Metabolomics Tools Assisting Classic Screening Methods in Discovering New Antibiotics from Mangrove Actinomycetia in Leizhou Peninsula.

Mangrove actinomycetia are considered one of the promising sources for discovering novel biologically active compounds. Traditional bioactivity- and/or taxonomy-based methods are inefficient and usually result in the re-discovery of known metabolites. Thus, improving selection efficiency among strain candidates is of interest especially in the early stage of the antibiotic discovery program. In this study, an integrated strategy of combining phylogenetic data and bioactivity tests with a metabolomics-based dereplication approach was applied to fast track the selection process. A total of 521 actinomycetial strains affiliated to 40 genera in 23 families were isolated from 13 different mangrove soil samples by the culture-dependent method. A total of 179 strains affiliated to 40 different genera with a unique colony morphology were selected to evaluate antibacterial activity against 12 indicator bacteria. Of the 179 tested isolates, 47 showed activities against at least one of the tested pathogens. Analysis of 23 out of 47 active isolates using UPLC-HRMS-PCA revealed six outliers. Further analysis using the OPLS-DA model identified five compounds from two outliers contributing to the bioactivity against drug-sensitive A. baumannii. Molecular networking was used to determine the relationship of significant metabolites in six outliers and to find their potentially new congeners. Finally, two Streptomyces strains (M22, H37) producing potentially new compounds were rapidly prioritized on the basis of their distinct chemistry profiles, dereplication results, and antibacterial activities, as well as taxonomical information. Two new trioxacarcins with keto-reduced trioxacarcinose B, gutingimycin B (16) and trioxacarcin G (20), together with known gutingimycin (12), were isolated from the scale-up fermentation broth of Streptomyces sp. M22. Our study demonstrated that metabolomics tools could greatly assist classic antibiotic discovery methods in strain prioritization to improve efficiency in discovering novel antibiotics from those highly productive and rich diversity ecosystems.

Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site.

Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP-N-acetylglucosamine to yield corresponding midecamycin 2'-O-glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP-N-acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2'-O-glucopyranoside, midecamycin 2'-O-GlcNAc and midecamycin 2'-O-xylopyranoside. In contrast to midecamycin, these midecamycin 2'-O-glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.

Diversity, novelty, antimicrobial activity, and new antibiotics of cultivable endophytic actinobacteria isolated from psammophytes collected from Taklamakan Desert.

Three hundred and twenty endophytic actinobacterial strains were isolated from psammophytes collected from Taklamakan Desert and identified. Among them, three strains already had been identified as new species of two genera and sixteen isolates showed relatively low 16S rRNA similarities < 98.6% to validly described species. Seventy-five of the isolates were selected as representative strains to screen antibacterial activity and mechanism. Forty-seven strains showed antagonistic activity against at least one of the indicator bacteria. Two Streptomyces strains produced bioactive compounds inducing DNA damage, and two Streptomyces strains produced bioactive compounds with inhibitory activity on protein biosynthesis. Notably, the strain Streptomyces sp. 8P21H-1 that demonstrated both strong antibacterial activity and inhibitory activity on protein biosynthesis was prioritized for exploring new antibiotics. Under the strategy of integrating genetics-based discovery program and MS/MS-based molecular networking, two new streptogramin-type antibiotics, i.e., acetyl-griseoviridin and desulphurizing griseoviridin, along with known griseoviridin, were isolated from the culture broth of strain 8P21H-1. Their chemical structures were determined by HR-MS, and 1D and 2D NMR. Desulphurizing griseoviridin and griseoviridin exhibited antibacterial activities by inhibiting translation.

Asymmetric Total Synthesis of Hetiamacins A-F.

Herein, we report a concise and stereoselective approach for the asymmetric total synthesis of hetiamacins A-F on the basis of the total synthesis of amicoumacin C, which could be synthesized from a known l-aspartic acid derivative. The synthesis of hetiamacin A was accomplished by an 11-step sequence that featured 1,3-oxazinane ring formation of amicoumacin B followed by amidation in one pot. Hetiamacins B-F were synthesized from amicoumacin A in only one step.

Bioprospecting of Soil-Derived Actinobacteria Along the Alar-Hotan Desert Highway in the Taklamakan Desert.

