Article

Nocardiopsis ansamitocini sp. Nov., a new producer of ansamitocin P-3 of the genus Nocardiopsis

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Abstract

An alkalitolerant actinomycete strain, designated EGI 80425T, capable of producing ansamitocin P-3, was isolated from a saline-alkali soil sample of Xinjiang province, north-west China and subjected to a polyphasic taxonomic characterization. Strain EGI 80425T formed non-fragmented substrate mycelia and white-colored aerial hyphae with long spore chains. Whole-cell hydrolysates of the isolate contained meso-diaminopimelic acid as the diagnostic diamino acid and rhamnose as the major sugar. The major fatty acids identified were anteiso-C17:0, iso-C16:0 and C18:1 (ω9c). The predominant menaquinones detected were MK-10(H4), MK-10(H6), MK-10(H8) and MK-9(H4). The G+C content of the genomic DNA of strain EGI 80425T was 70.2 mol%. Strain EGI 80425T showed the highest 16S rRNA gene sequence similarity of to Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111T (96.44 %). Phylogenetic analysis showed that the strain EGI 80425T clustered with the members of the genus Nocardiopsis. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, the strain EGI 80425T represents a novel species of the genus Nocardiopsis, for which the name Nocardiopsis ansamitocini sp. nov. (Type strain EGI 80425T =CGMCC 9969T=KCTC 39605T) is proposed.

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... Recently, two new members of the genus Nocardiopsis were reported, Nocardiopsis mwathae which was isolated from haloalkaline lake Elmenteita (Akhwale et al., 2016), and Nocardiopsis ansamitocini that was isolated from a salinealkali soil sample (Zhang et al., 2016). Species of the genus Nocardiopsis harbour distinct features from other genera, such as variably hydrogenated menaquinones with ten isoprene units as major menaquinones phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol as main polar lipids and no diagnostic cell-wall amino acids (Hozzein & Trujillo, 2012). ...
... Members of the genus Nocardiopsis are widely distributed in nature, especially in hypersaline or alkaline soils (Hozzein & Trujillo, 2012), and have great potential in biotechnology (Bennur et al., 2015). Alkaliphilic or alkalitolerant species of the genus Nocardiopsis have great potential to produce bioactive pharmaceuticals (Horikoshi, 1999;Ding et al., 2010Ding et al., , 2012Zhang et al., 2016). During a study of the diversity of culturable actinobacteria, strain EGI 80674 T was isolated from rhizosphere soil of Halocnermum strobilaceum (Pall.) ...
Article
An alkalitolerant actinomycete strain, designated EGI 80674T, was isolated from a rhizosphere soil of Halocnermum strobilaceum (Pall.) Bieb of Xinjiang, north-west China and subjected to a polyphasic taxonomic characterization. Strain EGI 80674T formed white-colored aerial hyphae with long spore chains. Whole-cell hydrolysates of the isolate contained meso-diaminopimelic acid as the diagnostic diamino acid with no diagnostic sugars. The major fatty acids identified were iso-C16:0, anteiso-C17:0 and 10-methyl-C18:0TBSA. The predominant menaquinones detected were MK-10(H8) and MK-10(H6). The G+C content of the genomic DNA of strain EGI 80674T was 70.9 mol%. Strain EGI 80674T showed the highest 16S rRNA gene sequence similarity to Nocardiopsis nikkonensis NBRC 102170T (97.24 %). The DNA-DNA relatedness value of strain EGI 80674T and N. nikkonensis NBRC 102170T was 18.4 ± 1.3 %. Phenotypical, chemotaxonomic and phylogenetic characterstics and DNA-DNA hybridization data suggest that strain EGI 80674T represents a novel species of the genus Nocardiopsis, for which the name Nocardiopsis rhizosphaerae sp. nov. (Type strain EGI 80674T =CGMCC 4.7228T=KCTC 39673T) is proposed.
... auranticum ATCC 31565 (Yu et al. 2002). Later on, several other actinobacterial strains were identified as AP-3 producers too (Li et al. 2015;Ning et al. 2017;Zhang et al. 2016;Zhong et al. 2019). Except for AP-3, other macro-lactam compounds such as pretilactam and mecbecin were also detected in Actinosynnema pretiosum (Tanida et al. 1980;Wang et al. 2019). ...
