clear whether Ms1 RNA is present also in other Actinobacteria species. Here, using a computational search based on secondary structure similarities combined with a linguistic gene synteny approach, we identified Ms1 RNA in Streptomyces. In S. coelicolor, Ms1 RNA overlaps with the previously annotated scr3559 sRNA with an unknown function. We experimentally confirmed that Ms1 RNA/scr3559 associates with the RNAP core without the primary sigma factor HrdB in vivo. Subsequently, we applied the computational approach to other Actinobacteria and identified Ms1 RNA candidates in 824 Actinobacteria species, revealing Ms1 RNA as a widespread class of RNAP binding sRNAs, and demonstrating the ability of our multifactorial computational approach to identify weakly conserved sRNAs in evolutionarily distant genomes.
- Publikační typ
- časopisecké články MeSH
RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.
- MeSH
- bakteriální proteiny chemie metabolismus ultrastruktura MeSH
- DNA bakterií chemie metabolismus MeSH
- DNA řízené RNA-polymerasy chemie metabolismus ultrastruktura MeSH
- elektronová kryomikroskopie MeSH
- katalytická doména MeSH
- molekulární modely MeSH
- Mycobacterium smegmatis enzymologie MeSH
- nukleové kyseliny metabolismus MeSH
- proteinové domény MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Specific quantification of root-colonizing arbuscular mycorrhizal fungi (AMF) by quantitative real-time PCR is a high-throughput technique, most suitable for determining abundances of AMF species or isolates in previously characterized experimental systems. The principal steps are the choice and validation of an appropriate assay to specifically amplify a gene fragment of the target AMF, preparation of templates from root samples, and quantification of the fungal gene copy numbers in these templates. The use of a suitable assay is crucial for a correct data collection but also highly specific for each experimental system and is therefore covered by general recommendations. Subsequently, specific steps are described for the validation of the assay using a standard dilution series, the determination of appropriate dilutions of DNA extracts from roots, and the quantification of the gene copy numbers in samples including calculations.
- MeSH
- DNA fungální genetika izolace a purifikace MeSH
- genová dávka genetika MeSH
- kořeny rostlin genetika mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- mykorhiza genetika izolace a purifikace MeSH
- půda MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, PMs1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding β and β' subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.
- MeSH
- bakteriální RNA metabolismus MeSH
- delece genu MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- malá nekódující RNA genetika metabolismus MeSH
- Mycobacterium smegmatis enzymologie genetika růst a vývoj metabolismus MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The relationship between mycorrhiza functioning and composition of arbuscular mycorrhizal (AM) fungal communities is an important but experimentally still rather little explored topic. The main aim of this study was thus to link magnitude of plant benefits from AM symbiosis in different abiotic contexts with quantitative changes in AM fungal community composition. A synthetic AM fungal community inoculated to the model host plant Medicago truncatula was exposed to four different abiotic contexts, namely drought, elevated phosphorus availability, and shading, as compared to standard cultivation conditions, for two cultivation cycles. Growth and phosphorus uptake of the host plants was evaluated along with the quantitative composition of the synthetic AM fungal community. Abiotic context consistently influenced mycorrhiza functioning in terms of plant benefits, and the effects were clearly linked to the P requirement of non-inoculated control plants. In contrast, the abiotic context only had a small and transient effect on the quantitative AM fungal community composition. Our findings suggest no relationship between the degree of mutualism in AM symbiosis and the relative abundances of AM fungal species in communities in our simplified model system. The observed progressive dominance of one AM fungal species indicates an important role of different growth rates of AM fungal species for the establishment of AM fungal communities in simplified systems such as agroecosystems.