Taklamakan desert is known as the largest dunefield in China and as the second largest shifting sand desert in the world. Although with long history and glorious culture, the Taklamakan desert remains largely unexplored and numerous microorganisms have not been harvested in culture or taxonomically identified yet. The main objective of this study is to explore the diversity, novelty, and pharmacological potential of the cultivable actinomycetes from soil samples at various sites along the Alar-Hotan desert highway in the Taklamakan desert. A total of 590 actinobacterial strains were recovered by the culture-dependent approach. Phylogenetic analysis based on 16S ribosomal RNA (rRNA) gene sequences unveiled a significant level of actinobacterial diversity with 55 genera distributed in 27 families of 12 orders. Thirty-six strains showed relatively low 16S rRNA similarities (<98.65%) with validly described species, among which four strains had already been characterized as novel taxa by our previous research. One hundred and forty-six actinobacterial isolates were selected as representatives to evaluate the antibacterial activities and mechanism of action by the paper-disk diffusion method and a double fluorescent protein reporter "pDualrep2" system, respectively. A total of 61 isolates exhibited antagonistic activity against the tested "ESKAPE" pathogens, among which seven strains could produce bioactive metabolites either to be able to block translation machinery or to induce SOS-response in the pDualrep2 system. Notably, Saccharothrix sp. 16Sb2-4, harboring a promising antibacterial potential with the mechanism of interfering with protein translation, was analyzed in detail to gain deeper insights into its bioactive metabolites. Through ultra-performance liquid chromatography (UPLC)-quadrupole time-of-flight (QToF)-MS/MS based molecular networking analysis and databases identification, four families of compounds (1-16) were putatively identified. Subsequent bioassay-guided separation resulted in purification of four 16-membered macrolide antibiotics, aldgamycin H (8), aldgamycin K (9), aldgamycin G (10), and swalpamycin B (11), and their structures were elucidated by HR-electrospray ionization source (ESI)-MS and NMR spectroscopy. All compounds 8-11 displayed antibacterial activities by inhibiting protein synthesis in the pDualrep2 system. In conclusion, this work demonstrates that Taklamakan desert is a potentially unique reservoir of versatile actinobacteria, which can be a promising source for discovery of novel species and diverse bioactive compounds.

Beilunmycin, a new virginiamycins antibiotic from mangrove-derived Streptomyces sp. 2BBP-J2 and the antibacterial activity by inhibiting protein translation.

One new virginiamycin derivative, 'beilunmycin' (1), and three known virginiamycin antibiotics, 16-hydroxy-virginiamycin M1 (2), virginiamycin M2 (3), and virginiamycin M1 (4), were isolated from the culture of a mangrove-derived endophytic Streptomyces sp. 2BBP-J2. The structures were characterized on the basis of their spectroscopic data, and the absolute configuration of 1 was established by ECD calculations. Compounds 1-4 exhibited antibacterial activities against Gram-positive bacteria, with MIC values in the range of 0.5-16 µg/ml. All the compounds demonstrated strong protein translation-stalling activity, with minimal concentrations detected with pDualrep2 in the range of 1.9-5.9 nmol.

Induction of apoptosis by Xiakemycin A in human hepatoma HepG2 cells.

Xiakemycin A (XKA), a new antibiotic in the pyranonaphthoquinone family, shows antitumor activity. However, the type of cell death induced by XKA remains elusive. In this study, we aim to investigate the type of death induced by XKA in hepatic cancer.The apoptotic features, such as chromatic agglutination, reactive oxygen species generation and membrane potential of mitochondria, in HepG2 cells treated by XKA were measured by Hoechst 33342 staining and flow cytometry. Apoptosis of HepG2 cells treated with XKA was determined by Annexin V-FITC/propidium iodide double staining and Western blot analysis, respectively.XKA had a significant dose-dependent elevation of chromatic agglutination, reactive oxygen species generation, Annexin V and propidium iodide staining, decrease of membrane potential. Meanwhile, in apoptotic HepG2 cells induced by XKA, robust increment was noticed in p53 expression, cleavage of PARP, caspase-3, and caspase-9.XKA showed potent inhibitory effects on the proliferation of HepG2 cells. Such phenomenon may be related to activation of the apoptotic pathway.

Marmoricola mangrovicus sp. nov., an endophytic actinobacterium isolated from Kandelia candel.

