Pseudonocardia lacus sp. nov., An Actinomycete Isolated from a Lake Sediment Sample.

A novel actinomycete strain, designated H11425T, was isolated from a sediment sample collected from Baihua Lake, Guizhou Province, PR China, and a polyphasic approach was employed to determine its taxonomic position. 16S rRNA gene sequence comparisons showed that strain H11425T is most closely related to Pseudonocardia sulfidoxydans JCM 10411T (97.9%) and Pseudonocardia kunmingensis JCM 32122T (97.8%). Both of phylogenetic analysis based on 16S rRNA gene sequence and phylogenomic analysis based on whole-genome sequence showed that strain H11425T formed a separate clade within the genus Pseudonocardia. The draft genome had a length of 8,059,576 bp with a G + C content of 74.5%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain H11425T and its closely related Pseudonocardia species were 76.8-79.0%, 64.8-69.9% and 21.7-23.3%, respectively, which were significantly lower than the widely accepted species-defined threshold. Strain H11425T contained meso-diaminopimelic acid, arabinose, galactose, glucose and ribose in its whole-cell hydrolysates. Mycolic acids were absent. The menaquinone was identifed as MK-8(H4). The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylcholine, an unknown phospholipid and four unidentified aminophospholipids. The major fatty acids were iso-C16:0, iso-C14:0, iso H-C16:1 and iso-C16:0 2OH. On the basis of the taxonomic evidence, strain H11425T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia lacus sp. nov. is proposed. The type strain is H11425T (= JCM 34851T = CICC 25118T).

Genome Mining and Metabolomics Unveil Pseudonochelin: A Siderophore Containing 5-Aminosalicylate from a Marine-Derived Pseudonocardia sp. Bacterium.

Pseudonochelin (1), a siderophore from a marine-derived Pseudonocardia sp. bacterium, was discovered using genome mining and metabolomics technologies. A 5-aminosalicylic acid (5-ASA) unit, not previously found in siderophore natural products, was identified in 1. Annotation of a putative psn biosynthetic gene cluster combined with bioinformatics and isotopic enrichment studies enabled us to propose the biosynthesis of 1. Moreover, 1 was found to display in vitro and in vivo antibacterial activity in an iron-dependent fashion.

Pseudonocardia terrae sp. nov., an actinobacterium isolated from rice rhizosphere soil in Thailand.

A novel actinomycete, designated strain RS11V-5T, was isolated from rhizosphere soil of Oryza sativa L. collected from Roi Et Province, Thailand, and its taxonomic position was evaluated. Cells of strain RS11V-5T were Gram-stain-positive, aerobic, and non-motile. Whole-cell hydrolysates contained meso-diaminopimelic acid, arabinose, galactose, glucose and ribose. MK-8(H4) was detected as the predominant menaquinone of this strain. The polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, hydroxy-phosphatidylmethylethanolamine, hydroxy-phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid and an unidentified glycolipid. The major fatty acids were iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c. Phylogenetic analyses based on the 16S rRNA gene sequences showed that strain RS11V-5T belonged to the genus Pseudonocardia and had high 16S rRNA sequence similarity of 99.3 % to Pseudonocardia kujensis KCTC 29062T and less than 98.4 % to other members of the genus Pseudonocardia. The DNA G+C content of the strain RS11V-5T was 73.3 mol%. Strain RS11V-5T showed 46.5 % digital DNA-DNA hybridization, 92.2 % orthologous average nucleotide identity (OrthoANI), 90.2 % ANI based on blast and 92.7 % ANI based on MUMmer to P. kujensis KCTC 29062T. Based its phenotypic, genotypic, phylogenetic and chemotaxonomic characteristics, strain RS11V-5T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia terrae sp. nov. is proposed. The type strain is RS11V-5T (=TBRC 15286T=NBRC 115296T).

Antifungal Macrolides Kongjuemycins from Coral-Associated Rare Actinomycete Pseudonocardia kongjuensis SCSIO 11457.

