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1.
Artigo em Inglês | MEDLINE | ID: mdl-34582326

RESUMO

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.


Assuntos
Actinobacteria , Actinobacteria/genética , Actinomyces , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Oceanos e Mares , Filogenia , Pseudonocardia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análise
2.
Arch Microbiol ; 203(6): 3407-3413, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33890128

RESUMO

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.


Assuntos
Cupressaceae , Raízes de Plantas , Pseudonocardia , Cupressaceae/microbiologia , Ácidos Graxos/análise , Hibridização de Ácido Nucleico , Filogenia , Raízes de Plantas/microbiologia , Pseudonocardia/classificação , Pseudonocardia/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da Espécie
3.
Inorg Chem ; 60(8): 5432-5435, 2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33779143

RESUMO

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.


Assuntos
Compostos Férricos/metabolismo , Hidroliases/metabolismo , Sítios de Ligação , Compostos Férricos/química , Hidroliases/química , Estrutura Molecular , Pseudonocardia/enzimologia , Rhodococcus equi/enzimologia
4.
Antonie Van Leeuwenhoek ; 114(1): 23-35, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33230720

RESUMO

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).


Assuntos
Actinobacteria , Actinobacteria/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Estuários , Ácidos Graxos/análise , Hibridização de Ácido Nucleico , Filogenia , Pseudonocardia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
5.
FEBS J ; 288(11): 3570-3584, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33342083

RESUMO

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).


Assuntos
Esterases/ultraestrutura , Lipase/ultraestrutura , Conformação Proteica , Cristalografia por Raios X , Metagenoma/genética , Pseudonocardia/química , Pseudonocardia/genética , Pseudonocardia/ultraestrutura , Especificidade por Substrato/genética
6.
Nat Prod Res ; 35(2): 188-194, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31137966

RESUMO

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.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Indóis/química , Pseudonocardia/química , Uridina/química , Animais , Antozoários/microbiologia , Antibacterianos/química , Antineoplásicos/química , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Fermentação , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pseudonocardia/metabolismo
7.
Molecules ; 25(20)2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33086715

RESUMO

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.


Assuntos
Sequência de Aminoácidos/genética , Estabilidade Enzimática/genética , Hidroliases/química , Catálise , Hidroliases/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Pseudonocardia/química , Pseudonocardia/genética , Temperatura
8.
Curr Opin Chem Biol ; 59: 172-181, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32949983

RESUMO

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.


Assuntos
Formigas/microbiologia , Hypocreales/fisiologia , Actinobacteria/fisiologia , Animais , Interações Hospedeiro-Patógeno , Pseudonocardia/fisiologia , Streptomyces/fisiologia , Simbiose
9.
Int J Syst Evol Microbiol ; 70(11): 5648-5653, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32931406

RESUMO

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).


Assuntos
Filogenia , Pseudonocardia/classificação , Microbiologia do Solo , Áreas Alagadas , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Pseudonocardia/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Tailândia , Vitamina K 2/análogos & derivados , Vitamina K 2/química
10.
Sci Total Environ ; 744: 140823, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-32721670

RESUMO

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).


Assuntos
Água Subterrânea , Poluentes Químicos da Água , Actinobacteria , Biodegradação Ambiental , Dioxanos , Mycobacterium , Pseudonocardia , Rizosfera
11.
Biodegradation ; 31(3): 201-211, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32468172

RESUMO

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.


Assuntos
Água Subterrânea , Poluentes Químicos da Água , Actinobacteria , Biodegradação Ambiental , Dioxanos , Pseudonocardia
12.
Biotechnol Lett ; 42(9): 1707-1718, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32323078

RESUMO

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.


Assuntos
Amidoidrolases , Proteínas de Bactérias , Ácido Edético/química , Amidoidrolases/química , Amidoidrolases/genética , Amidoidrolases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Metais Pesados/química , Metais Pesados/metabolismo , Mutação , Pseudonocardia/enzimologia , Pseudonocardia/genética , Especificidade por Substrato
13.
J Nat Prod ; 83(3): 725-729, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-31961674

RESUMO

Fungus-growing ants and their microbial symbionts have emerged as a model system for understanding antibiotic deployment in an ecological context. Here we establish that bacterial symbionts of the ant Trachymyrmex septentrionalis antagonize their most likely competitors, other strains of ant-associated bacteria, using the thiopeptide antibiotic GE37468. Genomic analysis suggests that these symbionts acquired the GE37468 gene cluster from soil bacteria. This antibiotic, with known activity against human pathogens, was previously identified in a biochemical screen but had no known ecological role. GE37468's host-associated defense role in this insect niche intriguingly parallels the function of similar thiopeptides in the human microbiome.


Assuntos
Antibiose , Formigas/microbiologia , Peptídeos Cíclicos/química , Pseudonocardia/química , Tiazóis/química , Animais , Estrutura Molecular , Simbiose
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