A Gram-stain-positive, aerobic, motile, non-spore-forming and rod-shaped actinobacterium, designated strain 4Q3S-7T, was isolated from a piece of surface-sterilized bark of Kandelia candel collected at the Cotai Ecological Zone in Macao, China. Colonies were yellowish white, circular, smooth and convex. The 16S rRNA gene sequence of strain 4Q3S-7T exhibited highest similarities to Marmoricola ginsengisoli Gsoil 097T (97.6 %), Marmoricola solisilvae KIS18-7T (97.6 %) and Marmoricola pocheonensis Gsoil 818T (97.3 %). Phylogenetic analysis showed that strain 4Q3S-7T clustered with species of the genus Marmoricola and was clearly affiliated to the genus Marmoricola. Genomic analyses, including average nucleotide identity and DNA-DNA hybridization, clearly separated strain 4Q3S-7T from M. ginsengisoli Gsoil 097T, M. solisilvae KIS18-7T and M. pocheonensis Gsoil 818T with values below the thresholds for species delineation. Strain 4Q3S-7T had ll-2,6-diaminopimelic acid as the diagnostic diamino acid in the cell wall. The major fatty acids (>10 % of total fatty acids) were C18 : 0 10-methyl (TBSA), C18 : 1ω9c, iso-C16 : 0 and iso-C16 : 0 2-OH. The predominant menaquinone was MK-8(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and an unidentified phospholipid. The DNA G+C content of strain 4Q3S-7T was 72.0 mol% (draft genome sequence). Based on its phylogenetic, phenotypic and chemotaxonomic features, strain 4Q3S-7T is considered to represent a novel species of the genus Marmoricola, for which the name Marmoricola mangrovicus sp. nov. is proposed. The type strain is 4Q3S-7T (=KCTC 39790T=CGMCC 4.7424T).

Diversity, Novelty, and Antimicrobial Activity of Endophytic Actinobacteria From Mangrove Plants in Beilun Estuary National Nature Reserve of Guangxi, China.

Endophytic actinobacteria are one of the important pharmaceutical resources and well known for producing different types of bioactive substances. Nevertheless, detection of the novelty, diversity, and bioactivity on endophytic actinobacteria isolated from mangrove plants are scarce. In this study, five different mangrove plants, Avicennia marina, Aegiceras corniculatum, Kandelia obovota, Bruguiera gymnorrhiza, and Thespesia populnea, were collected from Beilun Estuary National Nature Reserve in Guangxi Zhuang Autonomous Region, China. A total of 101 endophytic actinobacteria strains were recovered by culture-based approaches. They distributed in 7 orders, 15 families, and 28 genera including Streptomyces, Curtobacterium, Mycobacterium, Micrococcus, Brevibacterium, Kocuria, Nocardioides, Kineococcus, Kytococcus, Marmoricola, Microbacterium, Micromonospora, Actinoplanes, Agrococcus, Amnibacterium, Brachybacterium, Citricoccus, Dermacoccus, Glutamicibacter, Gordonia, Isoptericola, Janibacter, Leucobacter, Nocardia, Nocardiopsis, Pseudokineococcus, Sanguibacter, and Verrucosispora. Among them, seven strains were potentially new species of genera Nocardioides, Streptomyces, Amnibacterium, Marmoricola, and Mycobacterium. Above all, strain 8BXZ-J1 has already been characterized as a new species of the genus Marmoricola. A total of 63 out of 101 strains were chosen to screen antibacterial activities by paper-disk diffusion method and inhibitors of ribosome and DNA biosynthesis by means of a double fluorescent protein reporter. A total of 31 strains exhibited positive results in at least one antibacterial assay. Notably, strain 8BXZ-J1 and three other potential novel species, 7BMP-1, 5BQP-J3, and 1BXZ-J1, all showed antibacterial bioactivity. In addition, 21 strains showed inhibitory activities against at least one "ESKAPE" resistant pathogens. We also found that Streptomyces strains 2BBP-J2 and 1BBP-1 produce bioactive compound with inhibitory activity on protein biosynthesis as result of translation stalling. Meanwhile, Streptomyces strain 3BQP-1 produces bioactive compound inducing SOS-response due to DNA damage. In conclusion, this study proved mangrove plants harbored a high diversity of cultivable endophytic actinobacteria, which can be a promising source for discovery of novel species and bioactive compounds.

