Multi-omics analysis of antagonistic interactions among free-living Pseudonocardia from diverse ecosystems.

Actinomycetes are a phylogenetically diverse bacterial group which are widely distributed across terrestrial and aquatic ecosystems. Within this order, the genus Pseudonocardia and their specialised metabolites have been the focus of previous ecological studies due to their antagonistic interactions with other microorganisms and their mutualistic interactions with insects. However, the chemical ecology of free-living Pseudonocardia remains understudied. This study applies a multi-omics approach to investigate the chemical ecology of free-living actinomycetes from the genus Pseudonocardia. In a comparative genomics analysis, it was observed that the biosynthetic gene cluster family distribution was influenced mainly by phylogenetic distance rather than the geographic or ecological origin of strains. This finding was also observed in the mass spectrometry-based metabolomic profiles of nine Pseudonocardia species isolated from marine sediments and two terrestrial species. Antagonist interactions between these 11 species were examined, and matrix-assisted laser desorption/ionisation-mass spectrometry imaging was used to examine in situ chemical interactions between the Southern Ocean strains and their phylogenetically close relatives. Overall, it was demonstrated that phylogeny was the main predictor of antagonistic interactions among free-living Pseudonocardia. Moreover, two features at m/z 441.15 and m/z 332.20 were identified as metabolites related to these interspecies interactions.

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.

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).

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.

An EDTA-resistant pyrazinamidase from non-pathogen Pseudonocardia carboxydivorans.

OBJECTIVES: To characterize a pyrazinamidase from non-pathogen Pseudonocardia carboxydivorans. RESULTS: A pyrazinamidase gene pncA encoding a 23-kDa protein PncA-Pse from P. carboxydivorans was over-expressed in Escherichia coli and characterized. This PncA-Pse can convert both pyrazinamide and nicotinamide efficiently with the optimal pH and temperature of pH 8.5 and 45 °C, respectively. Although ferrous iron and manganese were detected in PncA-Pse, the enzymatic activity is not affected by EDTA with the final concentration of 10 mM. Moreover, the enzymatic activity was not significantly affected with the addition of several metal ions, respectively. Based on the structure modeling, the 61st histidine which is associated with the metal binding, was mutated into alanine to get mutant H61A. No activity, iron and manganese were detected for H61A, which implies that PncA-Pse is a metal enzyme with resistance of the metal ion chelator EDTA, which is different from the previous reports. CONCLUSION: This is the first characterized pyrazinamidase from the genus Pseudonocardia, a non-pathogen.