Actinomycetospora aeridis sp. nov., Actinomycetospora flava sp. nov. and Actinomycetospora aurantiaca sp. nov., endophytic actinobacteria isolated from wild orchid (Aerides multiflora Roxb).

Three novel mycelium-forming actinobacteria, designated OC33-EN06T, OC33-EN07T, and OC33-EN08T, were isolated from wild orchid (Aerides multiflora Roxb), collected from a hill evergreen forest in Northern Thailand. Strains OC33-EN06T and OC33-EN07T showed the highest 16S rRNA gene similarity with Actinomycetospora lutea TT00-04T, 99.17 and 99.45%, respectively. Strain OC33-EN08T showed high similarity with four species, namely 'Actinomycetospora termitidis Odt1-22T' (99.37%), Actinomycetospora chiangmaiensis DSM 45062T (99.02%), Actinomycetospora corticicola 014-5T (99.02%), and Actinomycetospora soli SF1T (98.81%). Comparative genome analysis of OC33-EN06T, OC33-EN07T, and OC33-EN08T with the closely related type strains showed that average nucleotide identity (ANI) based on blast, ANI based on MUMmer, and average amino acid identity values were less than 95% and the digital DNA-DNA hybridization values were less than 70%, all below the thresholds for species demarcation. The digital G+C content of OC33-EN06T, OC33-EN07T, and OC33-EN08T were 74.5, 74, and 74 mol%, respectively. These three strains developed bud-like chains of non-motile cylindrical spores with a smooth surface. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars contained ribose, arabinose, and galactose. The predominant menaquinone was MK-8(H4). The phospholipid profile included phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. Based on comparative analysis of genotypic, phenotypic and chemotaxonomic data, strains OC33-EN06T (=TBRC 18349T=NBRC 116543T), OC33-EN07T (=TBRC 18350T=NBRC 116544T), and OC33-EN08T (=TBRC 18318T=NBRC 116542T) represent the type strains of three novel species of the genus Actinomycetospora for which the names Actinomycetospora aeridis sp. nov., Actinomycetospora flava sp. nov., and Actinomycetospora aurantiaca sp. nov., are proposed.

Actinomycetospora lemnae sp. nov., A Novel Actinobacterium Isolated from Lemna aequinoctialis Able to Enhance Duckweed Growth.

Duckweed-associated actinobacteria are co-existing microbes that affect duckweed growth and adaptation. In this study, we aimed to report a novel actinobacterium species and explore its ability to enhance duckweed growth. Strain DW7H6T was isolated from duckweed, Lemna aequinoctialis. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that the strain was most closely related to Actinomycetospora straminea IY07-55T (99.0%), Actinomycetospora chibensis TT04-21T (98.9%), Actinomycetospora lutea TT00-04T (98.8%) and Actinomycetospora callitridis CAP 335T (98.4%). Chemotaxonomic and morphological characteristics of strain DW7H6T were consistent with members of the genus Actinomycetospora, while average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between the draft genomes of this strain and its closely related type strains were below the proposed threshold values used for species discrimination. Based on chemotaxonomic, phylogenetic, phenotypic, and genomic evidence obtained, we describe a novel Actinomycetospora species, for which the name Actinomycetospora lemnae sp. nov. is proposed. The type strain is DW7H6T (TBRC 15165T, NBRC 115294T). Additionally, the duckweed-associated actinobacterium strain DW7H6T was able to enhance duckweed growth when compared to the control, in which the number of fronds and biomass dry weight were increased by up to 1.4 and 1.3 fold, respectively. Moreover, several plant-associated gene features in the genome of strain DW7H6T potentially involved in plant-microbe interactions were identified.

Actinomycetospora soli sp. nov., isolated from the rhizosphere soil of Averrhoa carambola L.