After abandonment of agricultural fields, some grassland plant species colonize these sites with a frequency equivalent to dry grasslands (generalists) while others are missing or underrepresented in abandoned fields (specialists). We aimed to understand the inability of specialists to spread on abandoned fields by exploring whether performance of generalists and specialists depended on soil abiotic and/or biotic legacy. We performed a greenhouse experiment with 12 species, six specialists and six generalists. The plants were grown in sterile soil from dry grassland or abandoned field inoculated with microbial communities from one or the other site. Plant growth, abundance of mycorrhizal structures and plant response to inoculation were evaluated. We focused on arbuscular mycorrhizal fungi (AMF), one of the most important parts of soil communities affecting plant performance. The abandoned field soil negatively affected plant growth, but positively affected plant response to inoculation. The AMF community from both sites differed in infectivity and taxa frequencies. The lower AMF taxa frequency in the dry grassland soil suggested a lack of functional complementarity. Despite the fact that dry grassland AMF produced more arbuscules, the dry grassland inoculum did not improve phosphorus nutrition of specialists contrary to the abandoned field inoculum. Inoculum origin did not affect phosphorus nutrition of generalists. The lower effectiveness of the dry grassland microbial community toward plant performance excludes its inoculation in the abandoned field soil as a solution to allow settlement of specialists. Still, the distinct response of specialists and generalists to inoculation suggested that they differ in AMF responsiveness.
- MeSH
- houby MeSH
- kořeny rostlin MeSH
- mikrobiota * MeSH
- mykorhiza * MeSH
- pastviny MeSH
- půda MeSH
- půdní mikrobiologie MeSH
- vývoj rostlin MeSH
- Publikační typ
- časopisecké články MeSH
The effects of inoculation with an arbuscular mycorrhizal (AM) fungus on Cd and Ni tolerance and uptake in Medicago sativa, an AM host, and Sesuvium portulacastrum, a non-host plant, were investigated in a greenhouse experiment. The plants were cultivated in sterilized sand in a two-compartmented system, which prevented root competition but enabled colonization of the whole substrate by AM fungal extraradical mycelium. M. sativa was either left non-inoculated or inoculated with the AM fungus Rhizophagus irregularis, and both plants were either cultivated without heavy metal (HM) addition or supplied with cadmium (Cd) or nickel (Ni), each in two doses. Additional pots with singly cultivated plants were established to control for the effect of the co-cultivation. AM significantly enhanced the growth of M. sativa and substantially increased its uptake of both HMs. The roots of S. portulacastrum became colonized by AM fungal hyphae and vesicles. The presence of the AM fungus in the cultivation system tended to increase the HM uptake of S. portulacastrum, but the effect was less consistent and pronounced than that in M. sativa. We conclude that AM fungal mycelium radiating from M. sativa did not negatively affect the growth and HM uptake of S. portulacastrum. On the contrary, we hypothesize that it stimulated the absorption and translocation of Cd and Ni in the non-host species. Thus, our results suggest that AM fungal mycelium radiating from mycorrhizal plants does not decrease the HM uptake of non-host plants, many of which are considered promising candidate plants for phytoremediation.
- MeSH
- Aizoaceae * metabolismus mikrobiologie MeSH
- Glomeromycota fyziologie MeSH
- kadmium metabolismus MeSH
- kořeny rostlin metabolismus MeSH
- Medicago sativa * metabolismus mikrobiologie MeSH
- mycelium fyziologie MeSH
- mykorhiza fyziologie MeSH
- nikl metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Quantification of carbon (C) fluxes in mycorrhizal plants is one of the important yet little explored tasks of mycorrhizal physiology and ecology. (13)CO2 pulse-chase labelling experiments are increasingly being used to track the fate of C in these plant-microbial symbioses. Nevertheless, continuous monitoring of both the below- and aboveground CO2 emissions remains a challenge, although it is necessary to establish the full C budget of mycorrhizal plants. Here, a novel CO2 collection system is presented which allows assessment of gaseous CO2 emissions (including isotopic composition of their C) from both belowground and shoot compartments. This system then is used to quantify the allocation of recently fixed C in mycorrhizal versus nonmycorrhizal Medicago truncatula plants with comparable biomass and mineral nutrition. Using this system, we confirmed substantially greater belowground C drain in mycorrhizal versus nonmycorrhizal plants, with the belowground CO2 emissions showing large variation because of fluctuating environmental conditions in the glasshouse. Based on the assembled (13)C budget, the C allocation to the mycorrhizal fungus was between 2.3% (increased (13)C allocation to mycorrhizal substrate) and 2.9% (reduction of (13)C allocation to mycorrhizal shoots) of the plant gross photosynthetic production. Although the C allocation to shoot respiration (measured during one night only) did not differ between the mycorrhizal and nonmycorrhizal plants under our experimental conditions, it presented a substantial part (∼10%) of the plant C budget, comparable to the amount of CO2 released belowground. These results advocate quantification of both above- and belowground CO2 emissions in future studies.
