Genome-wide identification and expression analysis of LBD transcription factor genes in Moso bamboo (Phyllostachys edulis).

BACKGROUND: Moso bamboo, the fastest growing plant on earth, is an important source for income in large areas of Asia, mainly cultivated in China. Lateral organ boundaries domain (LBD) proteins, a family of transcription factors unique to plants, are involved in multiple transcriptional regulatory pathways and play important roles in lateral organ development, pathogen response, secondary growth, and hormone response. The LBD gene family has not previously been characterized in moso bamboo (Phyllostachys edulis). RESULTS: In this study, we identified 55 members of the LBD gene family from moso bamboo and found that they were distributed non-uniformly across its 18 chromosomes. Phylogenetic analysis showed that the moso bamboo LBD genes could be divided into two classes. LBDs from the same class share relatively conserved gene structures and sequences encoding similar amino acids. A large number of hormone response-associated cis-regulatory elements were identified in the LBD upstream promoter sequences. Synteny analysis indicated that LBDs in the moso bamboo genome showed greater collinearity with those of O. sativa (rice) and Zea mays (maize) than with those of Arabidopsis and Capsicum annuum (pepper). Numerous segmental duplicates were found in the moso bamboo LBD gene family. Gene expression profiles in four tissues showed that the LBD genes had different spatial expression patterns. qRT-PCR assays with the Short Time-series Expression Miner (STEM) temporal expression analysis demonstrated that six genes (PeLBD20, PeLBD29, PeLBD46, PeLBD10, PeLBD38, and PeLBD06) were consistently up-regulated during the rapid growth and development of bamboo shoots. In addition, 248 candidate target genes that function in a variety of pathways were identified based on consensus LBD binding motifs. CONCLUSIONS: In the current study, we identified 55 members of the moso bamboo transcription factor LBD and characterized for the first time. Based on the short-time sequence expression software and RNA-seq data, the PeLBD gene expression was analyzed. We also investigated the functional annotation of all PeLBDs, including PPI network, GO, and KEGG enrichment based on String database. These results provide a theoretical basis and candidate genes for studying the molecular breeding mechanism of rapid growth of moso bamboo.

Long terminal repeats (LTR) and transcription factors regulate PHRE1 and PHRE2 activity in Moso bamboo under heat stress.

BACKGROUND: LTR retrotransposons play a significant role in plant growth, genome evolution, and environmental stress response, but their regulatory response to heat stress remains unclear. We have investigated the activities of two LTR retrotransposons, PHRE1 and PHRE2, of moso bamboo (Phyllostachys edulis) in response to heat stress. RESULTS: The differential overexpression of PHRE1 and PHRE2 with or without CaMV35s promoter showed enhanced expression under heat stress in transgenic plants. The transcriptional activity studies showed an increase in transposition activity and copy number among moso bamboo wild type and Arabidopsis transgenic plants under heat stress. Comparison of promoter activity in transgenic plants indicated that 5'LTR promoter activity was higher than CaMV35s promoter. Additionally, yeast one-hybrid (Y1H) system and in planta biomolecular fluorescence complementation (BiFC) assay revealed interactions of heat-dependent transcription factors (TFs) with 5'LTR sequence and direct interactions of TFs with pol and gag. CONCLUSIONS: Our results conclude that the 5'LTR acts as a promoter and could regulate the LTR retrotransposons in moso bamboo under heat stress.

Homo- and Hetero-Dimers of CAD Enzymes Regulate Lignification and Abiotic Stress Response in Moso Bamboo.

Lignin biosynthesis enzymes form complexes for metabolic channelling during lignification and these enzymes also play an essential role in biotic and abiotic stress response. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme that catalyses the reduction of aldehydes to alcohols, which is the final step in the lignin biosynthesis pathway. In the present study, we identified 49 CAD enzymes in five Bambusoideae species and analysed their phylogenetic relationships and conserved domains. Expression analysis of Moso bamboo PheCAD genes in several developmental tissues and stages revealed that among the PheCAD genes, PheCAD2 has the highest expression level and is expressed in many tissues and PheCAD1, PheCAD6, PheCAD8 and PheCAD12 were also expressed in most of the tissues studied. Co-expression analysis identified that the PheCAD2 positively correlates with most lignin biosynthesis enzymes, indicating that PheCAD2 might be the key enzyme involved in lignin biosynthesis. Further, more than 35% of the co-expressed genes with PheCADs were involved in biotic or abiotic stress responses. Abiotic stress transcriptomic data (SA, ABA, drought, and salt) analysis identified that PheCAD2, PheCAD3 and PheCAD5 genes were highly upregulated, confirming their involvement in abiotic stress response. Through yeast two-hybrid analysis, we found that PheCAD1, PheCAD2 and PheCAD8 form homo-dimers. Interestingly, BiFC and pull-down experiments identified that these enzymes form both homo- and hetero- dimers. These data suggest that PheCAD genes are involved in abiotic stress response and PheCAD2 might be a key lignin biosynthesis pathway enzyme. Moreover, this is the first report to show that three PheCAD enzymes form complexes and that the formation of PheCAD homo- and hetero- dimers might be tissue specific.

Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element.

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR's affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5-2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

Distinct Anaerobic Bacterial Consumers of Cellobiose-Derived Carbon in Boreal Fens with Different CO2/CH4 Production Ratios.

Northern peatlands in general have high methane (CH4) emissions, but individual peatlands show considerable variation as CH4 sources. Particularly in nutrient-poor peatlands, CH4 production can be low and exceeded by carbon dioxide (CO2) production from unresolved anaerobic processes. To clarify the role anaerobic bacterial degraders play in this variation, we compared consumers of cellobiose-derived carbon in two fens differing in nutrient status and the ratio of CO2 to CH4 produced. After [13C]cellobiose amendment, the mesotrophic fen produced equal amounts of CH4 and CO2 The oligotrophic fen had lower CH4 production but produced 3 to 59 times more CO2 than CH4 RNA stable-isotope probing revealed that in the mesotrophic fen with higher CH4 production, cellobiose-derived carbon was mainly assimilated by various recognized fermenters of Firmicutes and by Proteobacteria The oligotrophic peat with excess CO2 production revealed a wider variety of cellobiose-C consumers, including Firmicutes and Proteobacteria, but also more unconventional degraders, such as Telmatobacter-related Acidobacteria and subphylum 3 of Verrucomicrobia Prominent and potentially fermentative Planctomycetes and Chloroflexi did not appear to process cellobiose-C. Our results show that anaerobic degradation resulting in different levels of CH4 production can involve distinct sets of bacterial degraders. By distinguishing cellobiose degraders from the total community, this study contributes to defining anaerobic bacteria that process cellulose-derived carbon in peat. Several of the identified degraders, particularly fermenters and potential Fe(III) or humic substance reducers in the oligotrophic peat, represent promising candidates for resolving the origin of excess CO2 production in peatlands. IMPORTANCE: Peatlands are major sources of the greenhouse gas methane (CH4), yet in many peatlands, CO2 production from unresolved anaerobic processes exceeds CH4 production. Anaerobic degradation produces the precursors of CH4 production but also represents competing processes. We show that anaerobic degradation leading to high or low CH4 production involved distinct sets of bacteria. Well-known fermenters dominated in a peatland with high CH4 production, while novel and unconventional degraders could be identified in a site where CO2 production greatly exceeds CH4 production. Our results help identify and assign functions to uncharacterized bacteria that promote or inhibit CH4 production and reveal bacteria potentially producing the excess CO2 in acidic peat. This study contributes to understanding the microbiological basis for different levels of CH4 emission from peatlands.

Secondary successional trajectories of structural and catabolic bacterial communities in oil-polluted soil planted with hybrid poplar.

Poplars have widely been used for rhizoremediation of a broad range of organic contaminants for the past two decades. Still, there is a knowledge gap regarding the rhizosphere-associated bacterial communities of poplars and their dynamics during the remediation process. It is envisaged that a detailed understanding of rhizosphere-associated microbial populations will greatly contribute to a better design and implementation of rhizoremediation. To investigate the long-term succession of structural and catabolic bacterial communities in oil-polluted soil planted with hybrid poplar, we carried out a 2-year field study. Hybrid aspen (Populus tremula × Populus tremuloides) seedlings were planted in polluted soil excavated from an accidental oil-spill site. Vegetated and un-vegetated soil samples were collected for microbial community analyses at seven different time points during the course of 2 years and sampling time points were chosen to cover the seasonal variation in the boreal climate zone. Bacterial community structure was accessed by means of 16S rRNA gene amplicon pyrosequencing, whereas catabolic diversity was monitored by pyrosequencing of alkane hydroxylase and extradiol dioxygenase genes. We observed a clear succession of bacterial communities on both structural and functional levels from early to late-phase communities. Sphingomonas type extradiol dioxygenases and alkane hydroxylase homologs of Rhodococcus clearly dominated the early-phase communities. The high-dominance/low-diversity functional gene communities underwent a transition to low-dominance/high-diversity communities in the late phase. These results pointed towards increased catabolic capacities and a change from specialist to generalist strategy of bacterial communities during the course of secondary succession.

