Designing amendments to improve plant performance for mine tailings revegetation.

To provide recommendations for establishment of plants on low-pH Formosa Mine tailings, two greenhouse experiments were conducted to evaluate the use of remedial amendments to improve the survival and growth of Douglas fir (Pseudotsuga menziesii) seedlings. A preliminary experiment indicated that 1% lime (by weight) raised tailings pH, permitting seedling survival. However, high rates of biosolid application (BS; 2% by weight) added to supply nutrients were phytotoxic when added with lime. A gasified conifer biochar (BC) added to tailings at 1%, 2.5%, or 5% (by weight), along with lime and BS, caused an additional increase in pH, decreased electrical conductivity (EC), and tended to increase the survival of Douglas fir. The addition of a locally sourced microbial inoculum (LSM) did not affect survival. A subsequent experiment expanded our experimental design by testing multiple levels of amendments that included lime (0.5% and 1% by weight), three application rates (0.2%, 0.5%, and 2%) of two nutrient sources (BS or mineral fertilizer), BC (0% and 2.5%), and with or without LSM. There were many interactions among amendments. In general, Douglas fir survival was enhanced when lime and BC were added. These experiments suggest that amending with lime, a nutrient source, and BC would enhance revegetation on low-pH, metal-contaminated mine tailings.

Biosynthetic Origin of Formylaminooxyvinylglycine and Characterization of the Formyltransferase GvgI.

4-Formylaminooxyvinylglycine (FVG) is an herbicidal and antibacterial nonproteinogenic amino acid produced by several strains of the Pseudomonas fluorescens species complex. It contains a unique vinyl alkoxyamine moiety with an O-N bond, and its biosynthetic origin remains unknown. Here, we show that the gvg cluster from P. fluorescens WH6 is responsible for the biosynthesis of FVG and two additional O-N bond-containing oxyvinylglycines, guanidinooxyvinylglycine and aminooxyvinylglycine. Feeding studies in the producing bacteria indicate that these compounds originate from homoserine. We identify a formyltransferase gvgI that is required for the production of FVG and characterize the activity of this enzyme in vitro toward amino acids with a side chain amine. Sequence similarity network analysis reveals that GvgI and homologues make up a distinct group from the main classes of formyltransferases.

Absence of 4-Formylaminooxyvinylglycine Production by Pseudomonas fluorescens WH6 Results in Resource Reallocation from Secondary Metabolite Production to Rhizocompetence.

Pseudomonas fluorescens WH6 produces the non-proteinogenic amino acid 4-formylaminooxyvinylglycine (FVG), a secondary metabolite with antibacterial and pre-emergent herbicidal activities. The gvg operon necessary for FVG production encodes eight required genes: one regulatory (gvgR), two of unknown functional potential (gvgA and C), three with putative biosynthetic function (gvgF, H, and I), and two small ORFs (gvgB and G). To gain insight into the role of GvgA and C in FVG production, we compared the transcriptome of knockout (KO) mutants of gvgR, A, and C to wild type (WT) to test two hypotheses: (1) GvgA and GvgC play a regulatory role in FVG production and (2) non-gvg cluster genes are regulated by GvgA and GvgC. Our analyses show that, collectively, 687 genes, including the gvg operon, are differentially expressed in all KO strains versus WT, representing >10% of the genome. Fifty-one percent of these genes were similarly regulated in all KO strains with GvgC having the greatest number of uniquely regulated genes. Additional transcriptome data suggest cluster regulation through feedback of a cluster product. We also discovered that FVG biosynthesis is regulated by L-glu, L-asp, L-gln, and L-asn and that resources are reallocated in KO strains to increase phenotypes involved in rhizocompetence including motility, biofilm formation, and denitrification. Altogether, differential transcriptome analyses of mutants suggest that regulation of the cluster is multifaceted and the absence of FVG production or its downregulation can dramatically shift the lifestyle of WH6.

Unexpected distribution of the 4-formylaminooxyvinylglycine (FVG) biosynthetic pathway in Pseudomonas and beyond.

The biological herbicide and antibiotic 4-formylaminooxyvinylglycine (FVG) was originally isolated from several rhizosphere-associated strains of Pseudomonas fluorescens. Biosynthesis of FVG is dependent on the gvg biosynthetic gene cluster in P. fluorescens. In this investigation, we used comparative genomics to identify strains with the genetic potential to produce FVG due to presence of a gvg gene cluster. These strains primarily belong to two groups of Pseudomonas, P. fluorescens and P. syringae, however, a few strains with the gvg cluster were found outside of Pseudomonas. Mass spectrometry confirmed that all tested strains of the P. fluorescens species group produced FVG. However, P. syringae strains did not produce FVG under standard conditions. Several lines of evidence regarding the transmission of the gvg cluster including a robust phylogenetic analysis suggest that it was introduced multiple times through horizontal gene transfer within the Pseudomonas lineage as well as in select lineages of Thiomonas, Burkholderia and Pantoea. Together, these data broaden our understanding of the evolution and diversity of FVG biosynthesis. In the course of this investigation, additional gene clusters containing only a subset of the genes required to produce FVG were identified in a broad range of bacteria, including many non-pseudomonads.