Article
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Ansamitocin P-3 (AP-3) exhibits potent biological activities against various tumor cells. As an important drug precursor, reliable supply of AP-3 is limited by low fermentation yield. Although different strategies have been implemented to improve AP-3 yield, few have investigated the impact of efflux on AP-3 production. In this study, AP-3 efflux genes were identified through combined analysis of two sets of transcriptomes. The production-based transcriptome was implemented to search for efflux genes highly expressed in response to AP-3 accumulation during the fermentation process, while the resistance-based transcriptome was designed to screen for genes actively expressed in response to the exogenous supplementation of AP-3. After comprehensive analysis of two transcriptomes, six efflux genes outside the ansamitocin BGC were identified. Among the six genes, individual deletion of APASM_2704, APASM_6861, APASM_3193, and APASM_2805 resulted in decreased AP-3 production, and alternative overexpression led to AP-3 yield increase from 264.6 to 302.4, 320.4, 330.6, and 320.6 mg/L, respectively. Surprisingly, APASM_2704 was found to be responsible for exportation of AP-3 and another macro-lactam antibiotic pretilactam. Furthermore, growth of APASM_2704, APASM_3193, or APASM_2805 overexpression mutants was obviously improved under 300 mg/L AP-3 supplementation. In summary, our study has identified AP-3 efflux genes outside the ansamitocin BGC by comparative transcriptomic analysis, and has shown that enhancing the transcription of transporter genes can improve AP-3 production, shedding light on strategies used for exporter screening and antibiotic production improvement. Key points • AP-3-related efflux genes were identified by transcriptomic analysis. • Deletion of the identified efflux genes led in AP-3 yield decrease. • Overexpression of the efflux genes resulted in increased AP-3 production.
... Members of this genus are widely distributed in hypersaline environments and produce very distinct secondary metabolites (Tsujibo et al., 2003;Sun et al., 2015Sun et al., , 2017Sharma and Singh, 2016). In past few years, attempts to isolate novel Nocardiopsis strains were made and many novel species such as N. algeriensis (Bouras et al., 2015), N. mangrovei (Huang et al., 2015), N. oceani and N. nanhaiensis (Pan et al., 2015), N. ansamitocini (Zhang et al., 2016b), N. sediminis (Muangham et al., 2016), N. rhizosphaerae (Zhang et al., 2016c), and N. akesuensis (Gao et al., 2016) have been reported. At present, the genus comprises 53 species and 5 subspecies ( 1 2017). ...
Article
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The genus Nocardiopsis is an unique actinobacterial group that widely distributed in hypersaline environments. In this study, we investigated the growth conditions, transcriptome analysis, production and accumulation of ectoine by Nocardiopsis gilva YIM 90087 T under salt stress. The colony color of N. gilva YIM 90087 T changed from yellow to white under salt stress conditions. Accumulation of ectoine and hydroxyectoine in cells was an efficient way to regulate osmotic pressure. The ectoine synthesis was studied by transferring the related genes (ectA, ectB, and ectC) to Escherichia coli. Transcriptomic analysis showed that the pathways of ABC transporters (ko02010) and glycine, serine, and threonine metabolism (ko00260) played a vital role under salt stress environment. The ectABC from N. gilva YIM 90087 T was activated under the salt stress. Addition of exogenous ectoine and hydroxyectoine were helpful to protect N. gilva YIM 90087 T from salt stress.
... Members of this genus are widely distributed in hypersaline environments and produce very distinct secondary metabolites (Tsujibo et al., 2003;Sun et al., 2015Sun et al., , 2017Sharma and Singh, 2016). In past few years, attempts to isolate novel Nocardiopsis strains were made and many novel species such as N. algeriensis (Bouras et al., 2015), N. mangrovei (Huang et al., 2015), N. oceani and N. nanhaiensis (Pan et al., 2015), N. ansamitocini (Zhang et al., 2016b), N. sediminis (Muangham et al., 2016), N. rhizosphaerae (Zhang et al., 2016c), and N. akesuensis (Gao et al., 2016) have been reported. At present, the genus comprises 53 species and 5 subspecies ( 1 2017). ...
Article
The genus Nocardiopsis is an unique actinobacterial group that widely distributed in hypersaline environments. In this study, we investigated the growth conditions, transcriptome analysis, production and accumulation of ectoine by Nocardiopsis gilva YIM 90087 T under salt stress. The colony color of N. gilva YIM 90087 T changed from yellow to white under salt stress conditions. Accumulation of ectoine and hydroxyectoine in cells was an efficient way to regulate osmotic pressure. The ectoine synthesis was studied by transferring the related genes (ectA, ectB, and ectC) to Escherichia coli. Transcriptomic analysis showed that the pathways of ABC transporters (ko02010) and glycine, serine, and threonine metabolism (ko00260) played a vital role under salt stress environment. The ectABC from N. gilva YIM 90087 T was activated under the salt stress. Addition of exogenous ectoine and hydroxyectoine were helpful to protect N. gilva YIM 90087 T from salt stress.