Total Synthesis of Originally Proposed and Revised Structure of Hetiamacin A.

The first total synthesis of the originally proposed and correct structures of hetiamacin A has been accomplished via Wittig olefination and Sharpless asymmetric dihydroxylation reaction. These total syntheses culminated in the stereostructural confirmation of the reassignment of hetiamacin A.

Lower endogenous p53 levels and degradation of AKT protein contribute to potent suppression of the new antibiotic Xiakemycin A on tumor cells.

Xiakemycin A (XKA), a new pyranonaphthoquinone antibiotic, is isolated from the fermentation broth of Streptomyces sp. CC8-201. It exerts potent suppression of cell proliferation on some types of tumor cells. In the present study, its underlying mechanism on tumor cells has been investigated. In contrast to the specific AKT inhibitor triciribine hydrate, XKA demonstrated a weak inhibition of the AKT kinase activity in vitro. Knockdown of AKT protein levels reduced XKA-inhibitory action on prostate carcinoma PC-3 cells. Degradation of AKT protein was markedly observed in the XKA-treated PC-3 cells in comparison with triciribine hydrate treatment. There was no typical apoptosis induced by XKA in PC-3 cells. The propidium iodide-stained cells increased concentration-dependently when the cells were treated with XKA. Degradation of apoptosis-related proteins, such as p53 and PARP-1, was also detected in the XKA-treated PC-3 cells. Knockdown of p53 protein levels potentiated XKA action on non-small lung cancer A549 cells. Collectively, the mechanism of XKA potent inhibition was due to degradation of AKT protein and low endogenous p53 levels. As a leading compound, new derivatives based on XKA will be developed to precisely treat tumor cells which have high AKT and low p53 protein levels.

Marmoricola endophyticus sp. nov., an endophytic actinobacterium isolated from Thespesia populnea.

A novel endophytic actinobacterium, designated strain 8BXZ-J1T, was isolated from surface-sterilized branches of Thespesia populnea collected from Beilun Estuary Mangrove Forest National Nature Reserve in Guangxi, China, and examined by a polyphasic approach to determine its taxonomic position. Cells of the isolate were Gram-stain-positive, aerobic, non-spore-forming, non-motile and short rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain 8BXZ-J1T belonged to the genus Marmoricola, sharing highest similarity with Marmoricola solisilvae DSM 27140T (96.9 %). The isolate grew at 10-35 °C (optimum, 28-30 °C), at pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 0-10 % (w/v) NaCl (optimum, 0-5.0 %). The organism contained ll-2,6-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan, MK-8(H4) as the major menaquinone, and a polar lipid profile including diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and two unknown lipids. The major fatty acids of strain 8BXZ-J1T were C18 : 0 10-methyl, iso-C16 : 0 and C16 : 0. The G+C content of the genomic DNA was 68.7 mol%. These data demonstrate that strain 8BXZ-J1T is representative of a novel species of the genus Marmoricola, for which the name Marmoricola endophyticus sp. nov. is proposed. The type strain is 8BXZ-J1T (=KCTC 39789T=CGMCC 1.16067T).

Hoyosella rhizosphaerae sp. nov., a halotolerant actinobacterium isolated from rhizosphere soil of Suaeda salsa, and emended description of the genus Hoyosella.

A halotolerant actinobacterium, designated J12GA03T, was isolated from a rhizosphere soil sample of Suaeda salsa collected from a dried saline lake in Hebei Province, China. Cells were Gram-staining-positive, non-motile and non-spore-forming cocci. Strain J12GA03T grew optimally at 28‒37 °C, 0‒3 % NaCl (w/v) and pH 6.5‒7.5. It contained meso-diaminopimelic acid as the diagnostic diamino acid and arabinose, galactose and ribose as the diagnostic whole-cell sugars. MK-8 and MK-7 were detected as predominant menaquinones. Major fatty acids were C17 : 1ω8c, C16 : 0 and C17 : 0. Polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, phosphoglycolipids, glycolipids, unidentified phospholipids and additional lipids. The muramyl residue was acetyl. Mycolic acids (34-38 carbon atoms) were present. The G+C content of the genomic DNA was 55.8 mol%. It shared the highest 16S rRNA gene sequence similarities with Amycolicicoccus subflavus DQS3-9A1T (98.18 %) and Hoyosella altamirensis OFN S31T (97.75 %). Phylogenetic trees showed that strain J12GA03T firmly formed a distinct monophyletic branch in the clade with A.subflavus DQS3-9A1T and H.altamirensis DSM 45258T. The levels of DNA-DNA relatedness with A.subflavus DSM 45089T and H.altamirensis DSM 45258T were 39.7±3.9 % and 35.7±3.0 %, respectively. Combining the evidence from the polyphasic taxonomic study, strain J12GA03T represents a novel species of the genus Hoyosella, for which the name Hoyosella rhizosphaerae sp. nov. is proposed. The type strain is J12GA03T (=DSM 101985T=CGMCC 1.15478T).