Phytostabilization of acidic mine tailings with biochar, biosolids, lime, and locally-effective microbes: Do amendment mixtures influence plant growth, tailing chemistry, and microbial composition?

Abandoned mine lands present persistent environmental challenges to ecosystems and economies; reclamation an important step for overcoming these challenges. Phytostabilization is an elegant and cost-effective reclamation strategy, however, establishing plants on severely degraded soils is problematic, often requiring soil amendment additions. We evaluated whether amendment mixtures composed of lime, biochar, biosolids, and locally effective microbes (LEM) could alleviate the constraints that hinder phytostabilization success. We hypothesized that 1) plants grown in tailings amended with lime, biochar, and biosolids (LBB) would establish faster and grow larger than plants grown in tailings amended with lime only, and 2) the LEM source would influence microbial community function and structure in amended mine tailings. We conducted a greenhouse study that simulated in situ conditions to measure the influence of LBB-LEM amendment blends on plant growth, plant nutrients, metal concentrations, microbial function, and microbial community structure. Blue wildrye [Elymus glaucus Buckley ssp. Jepsonii (Burtt Davy) Gould] was grown in tailings collected from the Formosa mine site amended with various combinations of LBB-LEM. The above and below ground biomass of plants grown in mine tailings amended with LBB was 3 to 4 times larger than the biomass of plants grown in tailings amended only with lime. Although the LEM addition did not influence immediate plant growth, it did affect nutrient content and altered the rhizosphere community membership. As such, it is not yet clear if LEM-driven alterations in microbial membership will advance mine reclamation strategies by improving long-term growth.

Microbial response to designer biochar and compost treatments for mining impacted soils.

The Oronogo-Duenweg mining belt is a designated United States Environmental Protection Agency Superfund site due to lead-contaminated soil and groundwater by former mining and smelting operations. Sites that have undergone remediation - in which the O, A, and B horizons have been removed alongside the lead contamination - have an exposed C horizon and are incalcitrant to revegetation efforts. Soils also continue to contain quantifiable Cd and Zn concentrations. In order to improve soil conditions and encourage successful site revegetation, our study employed three biochars, sourced from different feedstocks (poultry litter, beef lot manure, and lodge pole pine), at two rates of application (2.5%, and 5%), coupled with compost (0%, 2.5% and 5% application rates). Two plant species - switchgrass (Panicum virgatum) and buffalograss (Bouteloua dactyloides) - were grown in the amended soils. Amendment of soils with poultry litter biochar applied at 5% resulted in the greatest reduction of soil bioavailable Cd and Zn. Above ground biomass yields were greatest with beef lot manure biochar applied at 2.5% with 5% compost, or with 5% biochar at 2.5% and 5% compost rates. Maximal microbial biomass was achieved with 5% poultry litter biochar and 5% compost, and microbial communities in soils amended with poultry litter biochar distinctly clustered away from all other soil treatments. Additionally, poultry litter biochar amended soils had the highest enzyme activity rates for ß-glucosidase, N-acetyl-ß-D-glucosaminidase, and esterase. These results suggest that soil reclamation using biochar and compost can improve mine-impacted soil biogeophysical characteristics, and potentially improve future remediation efforts.

An examination of the role of biochar and biochar water-extractable substances on the sorption of ionizable herbicides in rice paddy soils.

The application of biochar as a soil amendment can increase concentrations of soil organic matter, especially water-extractable organic substances. Due to their mobility and reactivity, more studies are needed to address the potential impact of biochar water-extractable substances (BWES) on the sorption of herbicides in agricultural soils that are periodically flooded. Two paddy soils (100 and 700 years of paddy soil development), unamended or amended with raw (BC) or washed biochar (BCW), were used to test the influence of BWES on the sorption behavior of the herbicides azimsulfuron (AZ) and penoxsulam (PE). The adsorption of AZ to biochar was much stronger than that to the soils, and it was adsorbed to a much larger extent to BC than to BCW. The depletion of polar groups in the BWES from the washed biochar reduced AZ adsorption but had no effect on PE adsorption. The adsorption of AZ increased when the younger soil (P100) was amended with BC and decreased when it was amended with BCW. In P700, which has lower dissolved organic carbon (DOC) content than P100, the adsorption of AZ increased regardless of whether biochar was raw or washed. The adsorption of PE slightly decreased when P100 was amended with BC or BCW and slightly increased when P700 was amended with BC or BCW. In order to evaluate compositional differences in the biochar and BWES before and after the washing treatment, we performed solid-state 13C NMR spectroscopy of BC and BCW, and high resolution mass spectrometry of BWES. Our observations stress the importance of proper consideration of soil and biochar properties before their incorporation into paddy soils, since biochar may reduce or increase the mobility of AZ and PE depending on soil properties and time of application.