Production of EPS under Cr(VI) challenge in two indigenous bacteria isolated from a tannery effluent.

Indigenous Cr(VI) reducing bacterial strains Pseudomonas aeruginosa Rb-1 and Ochrobactrum intermedium Rb-2 were evaluated for EPS production under Cr(VI) challenged and free conditions. Strain Rb-2 was more efficient in total EPS production (13.63 mg g(-1)) than Rb-1 (4.15 mg g(-1)) under Cr(VI) stress. Thick covering of capsular material around the cells of both bacterial strains was detected by electron microscopy. Transmission electron micrographs showed the appearance of pilli like structures under chromium stress by two bacteria suggested the possible involvement of this in exchange of hereditary material to increase their chances of survival under stress conditions. FTIR study showed involvement of sulphonate and hydroxyl groups in the binding with Cr(VI) ions. Solid-state (13) C NMR spectra revealed that EPS produced by both strains exhibited structural similarity with the glucan. The partial psl gene sequences of Rb-1 and Rb-2 showed homology with psl gene of Pseudomonas aeruginosa PAO1 and capsular polysaccharide biosynthesis protein of various strains of Pseudomonas. This is the first report on the identification of psl gene from Ochrobacterum in NCBI GenBank database up to our knowledge.

Cross-site soil microbial communities under tillage regimes: fungistasis and microbial biomarkers.

The exploitation of soil ecosystem services by agricultural management strategies requires knowledge of microbial communities in different management regimes. Crop cover by no-till management protects the soil surface, reducing the risk of erosion and nutrient leaching, but might increase straw residue-borne and soilborne plant-pathogenic fungi. A cross-site study of soil microbial communities and Fusarium fungistasis was conducted on six long-term agricultural fields with no-till and moldboard-plowed treatments. Microbial communities were studied at the topsoil surface (0 to 5 cm) and bottom (10 to 20 cm) by general bacterial and actinobacterial terminal restriction fragment length polymorphism (T-RFLP) and phospholipid fatty acid (PLFA) analyses. Fusarium culmorum soil fungistasis describing soil receptivity to plant-pathogenic fungi was explored by using the surface layer method. Soil depth had a significant impact on general bacterial as well as actinobacterial communities and PLFA profiles in no-till treatment, with a clear spatial distinction of communities (P < 0.05), whereas the depth-related separation of microbial communities was not observed in plowed fields. The fungal biomass was higher in no-till surface soil than in plowed soil (P < 0.07). Soil total microbial biomass and fungal biomass correlated with fungistasis (P < 0.02 for the sum of PLFAs; P < 0.001 for PLFA 18:2ω6). Our cross-site study demonstrated that agricultural management strategies can have a major impact on soil microbial community structures, indicating that it is possible to influence the soil processes with management decisions. The interactions between plant-pathogenic fungi and soil microbial communities are multifaceted, and a high level of fungistasis could be linked to the high microbial biomass in soil but not to the specific management strategy.

Methane-cycling microbial communities and methane emission in natural and restored peatlands.

We addressed how restoration of forestry-drained peatlands affects CH(4)-cycling microbes. Despite similar community compositions, the abundance of methanogens and methanotrophs was lower in restored than in natural sites and correlated with CH(4) emission. Poor establishment of methanogens may thus explain low CH(4) emissions on restored peatlands even 10 to 12 years after restoration.

Bamboo Transposon Research: Current Status and Perspectives.

Bamboo, a fast-growing non-timber forest plant with many uses, is a valuable species for green development. However, bamboo flowering is very infrequent, extending, in general, for up to 120 years. Ecologically, bamboo species are generally better adapted to various environments than other grasses. Therefore, the species deserves a special status in what could be called Ecological Bioeconomy. An understanding of the genetic processes of bamboo can help us sustainably develop and manage bamboo forests. Transposable elements (TEs), jumping genes or transposons, are major genetic elements in plant genomes. The rapid development of the bamboo reference genome, at the chromosome level, reveals that TEs occupy over 63.24% of the genome. This is higher than found in rice, Brachypodium, and sorghum. The bamboo genome contains diverse families of TEs, which play a significant role in bamboo's biological processes including growth and development. TEs provide important clues for understanding the evolution of the bamboo genome. In this chapter, we briefly describe the current status of research on TEs in the bamboo genome, their regulation, and transposition mechanisms. Perspectives for future research are also provided.