Four new macrolides, kongjuemycins A and B1-B3 (1-4), were isolated from a coral-associated actinomycete Pseudonocardia kongjuensis SCSIO 11457. Their structures were characterized by comprehensive spectroscopic analysis and single-crystal X-ray diffraction. The absolute configurations of 1 and 2 were established by electronic circular dichroism calculation and the modified Mosher's method. Kongjuemycins displayed antifungal activity against three phytopathogenic fungi.

Pseudonocardia humida sp. nov., an Actinomycete Isolated from Mangrove Soil Showing Distinct Distribution Pattern of Biosynthetic Gene Clusters.

A novel actinomycete strain, designated S2-4T, was isolated from a mangrove soil sample, and a polyphasic approach was employed to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene indicated that strain S2-4T formed a unique clade next to that harboring Pseudonocardia dioxanivorans CB1190T, which shared the highest sequence similarity (98.20%) with the new isolate. Phylogenetic analysis based on core genes of genomic sequences displayed a different scenario, exhibiting closer phylogenetic relationship of strain S2-4T with several species with 16S rRNA gene sequence similarities ranging from 96.95 to 98.06%, which was confirmed by the phylogenetic tree reconstructed based on genomic sequences. Further, substantial differences between the genotypic properties of strain S2-4T and its closest neighbors, including digital DNA-DNA hybridization, average nucleotide identity, and distribution patterns of biosynthetic gene clusters (BGC), indicated the taxonomic position of strain S2-4T as a novel species of the genus Pseudonocardia. Accordingly, strain S2-4T was observed to show a different distribution pattern of a predicted BGC encoding ectoine by comparative genomic analysis, which could be strongly linked to its unique habitat distinct from where its close neighbors were isolated. The major cellular fatty acids were iso-C15:0, C21:0, and iso-C16:0. The predominant menaquinone was MK-8(H4). The polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidyl-N-monomethylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylinositol mannosides, and two unidentified glycolipids. Here, we propose a novel species of the genus Pseudonocardia: Pseudonocardia humida sp. nov. with the type strain S2-4T (= JCM 34291T = CGMCC 4.7706T).

Genus Pseudonocardia: What we know about its biological properties, abilities and current application in biotechnology.

The genus Pseudonocardia belongs to a group of Actinomycetes, and is a member of the family Pseudonocardiacea. The members of this genus are aerobic, Gram-positive, non-motile bacteria that are commonly found in soil, plant and environment. Although this genus has a low clinical significance; however, it has an important role in biotechnology due to the production of secondary metabolites, some of which have anti-bacterial, anti-fungal and anti-tumour effects. The use of phenotypic tests, such as gelatinase activity, starch hydrolysis, catalase and oxidase tests, as well as molecular methods, such as polymerase chain reaction, are necessary for Pseudonocardia identification at the genus and species levels.

Genome analysis and plant growth promoting activity of Pseudonocardia strain DR1-2 from the root of Dendrobium christyanum Rchb.f

Aims@#The objective of this study was to analyze the genome of endophytic actinomycete associated with orchids and evaluate its plant hormone activities, including phytohormone, siderophore, ammonia production, zinc and phosphate solubilization.@*Methodology and results@#Strain DR1-2 isolated from the roots of the Thai orchid, Dendrobium christyanum Rchb.f., was closely related to Pseudonocardia alni DSM 44104T, P. antarctica DSM 44749T and P. carboxydivorans Y8T (99.93-100% similarity) based 16S rRNA gene sequence. This strain exhibited IAA production (294.10 ± 12.17 μg/mL), phosphate solubilization (2.20 ± 0.08 solubilization Index, SI), positive for siderophore production and ammonia production (36.99 ± 2.24 μg/mL). It showed a maximum IAA of 489.73 ± 8.90 μg/mL, when optimized using 0.5% Ltryptophan, pH 6 and incubated at 30 °C for 7 days. The IAA of strain enhanced the root length, shoot length, number of roots and fresh weight of rice seedlings (Oryza sativa L. cv. RD49). The draft genome of strain DR1-2 was 6,077,423 bp in 23 contigs with G+C content of 74.6%. The average nucleotide identity-Blast (ANIb) and average nucleotide identity-MUMmer (ANIm) values of strain DR1-2 and related type strains were 95.81 to 97.25% and the digital DNA-DNA hybridization (dDDH) values were 72.60 to 74.00%, respectively. Genomic analysis of strain DR1-2 revealed that the gene encodes the enzyme involved in the phytohormones biosynthesis and gene clusters involved in the biosynthesis of bioactive metabolites.@*Conclusion, significance and impact of study@#Endophytic actinomycete, Pseudonocardia strain DR1-2 from Thai orchid, D. christyanum Rchb.f., exhibited significant IAA production and affected the growth of the plant, which was the potential source of plant hormones for agricultural applications.