Detection of 4-formylaminooxyvinylglycine in culture filtrates of Pseudomonas fluorescens WH6 and Pantoea ananatis BRT175 by laser ablation electrospray ionization-mass spectrometry.

The oxyvinylglycine 4-formylaminooxyvinylglycine (FVG) arrests the germination of weedy grasses and inhibits the growth of the bacterial plant pathogen Erwinia amylovora. Both biological and analytical methods have previously been used to detect the presence of FVG in crude and extracted culture filtrates of several Pseudomonas fluorescens strains. Although a combination of these techniques is adequate to detect FVG, none is amenable to high-throughput analysis. Likewise, filtrates often contain complex metabolite mixtures that prevent the detection of FVG using established chromatographic techniques. Here, we report the development of a new method that directly detects FVG in crude filtrates using laser ablation electrospray ionization-mass spectrometry (LAESI-MS). This approach overcomes limitations with our existing methodology and allows for the rapid analysis of complex crude culture filtrates. To validate the utility of the LAESI-MS method, we examined crude filtrates from Pantoea ananatis BRT175 and found that this strain also produces FVG. These findings are consistent with the antimicrobial activity of P. ananatis BRT175 and indicate that the spectrum of bacteria that produce FVG stretches beyond rhizosphere-associated Pseudomonas fluorescens.

Remediation of an acidic mine spoil: Miscanthus biochar and lime amendment affects metal availability, plant growth, and soil enzyme activity.

Biochar may be a tool for mine spoil remediation; however, its mechanisms for achieving this goal remain unclear. In this study, Miscanthus (Miscanthus giganteus) biochar was evaluated for its ability to reclaim acidic mine spoils (pH < 3) through reducing metal availability, improving soil microbial enzymatic activity, and initial growth of grass seedlings. Biochar was applied at 0, 1, 2.5 and 5% (w/w) along with lime/no lime and fertilizer additions. Blue Wildrye (Elymus glaucus cv. 'Elkton') was planted and later the shoots and roots were collected and metal concentrations determined. Afterwards, each pot was leached with deionized water, and the leachate analyzed for pH, electrical conductivity (EC), dissolved organic carbon (DOC) and soluble metal concentrations. After drying, the spoil was extracted with 0.01 M CaCl2 and Mehlich 3 (M3) to determine extractable Al, Cu, and Zn concentrations. Additionally, microbial activity was measured using a fluorescent ß-glucosidase and N-acetyl-ß-d-glucosaminidase assay. Spoil treated with lime and biochar had significantly greater pH and EC values. Significantly greater ß-glucosidase activity occurred only in the 5% biochar plus lime treatment, while N-acetyl-ß-d-glucosaminidase activities were not altered. Metal concentrations in rye shoot and roots were mixed. Lime additions significantly reduced extractable metal concentrations. Increasing biochar rates alone significantly reduced leachate DOC concentrations, and subsequently reduced leachable metal concentrations. Surprisingly, miscanthus biochar, by itself, was limited at mitigation, but when combined with lime, the combination was capable of further reducing extractable metal concentrations and improving ß-glucosidase enzyme activity.

Draft Genome Sequences of Seven 4-Formylaminooxyvinylglycine Producers Belonging to the Pseudomonas fluorescens Species Complex.

Vinylglycines are nonproteinogenic amino acids that inhibit amino acid metabolism and ethylene production. Here, we report the draft genome sequences of seven isolates of Pseudomonas that produce 4-formylaminooxyvinylglycine, a compound known to inhibit the germination of grasses and the growth of specific plant-pathogenic bacteria.

Functional analysis of a biosynthetic cluster essential for production of 4-formylaminooxyvinylglycine, a germination-arrest factor from Pseudomonas fluorescens WH6.

