Zhong-Ke Jiang

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)).

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)).

PJS, a novel isocoumarin with hexahydropyrimidine ring from Bacillus subtilis PJS

Amicoumacin group of antibiotics, such as baciphelacin,1 amicoumacins,2 AI-77s,3 xenocoumacins,4 Y-05460M-A,5 PM94128,6 Sg17-1-4,7 bacilosarcins A, B8 and lipoamicoumacins A–D,9 is a small family of isocoumarin, which possesses the common chromophore, 3, 4-dihydro-8-hydroxyisocoumarin. Most members of amicoumacin group of antibiotics are produced by the genus Bacillus and exhibit various important bioactivities. The chromophore shows specific UV absorbance at 247 and 314 nm in methanol. Thus, a project based on HPLC-diode array screening and HPLC-MS dereplication was carried out to find new amicoumacin analogues from the secondary metabolites of Bacillus spp. As a result, PJS (1), a new isocoumarin antibiotic was discovered from the fermentation broth of Bacillus subtilis PJS. In this study, we wish to report the fermentation, isolation, physico chemical properties, structural elucidation and biological activities of (1) (Figure 1). The producing strain Bacillus subtilis PJS was isolated from the leaf of an unidentified plant collected at Luopu county, Hetian area, Xinjiang Province, People’s Republic of China. On the basis of analysis of 16S rRNA, it was identified as Bacillus subtilis subsp. inaquosorum. A stock culture of the strain Bacillus subtilis PJS was maintained on modified Gause’s no. 1 agar slant consisting of soluble starch (Beijing Qi Te Xin Chemical Co. Ltd. China) 20.0 g, NaCl 50.0 g, K2HPO4 0.5 g, KNO3 1.0 g, MgSO4 1.0 g, FeSO4 0.02 g, glucose 1.0 g, peptone 0.5 g, tryptone 0.3 g and agar 20.0 g in 1.0 l distilled water (pH 8.0) at 4 1C. The stock culture was inoculated into 250 ml Erlenmeyer flasks containing 50 ml of seed medium, which was the same modified Gause’s no.1 liquid medium as above, but no agar. The culture was incubated on a rotary shaker (180 r.p.m.) at 28 1C for 24 h. Fifty millilitres of the seed culture was transferred to a 5000 ml Erlenmeyer flask containing 1000 ml of the producing medium, which was the same as the seed medium. The fermentation was carried out at 28 1C for 48 h on a rotary shaker (180 r.p.m.). Eighty liters of the fermentation broth was centrifuged at 4500 r.p.m. for 20 min, then, the supernatant was obtained and was extracted twice with 40 l of ethyl acetate each time. The organic layer was pooled and concentrated under reduced pressure at 37 1C to give yellow syrup (2.3 g). It was then separated by preparative thin layer chromatography on 10 10 cm plates (silica gel 60F254, Merck KGaA, Darmstadt, Germany) using CHCl3-MeOH, 65:35 (v/v) as the developing solvent. Under UV 365 nm, bands with light blue fluorescence at Rf 1⁄4 0.35 were scraped and then eluted with methanol to yield a semipurified sample (260 mg). After dissolved in 1 ml methanol, the sample was filtered through a 0.22mm membrane and was further purified by HPLC on a shim-Pack PRC-ODS column (250 20 mm, Shimadzu Corp., Tokyo, Japan) with MeOH-H2O, 55:45 (v/v) at 2 ml min 1. Peak at Rt1⁄4 26 min, showed UV absorbance at 247 and 314 nm detected by prominence diode array detector (SPD-M20A, Shimadzu), was collected and pooled to yield 10.4 mg of (1) as a white powder. Compound (1) was soluble in dimethyl sulfoxide (DMSO), MeOH, CHCl3 and pyridine. Its m.p. was 79–80 1C. MW of (1) was found to be 435 by high-resolution ESI-MS, which showed [MþH]þ at m/z 436.2089 (calcd 436.2079). The molecular formula was then established as C21H29O7N3, which has nine degrees of unsaturation. 13C-NMR and DEPT spectra of (1) indicated 21 carbon signals could be attributed to three carbonyl carbons, six aromatic carbons and 12 aliphatic carbons including six carbons bonded to nitrogen or oxygen. The UV absorption of (1) at lmax MeOH nm (e): 202(29 930), 246(6491) and 314(3491) was almost identical with that of isocoumarin compounds, such as Sg17-1-4, PM-94128, Y-05460M-A, bacilosarcins and AI-77-B. The IR spectrum (film) of (1) exhibited absorption bands at 3343, 2956, 1668, 1620, 1539, 1231, 807, 697 cm 1, which indicated the presence of a benzoic acid moiety with a phenolic hydroxyl group and an amide group. All data above revealed (1) had a

Xiakemycin A, a novel pyranonaphthoquinone antibiotic, produced by the Streptomyces sp. CC8-201 from the soil of a karst cave

Xiakemycin A, a novel pyranonaphthoquinone antibiotic, produced by the Streptomyces sp. CC8-201 from the soil of a karst cave

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)).

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).

Hetiamacin B-D, new members of amicoumacin group antibiotics isolated from Bacillus subtilis PJS.

Hetiamacin B–D, new members of amicoumacin group antibiotics isolated from Bacillus subtilis PJS

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).

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.

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).