Symbiont-Mediated Protection of Acromyrmex Leaf-Cutter Ants from the Entomopathogenic Fungus Metarhizium anisopliae.

Many fungus-growing ants engage in a defensive symbiosis with antibiotic-producing ectosymbiotic bacteria in the genus Pseudonocardia, which help protect the ants' fungal mutualist from a specialized mycoparasite, Escovopsis. Here, using germfree ant rearing and experimental pathogen infection treatments, we evaluate if Acromyrmex ants derive higher immunity to the entomopathogenic fungus Metarhizium anisopliae from their Pseudonocardia symbionts. We further examine the ecological dynamics and defensive capacities of Pseudonocardia against M. anisopliae across seven different Acromyrmex species by controlling Pseudonocardia acquisition using ant-nonnative Pseudonocardia switches, in vitro challenges, and in situ mass spectrometry imaging (MSI). We show that Pseudonocardia protects the ants against M. anisopliae across different Acromyrmex species and appears to afford higher protection than metapleural gland (MG) secretions. Although Acromyrmex echinatior ants with nonnative Pseudonocardia symbionts receive protection from M. anisopliae regardless of the strain acquired compared with Pseudonocardia-free conditions, we find significant variation in the degree of protection conferred by different Pseudonocardia strains. Additionally, when ants were reared in Pseudonocardia-free conditions, some species exhibit more susceptibility to M. anisopliae than others, indicating that some ant species depend more on defensive symbionts than others. In vitro challenge experiments indicate that Pseudonocardia reduces Metarhizium conidiospore germination area. Our chemometric analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) reveals that Pseudonocardia-carrying ants produce more chemical signals than Pseudonocardia-free treatments, indicating that Pseudonocardia produces bioactive metabolites on the Acromyrmex cuticle. Our results indicate that Pseudonocardia can serve as a dual-purpose defensive symbiont, conferring increased immunity for both the obligate fungal mutualist and the ants themselves. IMPORTANCE In some plants and animals, beneficial microbes mediate host immune response against pathogens, including by serving as defensive symbionts that produce antimicrobial compounds. Defensive symbionts are known in several insects, including some leaf-cutter ants where antifungal-producing Actinobacteria help protect the fungal mutualist of the ants from specialized mycoparasites. In many defensive symbioses, the extent and specificity of defensive benefits received by the host are poorly understood. Here, using "aposymbiotic" rearing, symbiont switching experiments, and imaging mass spectrometry, we explore the ecological and chemical dynamics of the model defensive symbiosis between Acromyrmex ants and their defensive symbiotic bacterium Pseudonocardia. We show that the defensive symbiont not only protects the fungal crop of Acromyrmex but also provides protection from fungal pathogens that infect the ant workers themselves. Furthermore, we reveal that the increased immunity to pathogen infection differs among strains of defensive symbionts and that the degree of reliance on a defensive symbiont for protection varies across congeneric ant species. Taken together, our results suggest that Acromyrmex-associated Pseudonocardia have evolved broad antimicrobial defenses that promote strong immunity to diverse fungal pathogens within the ancient fungus-growing ant-microbe symbiosis.

Pseudonocardia abyssalis sp. nov. and Pseudonocardia oceani sp. nov., two novel actinomycetes isolated from the deep Southern Ocean.