- MeSH
- fotosyntéza fyziologie MeSH
- Glomeromycota fyziologie MeSH
- kořeny rostlin metabolismus MeSH
- Medicago truncatula metabolismus mikrobiologie MeSH
- mykorhiza metabolismus MeSH
- oxid uhličitý chemie metabolismus MeSH
- uhlík metabolismus MeSH
- výhonky rostlin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Inoculation with arbuscular mycorrhizal fungi (AMF) may improve plant performance at disturbed sites, but inoculation may also suppress root colonization by native AMF and decrease the diversity of the root-colonizing AMF community. This has been shown for the roots of directly inoculated plants, but little is known about the stability of inoculation effects, and to which degree the inoculant and the inoculation-induced changes in AMF community composition spread into newly emerging seedlings that were not in direct contact with the introduced propagules. We addressed this topic in a greenhouse experiment based on the soil and native AMF community of a post-mining site. Plants were cultivated in compartmented pots with substrate containing the native AMF community, where AMF extraradical mycelium radiating from directly inoculated plants was allowed to inoculate neighboring plants. The abundances of the inoculated isolate and of native AMF taxa were monitored in the roots of the directly inoculated plants and the neighboring plants by quantitative real-time PCR. As expected, inoculation suppressed root colonization of the directly inoculated plants by other AMF taxa of the native AMF community and also by native genotypes of the same species as used for inoculation. In the neighboring plants, high abundance of the inoculant and the suppression of native AMF were maintained. Thus, we demonstrate that inoculation effects on native AMF propagate into plants that were not in direct contact with the introduced inoculum, and are therefore likely to persist at the site of inoculation.
Root colonization by arbuscular mycorrhizal fungi (AMF) can be quantified by different approaches. We compared two approaches that enable discrimination of specific AMF taxa and are therefore emerging as alternative to most commonly performed microscopic quantification of AMF in roots: quantitative real-time PCR (qPCR) using markers in nuclear ribosomal DNA (nrDNA) and mitochondrial ribosomal DNA (mtDNA). In a greenhouse experiment, Medicago truncatula was inoculated with four isolates belonging to different AMF species (Rhizophagus irregularis, Claroideoglomus claroideum, Gigaspora margarita and Funneliformis mosseae). The AMF were quantified in the root samples by qPCR targeted to both markers, microscopy and contents of AMF-specific phospholipid fatty acids (PLFA). Copy numbers of nrDNA and mtDNA were closely related within all isolates; however, the slopes and intercepts of the linear relationships significantly differed among the isolates. Across all isolates, a large proportion of variance in nrDNA copy numbers was explained by root colonization intensity or contents of AMF-specific PLFA, while variance in mtDNA copy numbers was mainly explained by differences among AMF isolates. We propose that the encountered inter-isolate differences in the ratios of mtDNA and nrDNA copy numbers reflect different physiological states of the isolates. Our results suggest that nrDNA is a more suitable marker region than mtDNA for the quantification of multiple AMF taxa as its copy numbers are better related to fungal biomass across taxa than are copy numbers of mtDNA.
- MeSH
- buněčné jádro genetika MeSH
- DNA fungální genetika MeSH
- Glomeromycota genetika MeSH
- kořeny rostlin mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce * MeSH
- Medicago truncatula mikrobiologie MeSH
- mitochondriální DNA genetika MeSH
- mykorhiza genetika MeSH
- Publikační typ
- časopisecké články MeSH