A novel actinobacterium, designated strain SF1T, was isolated from the rhizosphere soil of a star fruit plant (Averrhoa carambola L.) collected from Bangkok, Thailand, and its taxonomic position was evaluated. The strain showed morphological, chemotaxonomic and phylogenetic characteristics consistent with its classification in the genus Actinomycetospora. Strain SF1T was an aerobic, Gram-stain-positive and non-motile actinobacterium. Growth occurred at 15-35 °C, at pH 4.0-12.0 and in the presence of 0-10 % (w/v) NaCl. The 16S rRNA gene sequence of strain SF1T showed the highest similarity to Actinomycetospora chiangmaiensis YIM 0006T (99.5 %), Actinomycetospora corticicola 014-5T (98.8 %) and Actinomycetospora endophytica A-T 8314T (98.8 %). The genome sequencing revealed a genome size of 6.52 Mbp and a DNA G+C content of 74.0 %. In addition, the average nucleotide identity values between strain SF1T and reference strains, A. chiangmaiensis DSM 45062T, A. corticicola DSM 45772T and A. endophytica TBRC 5722T, were found to be 86.1, 86.5 and 79.7 %, respectively, and the level of digital DNA-DNA hybridization between them were 32.4, 32.4 and 23.3 %, respectively. The whole-cell hydrolysates of strain SF1T contained meso-diaminopimelic acid as the diagnostic diamino acid, with arabinose, galactose, glucose and ribose as whole-cell sugars. The predominant menaquinone was MK-8(H4). Major cellular fatty acids were iso-C16 : 0 and a summed feature consisting of C16 : 1 ω6c/C16 : 1 ω7c. Characterization based on chemotaxonomic, phylogenetic, phenotypic and genomic evidence demonstrated that strain SF1T represents a novel species of the genus Actinomycetospora, for which the name Actinomycetospora soli sp. nov. is proposed. The type strain is strain SF1T (=TBRC 15166T= NBRC 115295T).

Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA.

Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cß hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.

Actinomycetospora callitridis sp. nov., an endophytic actinobacterium isolated from the surface-sterilised root of an Australian native pine tree.

An endophytic actinobacterium, strain CAP 335T, was isolated from a root sample of a native pine tree growing on the Bedford Park campus of Flinders University, Adelaide, South Australia. The result of a polyphasic study showed that this strain was identified as a new member of the genus Actinomycetospora. This strain was observed to be a Gram stain-positive, aerobic actinobacterium with well-developed substrate mycelia and to form short chains of spores. Actinomycetospora chibensis TT04-21T and Actinomycetospora straminea IY07-55T were found to be close phylogenetic neighbours, each sharing 99.1% 16S rRNA gene similarity. Chemotaxonomic data including major fatty acids, cell wall components and major menaquinones confirmed the affiliation of strain CAP 335T to the genus Actinomycetospora. The phylogenetic analysis, physiological and biochemical studies and DNA-DNA hybridization, allowed the genotypic and phenotypic differentiation of strain CAP 335T and the closely related species with valid names. The name proposed for the new species is Actinomycetospora callitridis sp. nov. The type strain is CAP 335T (= DSM 101857T = NRRL B-65350T).

Actinomycetospora endophytica sp. nov., isolated from wild orchid (Podochilus microphyllus Lindl.) in Thailand.

A novel endophytic actinomycete, designated strain A-T 8314T, was isolated from a wild orchid, Podochilus microphyllus Lindl., collected from Trat Province, Thailand. The taxonomic position of strain A-T 8314T was established using a combination of genotypic and phenotypic analyses. The isolate was a Gram-positive bacterium that developed bud-like spore chains. Strain A-T 8314T grew aerobically at an optimum temperature of 20-25 °C and an optimal pH 6.0. The cell wall contained meso-diaminopimelic acid, and the whole-cell sugars were ribose, arabinose and galactose. The predominant menaquinone was MK-8 (H4). The polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylmonomethylethanolamine, hydroxy-phosphatidylmonomethylethanolamine and hydroxyl phosphatidylethanolamine. The predominant cellular fatty acid was iso-C16 : 0. The DNA G+C content of the genomic DNA was 73.2±0.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A-T 8314T belonged to the genus Actinomycetospora, and was most closely related to Actinomycetospora chiangmaiensis YIM 0006T (98.8 %) and Actinomycetosporacorticicola 014-5T (98.6 %). The DNA-DNA relatedness values that distinguished A-T 8314T from its closest species were below 70 %. Following an evaluation of phenotypic, chemotaxonomic and genotypic studies, it was concluded that the new isolate represents as a novel species, for which the name Actinomycetospora endophytica sp. nov is proposed. The type strain is A-T 8314T (=TBRC 5722T=NBRC 113235T).