The actinomycetes strains KRD168T and KRD185T were isolated from sediments collected from the deep Southern Ocean and, in this work, they are described as representing two novel species of the genus Pseudonocardia through a polyphasic approach. Despite sharing >99 % 16S rRNA gene sequence similarity with other members of the genus, comparative genomic analysis allowed species delimitation based on average nucleotide identity and digital DNA-DNA hybridization. The KRD168T genome is characterized by a size of 6.31 Mbp and a G+C content of 73.44 mol%, while the KRD185T genome has a size of 6.82 Mbp and a G+C content of 73.98 mol%. Both strains contain meso-diaminopimelic acid as the diagnostic diamino acid, glucose as the major whole-cell sugar, MK-8(H4) as a major menaquinone and iso-branched hexadecanoic acid as a major fatty acid. Biochemical and fatty acid analyses also revealed differences between these strains and their phylogenetic neighbours, supporting their status as distinct species. The names Pseudonocardia abyssalis sp. nov. (type strain KRD168T=DSM 111918T=NCIMB 15270T) and Pseudonocardia oceani (type strain KRD185T=DSM 111919T=NCIMB 15269T) are proposed.

Engineering of the thermophilic nitrile hydratase from Pseudonocardia thermophila JCM3095 for large-scale nicotinamide production based on sequence-activity relationships.

The green biocatalyst nitrile hydratase (NHase) is able to bio-transform 3-cyanopyridine into nicotinamide. As the NHase reaction is exothermic, an enzyme with high activity and stability is needed for nicotinamide production. In this study, we used sequence analysis and site-directed mutagenesis to generate a mutant of thermophilic NHase from Pseudonocardia thermophila JCM3095 with substantially enhanced activity and developed a powerful process for nicotinamide bio-production. The specific activity of αF126Y/αF168Y mutant was successfully increased by 3.98-fold over that of the wild-type enzyme. The half-life of such mutant was longer than 2 h, which was comparable to its parent enzyme. The relative activity of the αF126Y/αF168Y mutant after treatment with 1 M 3-cyanopyridine and 2 M nicotinamide was 73.2% and 63.7%, respectively, showing minor loss of its original stability. Structural analysis demonstrated that hydrogen bonds at the active site and α-ß subunit interface of the NHase contribute to the improved activity and the maintenance of stability. Escherichia coli transformant harboring the mutant NHase was used for nicotinamide bio-production, yielding a nicotinamide productivity of 251.1 g/(L·h), which is higher than the productivity obtained using other NHase-containing strains and transformants. The newly established variant is therefore a promising alternative for the industrial production of nicotinamides.

Pseudonocardia pini sp. nov., an endophytic actinobacterium isolated from roots of the pine tree Callitris preissii.

A Gram-positive, aerobic, actinobacterial strain with rod-shaped spores, CAP47RT, which was isolated from the surface-sterilized root of a native pine tree (Callitris preissii), grown in South Australia is described. The major cellular fatty acid of this strain was iso-H-C16:1 and major menaquinone was MK-8(H4). The diagnostic diamino acid in the cell-wall peptidoglycan was identified as meso-diaminopimelic acid. These chemotaxonomic data confirmed the affiliation of strain CAP47RT to the genus Pseudonocardia. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this strain in the family Pseudonocardiaceae, being most closely related to Pseudonocardia xishanensis JCM 17906T (98.8%), Pseudonocardia oroxyli DSM 44984T (98.7%), Pseudonocardia thailandensis CMU-NKS-70T (98.7%), and Pseudonocardia ailaonensis DSM 44979T (97.9%). The results of the polyphasic study which contain genome comparisons of ANIb, ANIm, and digital DNA-DNA hybridization revealed the differentiation of strain CAP47RT from the closest species with validated names. This strain represents a novel species and the name proposed for this microorganism is Pseudonocardia pini sp. nov., indicating the source of this actinobacterium from a pine tree. The type strain is CAP47RT (= DSM 108967T = NRRL B-65534T). Genome mining revealed that this strain contained a variety of genes encoding enzymes that can degrade hazardous chemicals.

Insight into the Maturation Process of the Nitrile Hydratase Active Site.