Genome-wide identification and analysis of the heat shock transcription factor family in moso bamboo (Phyllostachys edulis).

Heat shock transcription factors (HSFs) are central elements in the regulatory network that controls plant heat stress response. They are involved in multiple transcriptional regulatory pathways and play important roles in heat stress signaling and responses to a variety of other stresses. We identified 41 members of the HSF gene family in moso bamboo, which were distributed non-uniformly across its 19 chromosomes. Phylogenetic analysis showed that the moso bamboo HSF genes could be divided into three major subfamilies; HSFs from the same subfamily shared relatively conserved gene structures and sequences and encoded similar amino acids. All HSF genes contained HSF signature domains. Subcellular localization prediction indicated that about 80% of the HSF proteins were located in the nucleus, consistent with the results of GO enrichment analysis. A large number of stress response-associated cis-regulatory elements were identified in the HSF upstream promoter sequences. Synteny analysis indicated that the HSFs in the moso bamboo genome had greater collinearity with those of rice and maize than with those of Arabidopsis and pepper. Numerous segmental duplicates were found in the moso bamboo HSF gene family. Transcriptome data indicated that the expression of a number of PeHsfs differed in response to exogenous gibberellin (GA) and naphthalene acetic acid (NAA). A number of HSF genes were highly expressed in the panicles and in young shoots, suggesting that they may have functions in reproductive growth and the early development of rapidly-growing shoots. This study provides fundamental information on members of the bamboo HSF gene family and lays a foundation for further study of their biological functions in the regulation of plant responses to adversity.

The role of Sphagnum mosses in the methane cycling of a boreal mire.

Peatlands are a major natural source of atmospheric methane (CH4). Emissions from Sphagnum-dominated mires are lower than those measured from other mire types. This observation may partly be due to methanotrophic (i.e., methane-consuming) bacteria associated with Sphagnum. Twenty-three of the 41 Sphagnum species in Finland can be found in the peatland at Lakkasuo. To better understand the Sphagnum-methanotroph system, we tested the following hypotheses: (1) all these Sphagnum species support methanotrophic bacteria; (2) water level is the key environmental determinant for differences in methanotrophy across habitats; (3) under dry conditions, Sphagnum species will not host methanotrophic bacteria; and (4) methanotrophs can move from one Sphagnum shoot to another in an aquatic environment. To address hypotheses 1 and 2, we measured the water table and CH4 oxidation for all Sphagnum species at Lakkasuo in 1-5 replicates for each species. Using this systematic approach, we included Sphagnum spp. with narrow and broad ecological tolerances. To estimate the potential contribution of CH4 to moss carbon, we measured the uptake of delta13C supplied as CH4 or as carbon dioxide dissolved in water. To test hypotheses 2-4, we transplanted inactive moss patches to active sites and measured their methanotroph communities before and after transplantation. All 23 Sphagnum species showed methanotrophic activity, confirming hypothesis 1. We found that water level was the key environmental factor regulating methanotrophy in Sphagnum (hypothesis 2). Mosses that previously exhibited no CH4 oxidation became active when transplanted to an environment in which the microbes in the control mosses were actively oxidizing CH4 (hypothesis 4). Newly active transplants possessed a Methylocystis signature also found in the control Sphagnum spp. Inactive transplants also supported a Methylocystis signature in common with active transplants and control mosses, which rejects hypothesis 3. Our results imply a loose symbiosis between Sphagnum spp. and methanotrophic bacteria that accounts for potentially 10-30% of Sphagnum carbon.

Sphingobium sp. HV3 degrades both herbicides and polyaromatic hydrocarbons using ortho- and meta-pathways with differential expression shown by RT-PCR.

Sphingobium sp. HV3 described as an herbicide degrader harbours the pSKY4 plasmid, encoding an aromatic meta-pathway. The function of the plasmid was studied by Tn5 transposon mutagenesis and plasmid isolation and the degradation capacities of the HV3 strain were re-evaluated. Transcription of the tfdC from ortho-pathway was contrasted to the xylE and bphC of meta-pathway using real-time PCR. Cloning of the Tn5-insertion sites from the megaplasmid revealed genes for both aromatic and polyaromatic degradation. In the mutant Km24 strain the transposon was inserted to an ORF similar to the large subunit of ring hydroxylating dioxygenase, in the Km383 to a cis-biphenyl dihydrodiol dehydrogenase and in the Km187 and Km42 to a reductase component of a dioxygenase. A chlorocathecol ortho-pathway (10 kb) was amplified from the HV3 strain. The transcription of the tfdC was induced by 2,4-dichlorophenoxyacetic acid herbicide and m-xylene caused highest induction of both upper and lower aromatic meta-pathway genes. The detected novel degradation capacities (m-xylene, toluene, biphenyl, fluorene and phenanthrene) can be explained by the presence of functional meta-pathway genes in the pSKY4 megaplasmid. The characterization of the Sphingobium sp. HV3 improves our understanding of versatile catabolic bacteria unveiling roles of degradation pathways and plasmids in biodegradation.