... Members of this genus are widely distributed in hypersaline environments and produce very distinct secondary metabolites (Tsujibo et al., 2003;Sun et al., 2015Sun et al., , 2017Sharma and Singh, 2016). In past few years, attempts to isolate novel Nocardiopsis strains were made and many novel species such as N. algeriensis (Bouras et al., 2015), N. mangrovei (Huang et al., 2015), N. oceani and N. nanhaiensis (Pan et al., 2015), N. ansamitocini (Zhang et al., 2016b), N. sediminis (Muangham et al., 2016), N. rhizosphaerae (Zhang et al., 2016c), and N. akesuensis (Gao et al., 2016) have been reported. At present, the genus comprises 53 species and 5 subspecies ( 1 2017). ...
Article
Full-text available
The genus Nocardiopsis is an unique actinobacterial group that widely distributed in hypersaline environments. In this study, we investigated the growth conditions, transcriptome analysis, production and accumulation of ectoine by Nocardiopsis gilva YIM 90087T under salt stress. The colony color of N. gilva YIM 90087T changed from yellow to white under salt stress conditions. Accumulation of ectoine and hydroxyectoine in cells was an efficient way to regulate osmotic pressure. The ectoine synthesis was studied by transferring the related genes (ectA, ectB, and ectC) to Escherichia coli. Transcriptomic analysis showed that the pathways of ABC transporters (ko02010) and glycine, serine, and threonine metabolism (ko00260) played a vital role under salt stress environment. The ectABC from N. gilva YIM 90087T was activated under the salt stress. Addition of exogenous ectoine and hydroxyectoine were helpful to protect N. gilva YIM 90087T from salt stress.
... The biosynthetic process of AP-3, which is similar to 19-membered macrocyclic lactams, requires a starter unit, chain extension units and side chain modification [3]. Studies on AP-3 have been focusing on its production improvement by medium design [4], metabolic pathway manipulation [5], and genetic modification methods [6,7], as well as on its new microbial source [8], new derivatives [9], and its anti-tumor mechanism [10]. However, there is no proteomic publication on AP-3 fermentation under different culture conditions, although it might add helpful information to further pathway manipulation for its titer enhancement. ...
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Our previous work showed that the biosynthesis of ansamitocin P-3 (AP-3), an anti-tumor agent, by Actinosynnema pretiosum was depressed by ammonium but enhanced by isobutanol in the medium. Here we show proteomics analyses on A. pretiosum in different fermentation conditions with and without ammonium or isobutanol using two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization, and linear ion trap quadrupole mass spectrometry. Pairwise comparison of repetitive 2-DE maps was performed to find differentially expressed spots, and eight proteins were identified as functionally annotated ones. Among these proteins, D-3-phosphoglycerate dehydrogenase (PGDH) and glyceraldehyde 3-phosphate dehydrogenase showed statistically significant up-regulation in ammonium vs. basic or isobutanol medium, while fatty acid synthetase, histidine-tRNA ligase, transposase, molecular chaperone GroEL, SAM-dependent methyltransferase, and Crp/Fnr family transcriptional regulator were overexpressed in ammonium vs. basic medium. Based on the 2-DE data, exogenous L-serine which could inhibit the PGDH activity was added to the cultures with isobutanol, and a lower AP-3 production was confirmed under 2.5 mM serine addition (24 or 48 h).
... The natural habitats of strains belonging to the genus Nocardiopsis are distributed ubiquitously in the environment (Kroppenstedt & Evtushenko, 2006). Members of this genus have frequently been isolated from alkaline or saline soil and marine sediments (Chun et al., 2000;Al-Zarban et al., 2002;Sabry et al., 2004;Fang et al., 2011;Hamedi et al., 2011;Huang et al., 2015;Zhang et al., 2015). During our investigation of actinomycetes from mangrove in Ranong province, Thailand, strain 1SS5-02 T was isolated from sediment using the dilution-plate method. ...
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A method has been described for the isolation of DNA from micro-organisms which yields stable, biologically active, highly polymerized preparations relatively free from protein and RNA. Alternative methods of cell disruption and DNA isolation have been described and compared. DNA capable of transforming homologous strains has been used to test various steps in the procedure and preparations have been obtained possessing high specific activities. Representative samples have been characterized for their thermal stability and sedimentation behaviour.
