Enhanced degradation of phenoxyacetic acid in soil by horizontal transfer of the tfdA gene encoding a 2,4-dichlorophenoxyacetic acid dioxygenase

Few studies have investigated the possible impact of in situ gene transfer on the degradation of xenobiotic compounds in natural environments. In this work we showed that horizontal transfer of the tfdA gene, carried on plasmid pRO103, to phenol degrading recipient strains significantly increased the degradation rate of phenoxyacetic acid in sterile and non-sterile soil microcosms. The tfdA gene encodes a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase and by complementation with the phenol degradation pathway an expanded catabolic substrate range, now including phenoxyacetic acid, is evolved. Presence of selective pressure had a positive effect on the emergence of transconjugants. However, even in the absence of phenoxyacetic acid transconjugant populations were detected and were kept at a constant level throughout the experimental period. The residuesphere (interface between decaying plant material and soil matrix) of dry leaves of barley was shown to be a hot-spot for gene transfer and presence of barley straw increased the conjugation frequencies in soil microcosms to the same extent as presence of organic nutrients. The results of this study indicate that dissemination of catabolic plasmids is a possible mechanism of genetic adaptation to degradation of xenobiotic compounds in natural environments, and that complementation of catabolic pathways possibly plays an important role in the evolution of new degradative capabilities. The application of horizontal gene transfer as a possible tool in bioremediation of contaminated sites is discussed.     

Differential effects of AM fungal isolates on Medicago truncatula growth and metal uptake in a multimetallic (Cd, Zn, Pb) contaminated agricultural soil

Toxic metal accumulation in soils of agricultural interest is a serious problem needing more attention, and investigations on soil–plant metal transfer must be pursued to better understand the processes involved in metal uptake. Arbuscular mycorrhizal (AM) fungi are known to influence metal transfer in plants by increasing plant biomass and reducing metal toxicity to plants even if diverging results were reported. The effects of five AM fungi isolated from metal contaminated or non-contaminated soils on metal (Cd, Zn) uptake by plant and transfer to leachates was assessed with Medicago truncatula grown in a multimetallic contaminated agricultural soil. Fungi isolated from metal-contaminated soils were more effective to reduce shoot Cd concentration. Metal uptake capacity differed between AM fungi and depended on the origin of the isolate. Not only fungal tolerance and ability to reduce metal concentrations in plant but also interactions with rhizobacteria affected heavy metal transfer and plant growth. Indeed, thanks to association with nodulating rhizobacteria, one Glomus intraradices inoculum increased particularly plant biomass which allowed exporting twofold more Cd and Zn in shoots as compared to non-mycorrhizal treatment. Cd concentrations in leachates were variable among fungal treatments, but can be significantly influenced by AM inoculation. The differential strategies of AM fungal colonisation in metal stress conditions are also discussed.     

Bacterial conjugation: everybody's doin' it.

Recent evidence suggests that bacterial conjugation is ubiquitous in the bacterial world and that DNA transfer between different genera-kingdoms is possible. It has also been demonstrated that many bacterial gene transfer systems resemble each other at the molecular level and that others are a blend of two or more of these systems. Thus, in the absence of a sexual cycle, bacterial conjugation, along with bacteriophages, transposons, and natural transformation systems, forms a potent force for gene dissemination and repair in the eubacteria and simple eukaryotes.     

Gene transfer in the marine environment

Abstract This review summarises the literature on bacterial gene transfer in marine ecosystems. Relevant experiments carried out in model systems are also included. Prerequisites for the main gene transfer mechanisms, transformation, transduction and conjugation are discussed, such as concentrations of extracellular DNA in marine waters, numbers of bacteriophages in sea water and frequency of plasmids in marine bacteria. Transfer of chromosomal genes as well as plasmids are considered. We also discuss the possibility that gene transfer is more frequent in surface-associated bacterial communities. Examples of relevant studies using various solid surfaces and from the air-water interface are summarized. We suggest that there is a higher ‘flow-rate’ of genetic information through surface-associated communities compared to bulk water communities.     

Determination of plasmid transfer frequency in soil: Consequences of bacterial mating on selective agar media

The possible occurrence of bacterial matings on transconjugant-selective plates used in experiments aimed at assessing conjugal transfer in soil was investigated. Matings on transconjugant-selective plates (with rifampicin, streptomycin, tetracycline, and kanamycin) between donor and recipient cells were shown to occur depending on the number of parent cells. Temperature also influenced conjugation frequencies on agar plates, since a lower temperature resulted in a decreased number of transconjugants. The use of nalidixic acid instead of streptomycin in conjunction with rifampicin for donor counter selection inhibited conjugation on selective plates. Conjugation in soil was analyzed by plating on selective media with nalidixic acid or streptomycin. The results indicated that conjugation in bentonite- or nutrient-amended soil was readily detected; however, part of the transconjugants could be assigned to be the result of matings on the selective plates. Conjugation was also stimulated in the presence of plant roots. Colony hybridization experiments confirmed the presence of plasmid RP4 in the transconjugants.     