The metal binding motif of all nitrile hydratases (NHases, EC 4.2.1.84) is highly conserved (CXXCSCX) in the α-subunit. Accordingly, an eight amino acid peptide (VCTLCSCY), based on the metal binding motif of the Co-type NHase from Pseudonocardia thermophilia (PtNHase), was synthesized and shown to coordinate Fe(II) under anaerobic conditions. Parallel-mode EPR data on the mononuclear Fe(II)-peptide complex confirmed an integer-spin signal at g' ∼ 9, indicating an S = 2 system with unusually small axial ZFS, D = 0.29 cm-1 Exposure to air yielded a transient high-spin EPR signal most consistent with an intermediate/admixed S = 3/2 spin state, while the integer-spin signal was extinguished. Prolonged exposure to air resulted in the observation of EPR signals at g = 2.04, 2.16, and 2.20, consistent with the formation of a low-spin Fe(III)-peptide complex with electronic and structural similarity to the NHase from Rhodococcus equi TG328-2 (ReNHase). Coupled with MS data, these data support a progression for iron oxidation in NHases that proceeds from a reduced high spin to an oxidized high spin followed by formation of an oxidized low-spin iron center, something that heretofore has not been observed.

Crystal structures of a novel family IV esterase in free and substrate-bound form.

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme-substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV. DATABASE: Structural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo-PtEst1) and 6Z69 (PtEst1-inhibitor complex).

Pseudonocardia cytotoxica sp. nov., a novel actinomycete isolated from an Arctic fjord with potential to produce cytotoxic compound.

Herein we report the isolation of a novel actinomycete, strain MCCB 268T, from the sediment sample collected from a high Arctic fjord Kongsfjorden. MCCB 268T showed greater than 97% 16S rRNA gene sequence similarity with those of Pseudonocardia konjuensis LM 157T (98.06%), Pseudonocardia soli NW8-21 (97.22%) Pseudonocardia endophytica YIM 56035 (97.08%) and Pseudonocardia nantongensis KLBMP 1282 (97.34%) showing that the strain should be assigned to the genus Pseudonocardia. DNA-DNA hybridization with Pseudonocardia konjuensis LM 157T showed only 41.5% relatedness to strain MCCB 268T. The whole genome of the strain MCCB 268T was sequenced. Whole-genome average nucleotide identity, dDDH (%) and genome tree analysis demonstrated that strain significantly differed from other Pseudonocardia species. The G + C content was 70.5 mol%. MCCB 268T exhibited in vitro cytotoxicity and through bioassay guided fractionation followed by HPLC separation a cytotoxic compound (I) was isolated. The compound (I) was identified as 1-acetyl-ß-carboline through NMR spectra and high-resolution mass spectrometry. Compound (I) showed cytotoxicity against lung cancer cell line and mode of anticancer activity was found to be through the induction of apoptosis. Based on the genotypic and phenotypic features, MCCB 268T ought to be classified as a novel species under the genus Pseudonocardia for which the name Pseudonocardia cytotoxica sp. nov. is proposed (= CCUG72333T = JCM32718T).

A new uridine derivative and a new indole derivative from the coral-associated actinomycete Pseudonocardia sp. SCSIO 11457.

A new uridine derivative 11457 A (1), and a new indole derivative 11457B (2), together with a known compound 1H-indole-2-carbaldehyde (3), were characterized from the fermentation broth of the actinomycete Pseudonocardia sp. SCSIO 11457, an isolate associated with the scleractinian coral Galaxea fascicularis. Upon detailed spectroscopic analysis, 11457 A (1) was identified as a uridine analog, and 11457B (2) was elucidated as an indole derivative 2-hydroxy-1-(1H-indol-2-yl)pentane-1,4-dione. Biological evaluation indicated that none of compounds 1-3 showed antibacterial activities against pathogenic bacteria and cytotoxic activities against human cancer cell lines.

Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability.

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/ßA20P/ßP38L/ßF118W/ßS130Y/ßC189N/ßC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

Pseudonocardia acidicola sp. nov., a novel actinomycete isolated from peat swamp forest soil.