Bacterial succession in oil-contaminated soil under phytoremediation with poplars.

Petroleum hydrocarbons (PHCs) continue to be among the most common pollutants in soil worldwide. Phytoremediation has become a sustainable way of dealing with PHC contamination. We conducted the off-site phytoremediation of PHC-polluted soil from an oil tanker truck accident, where poplars were used for the phytoremediation of the oil-polluted soil in a boreal climate during a seven-year treatment. The succession of bacterial communities over the entire phytoremediation process was monitored using microbial ecological tools relying on high-throughput 16S rRNA gene sequencing. Upon the successful depletion of PHCs from soil, endophytic communities were analyzed in order to assess the complete plant-associated microbiome after the ecological recovery. The rhizosphere-associated soil exhibited different bacterial dynamics than unplanted soil, but both soils experienced succession of bacteria over time, with diversity being negatively correlated with PHC concentration. In the relatively short growing season in North Europe, seasonal variations in environmental conditions were identified that contributed to the dynamics of bacterial communities. Overall, our study proved that phytoremediation using poplar trees can be used to assist in the removal of PHCs from soils in boreal climate conditions and provides new insight into the succession patterns of bacterial communities associated with these plants.

Multi-omics analysis of cellular pathways involved in different rapid growth stages of moso bamboo.

Moso bamboo (Phyllostachys edulis (Carriere) J. Houzeau) is a rapidly growing grass of industrial and ecological importance. However, the molecular mechanisms of its remarkable growth are not well understood. In this study, we investigated the early-stage growth of moso bamboo shoots and defined three different growth stages based on histological and biochemical analyses, namely, starting of cell division (SD), rapid division (RD) and rapid elongation (RE). Further analyses on potentially relevant cellular pathways in these growth stages using multi-omics approaches such as transcriptomics and proteomics revealed the involvement of multiple cellular pathways, including DNA replication, repair and ribosome biogenesis. A total of 8045 differentially expressed genes (DEGs) and 1053 differentially expressed proteins (DEPs) were identified in our analyses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of detected DEGs identified several key biological pathways such as phytohormone metabolism, signal transduction, cell wall development and carbohydrate metabolism. The comparative analysis of proteins displayed that a total of 213 DEPs corresponded with DEGs and 3 significant expression profiles that could be promoting the fast growth of bamboo internodes. Moreover, protein-protein interaction network prediction analysis is suggestive of the involvement of five major proteins of signal transduction, DNA synthesis and RNA transcription, and may act as key elements responsible for the rapid shoot growth. Our work exploits multi-omics and bioinformatic approaches to unfurl the complexity of molecular networks involved in the rapid growth of moso bamboo and opens up questions related to the interactions between the functions played by individual molecular pathway.

The comparison of dissolved organic matter in hydrochars and biochars from pig manure.

Dissolved organic matter (DOM) has an important effect on soil fertility, activity of microorganisms and transport of contaminants. In this study, DOM released by the hydrochar and biochar prepared under various conditions from pig manure, was assessed using a combination of UV-Visible spectroscopy, fluorescence excitation-emission (EEM) spectrophotometry and 1H-nuclear magnetic resonance (1H NMR). The dissolved organic carbon (DOC) extracted from the hydrochar and biochar ranged from 3.34-11.96% and 0.38-0.48%, respectively, and the highest DOM was released by HCK0.5 (180 °C and 0.5% KOH). The aliphatic compounds were most common in DOM which mainly included three humic acid-like and one protein-like substance. The hydrochar-DOM had a larger molecular weight and lower aromaticity than biochar-DOM, but the effect of temperature on the DOM characteristics of hydrochar and biochar was opposite. The acidic treatment increased the content of functional groups containing oxygen and nitrogen in hydrochar-DOM, and alkaline treatment increased the content of aliphatic compounds. The results obtained are beneficial to select carbonation process and guide the rational application of hydrochar and biochar from pig manure in soil remediation field.