Gene transfer between Salmonella enterica serovar Typhimurium inside epithelial cells

Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed.     

Responses of soil bacterial and fungal communities to reciprocal transfers of soil between adjacent coniferous forest and meadow vegetation in the Cascade Mountains of Oregon

Little information exists on the responses of soil fungal and bacterial communities in high elevation coniferous forest/open meadow ecosystems of the northwest United States of America to treatments that impact vegetation and soil conditions. An experiment was conducted in which soil cores were reciprocally transplanted between immediately adjacent forests and meadows at two high elevation (∼1,600 m) sites (Carpenter and Lookout) in the H.J. Andrews Experimental Forest located in the Cascade Mountains of Oregon. Half of the cores were placed in PVC pipe (closed) to prevent new root colonization, whereas the other cores were placed in mesh bags (open) to allow recolonization by fine roots. A duplicate set of open and closed soil cores was not transferred between sites and was incubated in place. After 2 year, soil cores were removed and changes in fungal and bacterial biomasses determined using light microscopy, and changes in microbial community composition determined by PLFA analysis, and by length heterogeneity PCR of the internal transcribed spacer region of fungal ribosomal DNA. At both sites soil microbial community structures had responded to treatments after 2 year of incubation. At Carpenter, both fungal and bacterial community structures of forest soil changed significantly in response to transfer from forest to meadow, with the shift in fungal community structure being accompanied by a significant decrease in the PLFA biomarker of fungal biomass,18:2ω6,9. At Lookout, both fungal and bacterial community structures of forest soil changed significantly in response to open versus closed core treatments, with the shift in the fungal community being accompanied by a significant decrease in the 18:2ω6,9 content of closed cores, and the shift in the bacterial community structure being accompanied by a significant increase in bacterial biomass of closed cores. At both sites, fungal community structures of meadow soils changed differently between open and closed cores in response to transfer to forest, and were accompanied by increases in the18:2ω6,9 content of open cores. Although there were no significant treatment effects on the bacterial community structure of meadow soil at either site, bacterial biomass was significantly higher in closed versus open cores regardless of transfer.     

Interactions between biochar and mycorrhizal fungi in a water-stressed agricultural soil

Biochar may alleviate plant water stress in association with arbuscular mycorrhizal (AM) fungi but research has not been conclusive. Therefore, a glasshouse experiment was conducted to understand how interactions between AM fungi and plants respond to biochar application under water-stressed conditions. A twin chamber pot system was used to determine whether a woody biochar increased root colonisation by a natural AM fungal population in a pasture soil (‘field’ chamber) and whether this was associated with increased growth of extraradical AM fungal hyphae detected by plants growing in an adjacent (‘bait’) chamber containing irradiated soil. The two chambers were separated by a mesh that excluded roots. Subterranean clover was grown with and without water stress and harvested after 35, 49 and 63 days from each chamber. When biochar was applied to the field chamber under water-stressed conditions, shoot mass increased in parallel with mycorrhizal colonisation, extraradical hyphal length and shoot phosphorus concentration. AM fungal colonisation of roots in the bait chamber indicated an increase in extraradical mycorrhizal hyphae in the field chamber. Biochar had little effect on AM fungi or plant growth under well-watered conditions. The biochar-induced increase in mycorrhizal colonisation was associated with increased growth of extraradical AM fungal hyphae in the pasture soil under water-stressed conditions.     

High Diversity of Fungi may Mitigate the Impact of Pollution on Plant Litter Decomposition in Streams

We investigated how a community of microbial decomposers adapted to a reference site responds to a sudden decrease in the water quality. For that, we assessed the activity and diversity of fungi and bacteria on decomposing leaves that were transplanted from a reference (E1) to a polluted site (E2), and results were compared to those from decomposing leaves either at E1 or E2. The two sites had contrasting concentrations of organic and inorganic nutrients and heavy metals in the stream water. At E2, leaf decomposition rates, fungal biomass, and sporulation were reduced, while bacterial biomass was stimulated. Fungal diversity was four times lower at the polluted site. The structure of fungal community on leaves decomposing at E2 significantly differed from that decomposing at E1, as indicated by the principal response curves analysis. Articulospora tetracladia, Anguillospora filiformis, and Lunulospora curvula were dominant species on leaves decomposing at E1 and were the most negatively affected by the transfer to the polluted site. The transfer of leaves colonized at the reference site to the polluted site reduced fungal diversity and sporulation but not fungal biomass and leaf decomposition. Overall, results suggest that the high diversity on leaves from the upstream site might have mitigated the impact of anthropogenic stress on microbial decomposition of leaves transplanted to the polluted site.     

Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are mobile genetic elements that can transfer from one bacterial cell to another by conjugation. ICEBs1 is integrated into the trnS-leu2 gene of Bacillus subtilis and is regulated by the SOS response and the RapI-PhrI cell-cell peptide signaling system. When B. subtilis senses DNA damage or high concentrations of potential mating partners that lack the element, ICEBs1 excises from the chromosome and can transfer to recipients. Bacterial conjugation usually requires a DNA relaxase that nicks an origin of transfer (oriT) on the conjugative element and initiates the 5'-to-3' transfer of one strand of the element into recipient cells. The ICEBs1 ydcR (nicK) gene product is homologous to the pT181 family of plasmid DNA relaxases. We found that transfer of ICEBs1 requires nicK and identified a cis-acting oriT that is also required for transfer. Expression of nicK leads to nicking of ICEBs1 between a GC-rich inverted repeat in oriT, and NicK was the only ICEBs1 gene product needed for nicking. NicK likely mediates conjugation of ICEBs1 by nicking at oriT and facilitating the translocation of a single strand of ICEBs1 DNA through a transmembrane conjugation pore.     

Direct conjugal transfers of Ti plasmid to soil microflora

The bacterial species in soil that can receive a Ti plasmid by conjugation from Agrobacterium spp. were investigated. In order to have direct access to the potential reservoir of Ti plasmid amongst soil microflora, the conjugal system consisting of a multiply auxotrophic derivative of C58 (ST-96-4) and a derivative of pTiC58Delta(acc)R (pSTiEGK) containing a triple antibiotic-resistance cassette in traM was used to transfer the Ti plasmid in a complex soil microflora used as the recipient. Numerous transconjugants were obtained by this method but none was identified as Agrobacterium. This could be explained by the low density of Agrobacterium in the tested soil. As indicated by analysis of the ribosomal gene rrs, transconjugants recovered directly from soil were found to be new bacterial species which appeared to be closely related to Sinorhizobium spp.     

Fungal control of early-stage bacterial community development in decomposing wood

The earliest stages of bacterial colonisation of wood have received little attention, particularly with respect to how the colonisation process may be affected by the presence of wood-decay fungi. This study used 16s rRNA gene sequencing to examine the bacterial community in wood that had been incubated in the field for 14 or 84 d, either in wood uncolonised by fungi or pre-colonised by Vuilleminia comedens, Trametes versicolor or Hypholoma fasciculare. All three fungal species significantly delayed bacterial colonisation of the wood. V. comedens and H. fasciculare also reduced bacterial OTU richness and altered bacterial community composition, increasing the relative abundance of Burkholderiales and reducing the proportion of Enterobacteriaceae and Bacteroidetes. Wood that had not been pre-colonised showed seasonal differences between autumn and spring: bacterial richness increased between 14 d and 84 d in the spring, but not in the autumn. Community composition at 84 d in spring was also different to the other time points, with reduced dominance of Gamma-proteobacteria. Archaea were also detected in nearly a third of samples, but with no apparent pattern, and always at low abundances.     

沙坡头地区凋落物分解及其对土壤微生物群落的影响

以腾格里沙漠东南缘沙坡头人工固沙植被区典型植物种凋落物(小画眉草、藓类、油蒿叶片)为对象,运用凋落物分解袋法和高通量测序技术,分析了3种植物凋落物分解特征及其对土壤微生物群落的影响。结果表明: 分解时间和凋落物类型均显著影响分解速率,藓类分解最慢,13个月后质量损失比仅为15.4%,油蒿叶片和小画眉草的平均分解速率分别是藓类的4.9和3.4倍。经过11个月的分解,细菌群落的优势菌门为放线菌门和变形菌门,真菌群落的优势菌门是子囊菌门;藓类分解过程中,拟杆菌门和绿弯菌门的相对丰度显著增加,担子菌门的相对丰度显著降低。凋落物分解后,细菌和真菌群落物种多样性和丰富度显著增加,细菌群落组成在凋落物间变化不显著,真菌群落变化显著。凋落物的分解速率与细菌和真菌群落多样性及丰富度均呈负线性变化。植物多糖、全磷和土壤pH、微生物生物量氮、铵态氮含量是影响微生物群落结构的主要因子。凋落物分解改变了土壤微生物群落物种组成和种间相似性,显著增加了土壤中微生物群落的多样性和丰富度,促进了土壤生境的恢复。     