Lysinibacter cavernae gen. nov., sp. nov., a new member of the family Microbacteriaceae isolated from a karst cave.

A Gram-stain-positive, aerobic, straight or slightly bent rod-shaped, non-motile, non-spore-forming bacterium, designated strain CC5-806T, was isolated from a soil sample collected from a wild karst cave in the Wulong region, Chongqing, PR China and examined using a polyphasic approach to clarify its taxonomic position. This bacterium did not produce substrate mycelium or aerial hyphae, and no diffusible pigments were observed on the media tested. Strain CC5-806T grew optimally without NaCl at 20 °C and at pH 7.0. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain CC5-806T belonged to the family Microbacteriaceae and showed the highest levels of 16S rRNA gene sequence similarities with Frigoribacterium endophyticum EGI 6500707T (97.56 %), Frigoribacterium faeni 801T (97.53 %) and Glaciihabitans tibetensis MP203T (97.42 %). Phylogenetic trees revealed that strain CC5-806T did not show a clear affiliation to any genus within the family Microbacteriaceae. The DNA G+C content of strain CC5-806T was 62.6 mol%. The cell-wall peptidoglycan contained l-lysine as a diagnostic diamino acid. The predominant menaquinones were MK-11, MK-10 and MK-9. Phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid, four unidentified phospholipids and other polar lipids were detected in the polar lipid extracts. The major fatty acids were anteiso-C15 : 0, iso-C16 : 0 and iso-C14 : 0. On the basis of the phylogenetic analysis, and phenotypic and chemotaxonomic characteristics, strain CC5-806T was distinguishable from phylogenetically related genera in the family Microbacteriaceae. It represents a novel species of a novel genus, for which the name Lysinibacter cavernae gen. nov., sp. nov. is proposed. The type strain is CC5-806T ( = DSM 27960T = CGMCC 1.14983T).

Prauserella endophytica sp. nov., an endophytic actinobacterium isolated from Tamarix taklamakanensis.

A novel endophytic actinobacterium, designated strain SP28S-3(T), was isolated from a surface-sterilized stem of Tamarix taklamakanensis collected from the southern edge of Taklamakan desert, Xinjiang, China. Strain SP28S-3(T) was found to show chemotaxonomic and morphological properties consistent with its classification in the genus Prauserella. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphoglycolipid, phosphatidylcholine, phosphatidylinositol, a glycolipid, an aminolipid and unidentified phospholipids. The major fatty acids (>10 %) were identified as iso-C16:0 and C16:0. The genomic DNA G+C content was determined to be 69.7 mol%. Phylogenetic analysis of strain SP28S-3(T) clearly showed that the strain had the highest similarity of 16S rRNA gene sequence with Prauserella coralliicola SCSIO 11529(T) (99.9 %), followed by Prauserella marina DSM 45268(T) (97.0 %) and is affiliated with the genus Prauserella. The low level (47.8 ± 5.5 %) of DNA-DNA relatedness between strain SP28S-3(T) and P. coralliicola SCSIO 11529(T) combined with other polyphasic taxonomic evidence clearly support the conclusion that strain SP28S-3(T) represents a novel Prauserella species, for which the name Prauserella endophytica sp. nov. is proposed. The type strain is SP28S-3(T) (=DSM 46655(T) = CGMCC 4.7182 (T)).

Nocardioides deserti sp. nov., an actinobacterium isolated from desert soil.