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Bring in the new: Naphthospironone A (1), a novel polycyclic metabolite with an unprecedented spiro[bicyclo[3.2.1]octene-pyran]dione ring system, was isolated from alkalophilic Nocardiopsis sp. (YIM DT266). Its complete structural assignment with the absolute stereochemistry was elucidated by spectroscopic data, X-ray crystal diffraction, and calculation of optical rotation by using DFT methods. Naphthospironone A exhibited moderate cytotoxic and antibiotic bioactivities.
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A Gram-positive, non-motile, white-pigmented, short rod actinobacterium, designated YIM 90734T, was isolated from a saline soil sample collected from Ganjiahu Suosuo Forest National Nature Reserve in Xinjiang province, north-west China, and its taxonomic position was investigated by using a polyphasic approach. Strain YIM 90734T grew optimally at 28-37 degrees C and pH 6.0-8.0 and in 5% (w/v) NaCl. The peptidoglycan type was A4alpha, L-Lys-L-Ala-L-Glu and tyvelose and mannose were the major cell-wall sugars. The predominant menaquinones were MK-10 and MK-9. Major cellular fatty acids (>10% of total) were anteiso-C15:0 and anteiso-C17:0. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, one unknown phospholipid and two unknown glycolipids. The DNA G+C content was 70.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 90734T belonged to the genus Zhihengliuella. The 16S rRNA gene sequence similarity between strain YIM 90734T and the type strain of the only recognized Zhihengliuella species, Zhihengliuella halotolerans, was 97.7%. However, the level of DNA-DNA relatedness of the two strains was 41.4%. The DNA-DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain YIM 90734T could be differentiated from Z. halotolerans. On the basis of the data presented, strain YIM 90734T is considered to represent a novel species of the genus Zhihengliuella, for which the name Zhihengliuella alba sp. nov. is proposed. The type strain is YIM 90734T (=KCTC 19375T=DSM 21143T). The description of the genus Zhihengliuella has also been emended.
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WE have isolated a new group of ansamycin antibiotics with potent antitumour activity, from a fermentation broth of Nocardia sp. No. C-15003 (N-l) and have named it ansamitocin. Structures of ansamitocin were found to be similar to maytansine and related maytansinoids obtained from plant sources by Kupchan et al.1-4 and Wani et al.5. These comounds have strong antitumour activities, but development of production would be difficult, because plants containing maytansinoids are only harvested in tropical areas and their content in the plants is extremely low. Some attempts have been made to find a maytansinoid-producing microorganism6, but no success has been reported. We report here the microbial production, isolation and structural elucidation of these antibiotics and their antitumour activities against several experimental tumours in mice.
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Menaquinones were the only isoprenoid quinones found in 48 corynebacteria and actinomycete strains examined. Dihydromenaquinones having nine isoprene units were the main components isolated from Gordona, Mycobacterium, Corynebacterium bovis, Corynebacterium glutamicum and a strain labelled Nocardia farcinica, but dihydromenaquinones having eight isoprene units were characteristic of other Corynebacterium species and representatives of the 'rhodochrous' complex. Tetrahydromenaquinones having six and eight isoprene units were found in Nocardia strains and in a single strain of Micropolyspora brevicatena, which also contained mycolic acids similar in chain length to those of Nocardia. Menaquinones having nine isoprene units with from one to five double bonds hydrogenated were the main components in Actinomadura madurae, Actinomadura pelletieri, Micropolyspora faeni, Oerskovia turbata and Streptomyces strains. Actinomadura dassonvillei strains had a characteristic pattern of di-, tetra- and hexahydromenaquinones with 10 isoprene units which was slightly different from the pattern in mixtures of similar quinones from Actinomyces israelii and Actinomyces viscosus.
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A collection of 283 nocardioform bacteria was studied by means of 241 tests which included many biochemical, physiological and nutritional characters. The results were subjected to computer analysis in which two coefficients of association and two cluster-analysis techniques were used. Single linkage and average linkage cluster analysis gave almost identical results, but with the simple matching coefficient a few strains were grouped together on the basis of shared negative correlations. With the matching coefficient 15 clusters were formed whereas with the similarity coefficient there were 18. In each analysis the seven major clusters corresponded to (1) Nocardia asteroides, (2) Nocardia caviae, (3) Nocardia brasiliensis, (4) Actinomadura madurae, (5) Actinomadura pelletieri, (6) Oerskovia turbata, (7) the rhodochrous complex. The data revealed many new characters which could be weighted for identification. The clusters and subgroups were therefore readily distinguished from one another by several unrelated phenotypic characters.