Rhizosphere-associated Pseudomonas fluorescens WH6 produces the germination-arrest factor 4-formylaminooxyvinylglycine (FVG). FVG has previously been shown to both arrest the germination of weedy grasses and inhibit the growth of the bacterial plant pathogen Erwinia amylovora. Very little is known about the mechanism by which FVG is produced. Although a previous study identified a region of the genome that may be involved in FVG biosynthesis, it has not yet been determined which genes within that region are sufficient and necessary for FVG production. In the current study, we explored the role of each of the putative genes encoded in that region by constructing deletion mutations. Mutant strains were assayed for their ability to produce FVG with a combination of biological assays and TLC analyses. This work defined the core FVG biosynthetic gene cluster and revealed several interesting characteristics of FVG production. We determined that FVG biosynthesis requires two small ORFs of less than 150 nucleotides and that multiple transporters have overlapping but distinct functionality. In addition, two genes in the centre of the biosynthetic gene cluster are not required for FVG production, suggesting that additional products may be produced from the cluster. Transcriptional analysis indicated that at least three active promoters play a role in the expression of genes within this cluster. The results of this study enrich our knowledge regarding the diversity of mechanisms by which bacteria produce non-proteinogenic amino acids like vinylglycines.

Gasified Grass and Wood Biochars Facilitate Plant Establishment in Acid Mine Soils.

Heavy metals in exposed mine tailings threaten ecosystems that surround thousands of abandoned mines in the United States. Biochars derived from the pyrolysis or gasification of biomass may serve as a valuable soil amendment to revegetate mine sites. We evaluated the ability of two biochars, produced by gasification of either Kentucky bluegrass seed screenings (KB) or mixed conifer wood (CW), to support the growth of plants in mine spoils from the abandoned Formosa and Almeda Mines in Oregon. To evaluate the potential for plant establishment in mine tailings, wheat was grown in tailings amended with biochar at rates ranging from 0 to 9% (w/w). Both KB and CW biochars promoted plant establishment by increasing soil pH, increasing concentrations of macro- and micronutrients, and decreasing the solubility and plant uptake of heavy metals. Formosa tailings required at least 4% biochar and Almeda soil required at least 2% biochar to promote healthy wheat growth. A complimentary experiment in which mine spoils were leached with simulated precipitation indicated that biochar amendment rates ≥4% were sufficient to neutralize the elution pH and reduce concentrations of potentially toxic elements (Zn, Cu, Ni, Al) to levels near or below concern. These findings support the use of gasified biochar amendments to revegetate acid mine soils.

Negative regulation of germination-arrest factor production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor.

Pseudomonas fluorescens WH6 secretes a germination-arrest factor (GAF) that we have identified previously as 4-formylaminooxyvinylglycine. GAF irreversibly inhibits germination of the seeds of numerous grassy weeds and selectively inhibits growth of the bacterial plant pathogen Erwinia amylovora. WH6-3, a mutant that has lost the ability to produce GAF, contains a Tn5 insertion in prtR, a gene that has been described previously in some strains of P. fluorescens as encoding a transmembrane regulator. As in these other pseudomonads, in WH6, prtR occurs immediately downstream of prtI, which encodes a protein homologous to extracytoplasmic function (ECF) sigma factors. These two genes have been proposed to function as a dicistronic operon. In this study, we demonstrated that deletion of prtI in WT WH6 had no effect on GAF production. However, deletion of prtI in the WH6-3 mutant overcame the effects of the Tn5 insertion in prtR and restored GAF production in the resulting double mutant. Complementation of the double prtIR mutant with prtI suppressed GAF production. This overall pattern of prtIR regulation was also observed for the activity of an AprX protease. Furthermore, reverse transcription quantitative real-time PCR analysis demonstrated that alterations in GAF production were mirrored by changes in the transcription of two putative GAF biosynthetic genes. Thus, we concluded that PrtI exerted a negative regulatory effect on GAF production, although the mechanism has not yet been determined. In addition, evidence was obtained that the transcription of prtI and prtR in WH6 may be more complex than predicted by existing models.

RNAi silencing of a cytochrome P450 monoxygenase disrupts the ability of a filamentous fungus, Graphium sp., to grow on short-chain gaseous alkanes and ethers.

Graphium sp. (ATCC 58400), a filamentous fungus, is one of the few eukaryotes that grows on short-chain alkanes and ethers. In this study, we investigated the genetic underpinnings that enable this fungus to catalyze the first step in the alkane and ether oxidation pathway. A gene, CYP52L1, was identified, cloned and functionally characterized as an alkane-oxidizing cytochrome P450 (GSPALK1). Analysis of CYP52L1 suggests that it is a member of the CYP52 cytochrome P450 family, which is comprised of medium- and long-chain alkane-oxidizing enzymes found in yeasts. However, phylogenetic analysis of GSPALK1 with other CYP52 members suggests they are not closely related. Post-transcriptional ds-RNA-mediated gene silencing of CYP52L1 severely reduced the ability of this fungus to oxidize alkanes and ethers, however, downstream metabolic steps in these pathways were unaffected. Collectively, the results of this study suggest that GSPALK1 is the enzyme that catalyzes the initial oxidation of alkanes and ethers but is not involved in the later steps of alkane or ether metabolism.