Insights into selenite reduction and biogenesis of elemental selenium nanoparticles by two environmental isolates of Burkholderia fungorum.

Microorganisms capable of transforming toxic selenium oxyanions into non-toxic elemental selenium (Se°) may be considered as biocatalysts for the production of selenium nanoparticles (SeNPs), eventually exploitable in different biotechnological applications. Two Burkholderia fungorum strains (B. fungorum DBT1 and B. fungorum 95) were monitored during their growth for both capacity and efficiency of selenite (SeO32-) reduction and elemental selenium formation. Both strains are environmental isolates in origin: B. fungorum DBT1 was previously isolated from an oil refinery drainage, while B. fungorum 95 has been enriched from inner tissues of hybrid poplars grown in a soil contaminated by polycyclic aromatic hydrocarbons. Our results showed that B. fungorum DBT1 is able to reduce 0.5mM SeO32- to Se° when cultured aerobically in liquid medium at 27°C, while B. fungorum 95 can reduce more than 1mM SeO32- to Se° within 96h under the same growth conditions, with the appearance of SeNPs in cultures of both bacterial strains. Biogenic SeNPs were spherical, with pH-dependent charge and an average hydrodynamic diameter of 170nm and 200nm depending on whether they were produced by B. fungorum 95 or B. fungorum DBT1, respectively. Electron microscopy analyses evidenced that Se nanoparticles occurred intracellularly and extracellularly. The mechanism of SeNPs formation can be tentatively attributed to cytoplasmic enzymatic activation mediated by electron donors. Biogenic nanoparticles were then probably released outside the bacterial cells as a consequence of a secretory process or cell lysis. Nevertheless, formation of elemental selenium nanoparticles under aerobic conditions by B. fungorum DBT1 and B. fungorum 95 is likely due to intracellular reduction mechanisms. Biomedical and other high tech sectors might exploit these biogenic nanoparticles in the near future, once fully characterized and tested for their multiple properties.

Detection of methanogenic Archaea in peat: comparison of PCR primers targeting the mcrA gene.

Methanogens (domain Archaea) have a unique role in the carbon cycle as producers of the greenhouse gas methane (CH(4)). Methyl-coenzyme M reductase (MCR) is a vital enzyme in CH(4) production, and the mcrA gene coding for a subunit of MCR has been employed as a specific marker for the detection and differentiation of methanogen communities. A critical step in assessing environmental mcrA diversity is the selection of PCR primers. The objective of this study was to compare the diversity coverage of three published mcrA primer sets MCR, ME and ML (also known as MCR and Luton-mcrA) and their ability to discern methanogen communities in a drained peatland. The primers were applied to DNA extracts from unfertilised and ash-fertilised peat from two different depths. Amplified mcrA communities were cloned and sequenced, and the sequences were divided into operational taxonomic units (OTUs) by restriction fragment length polymorphism (RFLP) and sequence analysis. All primers recovered characteristic OTUs associated with the peat depths and treatments and confirmed a previous observation of low methanogen diversity. The minor differences in OTU ranges of the primers did not greatly affect the observed community composition. However, as the proportions of several OTUs varied strongly, the primers provided different quantitative representations of mcrA communities. We concluded that the ML and MCR primers had better amplification ranges than the ME set, but the use of MCR with peat samples was problematic due to poor amplification. Consequently, the ML primers were best suited for mcrA analysis of peatland methanogen communities.

Novel upper meta-pathway extradiol dioxygenase gene diversity in polluted soil.

For the determination of the catabolic community diversity that is related to biodegradation potential, we developed a protocol for the assessment of catabolic marker genes in polluted soils. Primers specific to upper pathway extradiol dioxygenase genes were designed which amplified a 469-bp product from Sphingomonas sp. HV3. The constructed primers were used in PCR amplification of upper pathway ring cleavage genes from DNA directly isolated from a mineral oil polluted landfill site, a mineral oil landfarming site and a birch rhizosphere-associated soil that was either artificially polluted with a PAH mixture or not polluted. Amplicons were cloned and subjected to restriction fragment length polymorphism analysis dividing the HhaI-digested products into operational taxonomic units. Altogether 26 different operational taxonomic units were detected with the sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Phylogenetic analysis divided the operational taxonomic units from the polluted soils into seven clusters. Two contained exclusively sequences with no close homologues in the database, therefore representing novel catabolic genes. This large proportion of novel extradiol sequences shows that there is an extensive unknown catabolic diversity in polluted environments.