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The application of maximum likelihood techniques to the estimation of evolutionary trees from nucleic acid sequence data is discussed. A computationally feasible method for finding such maximum likelihood estimates is developed, and a computer program is available. This method has advantages over the traditional parsimony algorithms, which can give misleading results if rates of evolution differ in different lineages. It also allows the testing of hypotheses about the constancy of evolutionary rates by likelihood ratio tests, and gives rough indication of the error of ;the estimate of the tree.
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The term "alkaliphile" is used for microorganisms that grow optimally or very well at pH values above 9 but cannot grow or grow only slowly at the near-neutral pH value of 6.5. Alkaliphiles include prokaryotes, eukaryotes, and archaea. Many different taxa are represented among the alkaliphiles, and some of these have been proposed as new taxa. Alkaliphiles can be isolated from normal environments such as garden soil, although viable counts of alkaliphiles are higher in samples from alkaline environments. The cell surface may play a key role in keeping the intracellular pH value in the range between 7 and 8.5, allowing alkaliphiles to thrive in alkaline environments, although adaptation mechanisms have not yet been clarified. Alkaliphiles have made a great impact in industrial applications. Biological detergents contain alkaline enzymes, such as alkaline cellulases and/or alkaline proteases, that have been produced from alkaliphiles. The current proportion of total world enzyme production destined for the laundry detergent market exceeds 60%. Another important application is the industrial production of cyclodextrin by alkaline cyclomaltodextrin glucanotransferase. This enzyme has reduced the production cost and paved the way for cyclodextrin use in large quantities in foodstuffs, chemicals, and pharmaceuticals. It has also been reported that alkali-treated wood pulp could be biologically bleached by xylanases produced by alkaliphiles. Other applications of various aspects of alkaliphiles are also discussed.
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A taxonomic study was performed on strain YIM 31775(T), which was isolated from a soil sample collected from Yunnan Province, China. The isolate was chemo-organotrophic, aerobic and Gram-negative. Cells were short rods and motile, with one or more polar flagella. Growth temperature and pH ranged from 4 to 55 degrees C and 6.5 to 12.0, respectively; the optimum growth temperature and pH were 28-37 degrees C and 7.0-9.0, respectively. Q-8 was the predominant respiratory lipoquinone. The major fatty acids were C(16 : 1)omega7c (42.4 %) and C(16 : 0) (28.1 %). The DNA G + C content was 62.4 +/ -0.3 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain YIM 31775(T) should be placed within the family 'Oxalobacteraceae', in which it formed a distinct lineage. Based on the high 16S rRNA gene sequence divergence and phenotypic characteristics, it is proposed that strain YIM 31775(T) should be classified as representing a novel member of the family 'Oxalobacteraceae', for which the name Naxibacter alkalitolerans gen. nov., sp. nov. is proposed. The type strain is YIM 31775(T) (= CCTCC AA 204003(T) = KCTC 12194(T)).
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A Gram-positive, motile, short-rod-shaped strain, designated YIM 004(T), was isolated from a forest-soil sample collected from Lijiang, Yunnan Province, China, and was investigated using a polyphasic taxonomic approach. The isolate contained chemotaxonomic markers that corresponded to those of its phylogenetic neighbour, Georgenia muralis, i.e. it possessed peptidoglycan type A4 alpha with lysine as the diagnostic cell-wall diamino acid, the predominant menaquinone was MK-8(H(4)) and the major fatty acid was ai-C(15 : 0). The G+C content of the genomic DNA was 72.9 mol%. Strain YIM 004(T) exhibited a 16S rRNA gene sequence similarity of 97.3 % and a DNA-DNA relatedness value of 18 % with respect to G. muralis DSM 14418(T). On the basis of the phenotypic and genotypic differences between the isolate and G. muralis, strain YIM 004(T) represents a novel species of the genus Georgenia, for which the name Georgenia ruanii sp. nov. is proposed. The type strain is YIM 004(T) (=CCTCC AB 204065(T)=DSM 17458(T)=KCTC 19029(T)). In addition, an emended description of the genus Georgenia is presented.
S2 Maximum-parsimony phylogenetic tree based on 16S rRNA gene sequences 43 showing the position of strain EGI 80425 T . Haloactinospora alba YIM 90648 T 44 (DQ923130) was used as an outgroup. Bootstrap values (expressed as percentages of 45 1,000 replications
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Fig. S2 Maximum-parsimony phylogenetic tree based on 16S rRNA gene sequences 43 showing the position of strain EGI 80425 T. Haloactinospora alba YIM 90648 T 44 (DQ923130) was used as an outgroup. Bootstrap values (expressed as percentages of 45 1,000 replications) of above 50 % are shown at the branch nodes.
Genus I. Nocardiopsis Meyer 1976, 487AL
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