A novel actinobacterium, designated strain K10HN5T, was isolated from a peat soil sample collected from Kantulee peat swamp forest, Surat Thani Province, Thailand and its taxonomic position was determined using a polyphasic approach. Strain K10HN5T contained meso-diaminopimelic acid, arabinose, galactose, glucose and ribose in its whole-cell hydrolysates. The predominant menaquinone was MK-8(H4). The major fatty acids were iso-C16 : 0, iso-C15 : 0 and iso-C16 : 1H. Mycolic acids were not present. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylethanolamine, hydroxyphosphatidylmethylethanolamine and phosphatidylinositol. The 16S rRNA gene sequence analysis indicated that it was closely related to Pseudonocardia bannensis DSM 45300T (97.9 %) and Pseudonocardia xinjiangensis JCM 11839T (97.9 %). Strain K10HN5T exhibited low average nucleotide identity and digital DNA-DNA hybridization values with P. bannensis DSM 45300T (82.6, 28.7 %) and P. xinjiangensis JCM11839T (76.3, 22.2 %). The DNA G+C content of strain K10HN5T was 72.4 mol%. Based on polyphasic data, strain K10HN5T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia acidicola sp. nov. is proposed. The type strain is K10HN5T (=TBRC 10048T=NBRC 113897T).

Chemical warfare between fungus-growing ants and their pathogens.

Fungus-growing attine ants are under constant threat from fungal pathogens such as the specialized mycoparasite Escovopsis, which uses combined physical and chemical attack strategies to prey on the fungal gardens of the ants. In defence, some species assemble protective microbiomes on their exoskeletons that contain antimicrobial-producing Actinobacteria. Underlying this network of mutualistic and antagonistic interactions are an array of chemical signals. Escovopsis weberi produces the shearinine terpene-indole alkaloids, which affect ant behaviour, diketopiperazines to combat defensive bacteria, and other small molecules that inhibit the fungal cultivar. Pseudonocardia and Streptomyces mutualist bacteria produce depsipeptide and polyene macrolide antifungals active against Escovopsis spp. The ant nest metabolome is further complicated by competition between defensive bacteria, which produce antibacterials active against even closely related species.

Bioaugmenting the poplar rhizosphere to enhance treatment of 1,4-dioxane.

1,4-Dioxane is a highly mobile and persistent groundwater pollutant that often forms large dilute plumes. Because of this, utilizing aggressive pump-and-treat and ex-situ technologies such as advanced oxidation can be prohibitively expensive. In this study, we bioaugmented the poplar rhizosphere with dioxane-degrading bacteria Mycobacterium dioxanotrophicus PH-06 or Pseudonocardia dioxanivorans CB1190 to enhance treatment of 1,4-dioxane in bench-scale experiments. All treatments tested removed 10 mg/L dioxane to near health advisory levels (<4 µg/L). However, PH-06-bioaugmented poplar significantly outperformed all other treatments, reaching <4 µg/L in only 13 days. Growth curve experiments confirmed that PH-06 could not utilize root extract as an auxiliary carbon source for growth. Despite this limitation, our findings suggest that PH-06 is a strong bioaugmentation candidate to enhance the treatment of dioxane by phytoremediation. In addition, we confirmed that CB1190 could utilize both 1,4-dioxane and root extract as substrates. Finally, we demonstrated the large-scale production of these two strains for use in the field. Overall, this study shows that combining phytoremediation and bioaugmentation is an attractive strategy to treat dioxane-contaminated groundwater to low risk-based concentrations (~1 µg/L).

Enhanced long-term attenuation of 1,4-dioxane in bioaugmented flow-through aquifer columns.

Long term natural attenuation of 1,4-dioxane (dioxane) and its enhanced biodegradation after bioaugmentation with Pseudonocardia dioxanivorans CB1190 were assessed using flow-through aquifer columns. Natural attenuation of dioxane was not observed even after 2 years of acclimation. However, dioxane removal was observed in the bioaugmented columns (34% when the influent was 200 µg/L and 92% for 5 mg/L). The thmA gene that encodes the tetrahydrofuran monooxygenase that initiates dioxane degradation by CB1190 was only detected at the inoculation port and persisted for months after inoculation, implying the resiliency of bioaugmentation and its potential to offer long-term enhanced biodegradation capabilities. However, due to extensive clumping and limited mobility of CB1190, the augmented catabolic potential may be restricted to the immediate vicinity of the inoculation port. Accordingly, bioaugmentation with CB1190 seems more appropriate for the establishment of biobarriers. Bioaugmentation efficiency was associated with the availability of oxygen. Aeration of the column influent to increase dissolved oxygen significantly improved dioxane removal (p < 0.05), suggesting that (for sites with oxygen-limiting conditions) bioaugmentation can benefit from engineered approaches for delivering additional oxygen.