A rod- or coccus-shaped, non-spore-forming actinobacterium, designated strain SC8A-24(T), was isolated from a soil sample collected from the rhizosphere of Alhagi sparsifolia on the southern edge of the Taklimakan desert, Xinjiang, China, and examined by a polyphasic approach to clarify its taxonomic position. This actinobacterium was Gram-staining-positive and aerobic. Substrate and aerial mycelia were not observed, and no diffusible pigments were observed on the media tested. Strain SC8A-24(T) grew optimally without NaCl at 28-30 °C and pH 7.0-8.0. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain SC8A-24(T) belonged to the genus Nocardioides and shared the highest 16S rRNA gene sequence similarity with Nocardioides salarius CL-Z59(T) (96.51%), N. pyridinolyticus OS4(T) (96.43%) and N. ginsengagri BX5-10(T) (96.37%). The DNA G+C content of strain SC8A-24(T) was 71 mol%. The cell-wall peptidoglycan contained ll-2,6-diaminopimelic acid, and MK-8(H4) was the predominant menaquinone. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid and an unidentified phospholipid. The major fatty acids were C17 : 1ω8c, 10-methyl C17 : 0 and C18 : 1ω9c. On the basis of phylogenetic analysis and phenotypic and chemotaxonomic characteristics, strain SC8A-24(T) represents a novel species of the genus Nocardioides , for which the name Nocardioides deserti sp. nov. is proposed. The type strain is SC8A-24(T) ( =DSM 26045(T)  = CGMCC 4.7183(T)).

Nesterenkonia populi sp. nov., an actinobacterium isolated from Populus euphratica.

An alkaliphilic and moderately halophilic actinobacterium, designated strain GP10-3(T), was isolated from Populus euphratica collected from the southern edge of Taklimakan desert, Xinjiang, China. Cells of this strain were Gram-stain-positive, non-motile and non-spore-forming short rods. Strain GP10-3(T) grew optimally at 37 °C on LB agar media in the presence of 5-10% (w/v) NaCl at pH 9.0. The menaquinones were MK-7, MK-8 and MK-9. The major fatty acids (>10%) were anteiso-C17 : 0, anteiso-C15 : 0 and iso-C16 : 0. The peptidoglycan type was variation A4α, L-Lys-L-Glu. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylglycolipid, phosphatidylcholine, phosphatidylinositol, glycolipid and an unidentified phospholipid. The DNA G+C content was 67.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain GP10-3(T) belonged to the genus Nesterenkonia , sharing 94.6-96.9% sequence similarity with the type strains of species within this genus with validly published names. Based on the evidence of the polyphasic taxonomic study, strain GP10-3(T) represents a novel species of the genus Nesterenkonia , for which the name Nesterenkonia populi sp. nov. is proposed. The type strain is GP10-3(T) ( = DSM 27959(T) = KCTC 29119(T)).

Allosalinactinospora lopnorensis gen. nov., sp. nov., a new member of the family Nocardiopsaceae isolated from soil.

A novel actinomycete, designated strain CA15-2(T), was isolated from a soil sample collected from the rhizosphere of tamarisk in the Lop Nor region, Xinjiang, China, and was characterized by using a polyphasic taxonomic approach. Optimal growth occurred at 37 °C and pH 7.5-8.0 and with 5% (w/v) NaCl. Strain CA15-2(T) formed white to pale-yellow branched substrate mycelium without fragmentation and sparse aerial mycelium with wavelike curves. Whole-cell hydrolysates of the isolate contained meso-diaminopimelic acid as the diagnostic diamino acid of the cell wall but no diagnostic sugars. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmethylethanolamine, phosphatidylethanolamine, one unidentified glycolipid, one unidentified phospholipid and other unidentified lipids. MK-9(H8), MK-10(H8) and MK-10(H6) were the predominant menaquinones. The major fatty acids were iso-C16:0 and C16:0. The G+C content of the genomic DNA was 69.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CA15-2(T) formed a distinct subclade in the family Nocardiopsaceae, with less than 95% 16S rRNA gene sequence similarity to all known members of the family Nocardiopsaceae. On the basis of the evidence from our polyphasic study, a novel genus, Allosalinactinospora gen. nov., is proposed, with the type species Allosalinactinospora lopnorensis gen. nov., sp. nov. The type strain of Allosalinactinospora lopnorensis is strain CA15-2(T) ( = DSM 45697(T) =CGMCC 4.7074(T)).