Bacterial and fungal communities in bulk soil and rhizospheres of aluminum-tolerant and aluminum-sensitive maize (Zea mays L.) lines cultivated in unlimed and limed Cerrado soil

Liming of acidic soils can prevent aluminum toxicity and improve crop production. Some maize lines show aluminum (Al) tolerance, and exudation of organic acids by roots has been considered to represent an important mechanism involved in the tolerance. However, there is no information about the impact of liming on the structures of bacterial and fungal communities in Cerrado soil, nor if there are differences between the microbial communities from the rhizospheres of Al-tolerant and Al-sensitive maize lines. This study evaluated the effects of liming on the structure of bacterial and fungal communities in bulk soil and rhizospheres of Al-sensitive and Al-tolerant maize (Zea mays L.) lines cultivated in Cerrado soil by PCR-DGGE, 30 and 90 days after sowing. Bacterial fingerprints revealed that the bacterial communities from rhizospheres were more affected by aluminum stress in soil than by the maize line (Al-sensitive or Al-tolerant). Differences in bacterial communities were also observed over time (30 and 90 days after sowing), and these occurred mainly in the Actinobacteria. Conversely, fungal communities from the rhizosphere were weakly affected either by liming or by the rhizosphere, as observed from the DGGE profiles. Furthermore, only a few differences were observed in the DGGE profiles of the fungal populations during plant development when compared with bacterial communities. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Cerrado bulk soil revealed that Actinomycetales and Rhizobiales were among the dominant ribotypes.     

农杆菌T-复合物的形成与转运研究进展

在简要介绍农杆菌T-DNA转运全过程的基础上,结合作者近年的工作,重点对T-复合物的形成和T-复合物在农杆菌细胞内的转运机理的最新进展进行归纳和评述．农杆菌能够将其Ti质粒上的一段DNA以单链DNA-蛋白质复合物(简称T-复合物)的形式,通过其细胞两端的四型分泌系统(typeⅣ secretion system,T4SS)转运到宿主植物中,并使宿主发生遗传转化,因而农杆菌介导的T-DNA转运技术已成为应用最广泛的植物转基因技术,同时,由于转运T-复合物的T4SS也是某些质粒接合转移和许多病源微生物分泌致病效应蛋白的通道,因此,农杆菌T-DNA转运机理的研究受到了广泛的重视和关注,使得这方面的研究进展非常迅速．     

MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.

Arbuscular mycorrhiza are mutualistic interactions formed between soil fungi and plant roots. This study presents the MycoRed system, which uses red plant pigments derived from beetroot to reveal how fungi establish symbiosis with living legume and wild tobacco roots.     

Effect of Bacterial Wilt on Fungal Community Composition in Rhizosphere Soil of Tobaccos in Tropical Yunnan

Bacterial wilt, which is a major soil-borne disease with widespread occurrence, poses a severe danger in the field of tobacco production. However, there is very limited knowledge on bacterial wilt-induced microecological changes in the tobacco root system and on the interaction between Ralstonia solanacearum and fungal communities in the rhizosphere soil. Thus, in this study, changes in fungal communities in the rhizosphere soil of tobaccos with bacterial wilt were studied by 18S rRNA gene sequencing. The community composition of fungi in bacterial wilt-infected soil and healthy soil in two tobacco areas (Gengma and Boshang, Lincang City, Yunnan Province, China) was studied through the paired comparison method in July 2019. The results showed that there were significant differences in fungal community composition between the rhizosphere soil of diseased plants and healthy plants. The changes in the composition and diversity of fungal communities in the rhizosphere soil of tobaccos are vital characteristics of tobaccos with bacterial wilt, and the imbalance in the rhizosphere microecosystem of tobacco plants may further aggravate the disease.     

Use of strontium isotopes and foliar K content to estimate weathering of biotite induced by pine seedlings colonised by ectomycorrhizal fungi from two different soils

Pinus sylvestris seedlings, colonised by ectomycorrhizal (EM) fungi from either of two different soils (untreated forest soil and a limed soil from a clear cut area), were grown with or without biotite as a source of K. The biotite was naturally enriched in ⁸⁷Sr and the ratio of ⁸⁷Sr/ ⁸⁶Sr in the plant biomass was estimated and used as a marker for biotite weathering and compared to estimates of weathering based on foliar content of K. Different nutrient regimes were used to expose the seedlings to deficiencies of K with and without an application of nitrogen (NH₄NO₃) in excess of seedling demand. The seedlings were grown for 220 days and the elemental composition of the shoots were analysed at harvest. The EM colonisation was followed by analysing the concentration of ergosterol in the roots and the soils. Bacterial activity of the soil was estimated by the thymidine incorporation technique. The concentration of organic acids in the soil solution was measured in the soil in which seedlings colonised by EM fungi from the untreated forest soil were grown. It was found that seedlings colonised by EM fungi from untreated forest soil had taken up more K in treatments with biotite addition compared to seedlings colonised by EM fungi from the limed forest soil (p<0.05). Seedlings from untreated forest soil had larger shoots and contained more K when grown with biotite compared to KCl as K source, indicating that biotite had a stimulatory effect on the growth of these seedlings which was not related to K uptake. Seedlings from the limed soil, on the other hand, had similar foliar K content when grown with either biotite or KCl as K source. The larger uptake of K in seedlings from untreated forest soil was not an effect of a more developed EM colonisation of the roots since seedlings from the limed soil had a higher ergosterol concentration both in the soil and in the roots. Nutrient regimes had no significant influence on the total uptake of K but the ⁸⁷Sr/ ⁸⁶Sr isotope ratio in the plant biomass indicated that seedlings grown with excess nitrogen supply had taken up proportionally less Sr from the biotite (1.8% of total Sr content) compared to seedlings grown with a moderate nitrogen supply (5.0%). Furthermore, seedlings grown with excess nitrogen supply had a reduced fungal colonisation of roots and soil and bacterial activity was lower in these soils. The ⁸⁷Sr/ ⁸⁶Sr ratio in the plant biomass was positively correlated with fungal colonisation of the roots (r ²=0.98), which may indicate that the fungus was involved in releasing Sr from the biotite. Uptake of K from biotite was not related to the amount of organic acids in the soil solution. Oxalic acid was positively related to the amount of ergosterol in the root, suggesting that oxalic acid in the soil solution originates from the EM symbionts. The accuracy of the estimations of biotite weathering based on K uptake by the seedlings in comparison with the ⁸⁷Sr/⁸⁶Sr isotope ratio measured in the shoots is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Horizontal DNA transfer between bacteria in the environment

In the environment horizontal DNA transfer between various bacterial species and genera takes place by transformation, transduction, but mainly by conjugation. Conjugation is responsible for the spread of genes coding for antibiotic resistance and xenobiotic degradation. Transfer events are reported in animal, rhizosphere and phylloplane ecosystems and in non polluted and polluted water and soil. Genetic exchange between Bacteria and Archaea is also observed. Evaluation of the extent of interspecies gene transfer is crucial in view of the deliberate release of a variety of unmodified and genetically modified microorganisms into the natural environments.     

Microbial Diversity of a Mediterranean Soil and Its Changes after Biotransformed Dry Olive Residue Amendment

The Mediterranean basin has been identified as a biodiversity hotspot, about whose soil microbial diversity little is known. Intensive land use and aggressive management practices are degrading the soil, with a consequent loss of fertility. The use of organic amendments such as dry olive residue (DOR), a waste produced by a two-phase olive-oil extraction system, has been proposed as an effective way to improve soil properties. However, before its application to soil, DOR needs a pre-treatment, such as by a ligninolytic fungal transformation, e.g. Coriolopsis floccosa. The present study aimed to describe the bacterial and fungal diversity in a Mediterranean soil and to assess the impact of raw DOR (DOR) and C. floccosa-transformed DOR (CORDOR) on function and phylogeny of soil microbial communities after 0, 30 and 60 days. Pyrosequencing of the 16S rRNA gene demonstrated that bacterial diversity was dominated by the phyla Proteobacteria, Acidobacteria, and Actinobacteria, while 28S-rRNA gene data revealed that Ascomycota and Basidiomycota accounted for the majority of phyla in the fungal community. A Biolog EcoPlate experiment showed that DOR and CORDOR amendments decreased functional diversity and altered microbial functional structures. These changes in soil functionality occurred in parallel with those in phylogenetic bacterial and fungal community structures. Some bacterial and fungal groups increased while others decreased depending on the relative abundance of beneficial and toxic substances incorporated with each amendment. In general, DOR was observed to be more disruptive than CORDOR.