FadR1, a pathway-specific activator of fidaxomicin biosynthesis in Actinoplanes deccanensis Yp-1.

Fidaxomicin, an 18-membered macrolide antibiotic, is highly active against Clostridium difficile, the most common cause of diarrhea in hospitalized patients. Though the biosynthetic mechanism of fidaxomicin has been well studied, little is known about its regulatory mechanism. Here, we reported that FadR1, a LAL family transcriptional regulator in the fidaxomicin cluster of Actinoplanes deccanensis Yp-1, acts as an activator for fidaxomicin biosynthesis. The disruption of fadR1 abolished the ability to synthesize fidaxomicin, and production could be restored by reintegrating a single copy of fadR1. Overexpression of fadR1 resulted in an approximately 400 % improvement in fidaxomicin production. Electrophoretic mobility shift assays indicated that fidaxomicin biosynthesis is under the control of FadR1 through its binding to the promoter regions of fadM, fadA1-fadP2, fadS2-fadC, and fadE-fadF, respectively. And the conserved binding sites of FadR1 within the four promoter regions were determined by footprinting experiment. All results indicated that fadR1 encodes a pathway-specific positive regulator of fidaxomicin biosynthesis and upregulates the transcription levels of most of genes by binding to the four above intergenic regions. In summary, we not only clearly elucidate the regulatory mechanism of FadR1 but also provide strategies for the construction of industrial high-yield strain of fidaxomicin.

Bidirectional Regulation of AdpAch in Controlling the Expression of scnRI and scnRII in the Natamycin Biosynthesis of Streptomyces chattanoogensis L10.

AdpA, an AraC/XylS family protein, had been proved as a key regulator for secondary metabolism and morphological differentiation in Streptomyces griseus. Here, we identify AdpAch, an ortholog of AdpA, as a "higher level" pleiotropic regulator of natamycin biosynthesis with bidirectional regulatory ability in Streptomyces chattanoogensis L10. DNase I footprinting revealed six AdpAch-binding sites in the scnRI-scnRII intergenic region. Further analysis using the xylE reporter gene fused to the scnRI-scnRII intergenic region of mutated binding sites demonstrated that the expression of scnRI and scnRII was under the control of AdpAch. AdpAch showed a bi-stable regulatory ability where it firstly binds to the Site C and Site D to activate the transcription of the two pathway-specific genes, scnRI and scnRII, and then binds to other sites where it acts as an inhibitor. When Site A and Site F were mutated in vivo, the production of natamycin was increased by 21% and 25%, respectively. These findings indicated an autoregulatory mechanism where AdpAch serves as a master switch with bidirectional regulation for natamycin biosynthesis.

Paracoccus siganidrum sp. nov., isolated from fish gastrointestinal tract.

A bacterial strain, designated M26(T), was isolated from a fish gastrointestinal tract, collected from Zhanjiang Port, South China. 16S rRNA gene sequence analysis indicated that strain M26(T) belongs to the subclass α-Proteobacteria, being related to the genus Paracoccus, and sharing highest sequence similarity with Paracoccus alcaliphilus JCM 7364(T) (98.1 %), Paracoccus huijuniae FLN-7(T) (97.3 %), Paracoccus stylophorae KTW-16(T) (97.1 %) and Paracoccus seriniphilus DSM 14827(T) (96.9 %). The major quinone was determined to be ubiquinone Q-10, with Q-9 and Q-8 as minor components. The major fatty acid was identified as C18:1ω7c, with smaller amounts of C18:0 and C16:0. The G+C content of the genomic DNA was determined to be 64.3 mol%. The DNA hybridization value between strain M26(T) and the most closely related type strain, P. alcaliphilus, was 29.0 ± 1.0 %. The results of physiological and biochemical tests and low DNA-DNA relatedness showed that the strain could be readily distinguished from closely related species. On the basis of these phenotypic and genotypic data, strain M26(T) is concluded to represent a novel species of the genus Paracoccus, for which the name Paracoccus siganidrum sp. nov. is proposed. The type strain is M26(T) (=CCTCC AB 2012865(T) = DSM 26381(T)).

Actinoallomurus acanthiterrae sp. nov., an actinomycete isolated from rhizosphere soil of the mangrove plant Acanthus ilicifolius.

A novel actinobacterium strain, 2614A723(T), was isolated from rhizosphere soil of mangrove plant Acanthus ilicifolius collected at Touyuan, Wenchang, Hainan province, China. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 2614A723(T) formed a distinct phyletic line in the genus Actinoallomurus, the 16S rRNA gene tree sharing similarities of 98.35%, 98.07% and 97.86% with Actinoallomurus spadix NBRC 14099(T), Actinoallomurus purpureus TTN02-30(T) and Actinoallomurus luridus TT02-15(T), respectively. Strain 2614A723(T) contained lysine and meso-diaminopimelic acid in the cell wall peptidoglycan and madurose, galactose and xylose in the whole-cell sugars. The predominant menaquinones were MK-9(H4) and MK-9(H6). The major polar phospholipids were phosphatidylglycerol and diphosphatidylglycerol. The predominant fatty acids were iso-C16 : 0 and anteiso-C17 : 0. These chemotaxonomic data confirmed the affiliation of strain 2614A723(T) to the genus Actinoallomurus. It is apparent from the combined phenotypic data, biochemical tests and DNA-DNA hybridization values that strain 2614A723(T) should be classified in the genus Actinoallomurus as a representative of a novel species. The name Actinoallomurus acanthiterrae sp. nov. is proposed with strain 2614A723(T) ( = CCTCC AA 2012001(T) = DSM 45727(T)) as the type strain.

Barrientosiimonas humi gen. nov., sp. nov., an actinobacterium of the family Dermacoccaceae.

Three novel actinobacteria, strains 39(T), 40 and 41, were isolated from soil collected from Barrientos Island in the Antarctic. The taxonomic status of these strains was determined using a polyphasic approach. Comparison of 16S rRNA gene sequences revealed that strain 39(T) represented a novel lineage within the family Dermacoccaceae and was most closely related to members of the genera Demetria (96.9 % 16S rRNA gene sequence similarity), Branchiibius (95.7 %), Dermacoccus (94.4-95.3 %), Calidifontibacter (94.6 %), Luteipulveratus (94.3 %), Yimella (94.2 %) and Kytococcus (93.1 %). Cells were irregular cocci and short rods. The peptidoglycan type was A4α with an L-Lys-L-Ser-D-Asp interpeptide bridge. The cell-wall sugars were galactose and glucose. The major menaquinone was MK-8(H(4)). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphoglycolipid, two glycolipids and one unknown phospholipid. The acyl type of the cell-wall polysaccharide was N-acetyl. The major cellular fatty acids were anteiso-C(17 : 0) (41.97 %), anteiso-C(17 : 1)ω9c (32.16 %) and iso-C(16 : 0) (7.68 %). The DNA G+C content of strain 39(T) was 68.4 mol%. On the basis of phylogenetic and phenotypic differences from other genera of the family Dermacoccaceae, a novel genus and species, Barrientosiimonas humi gen. nov., sp. nov., is proposed; the type strain of the type species is 39(T) (=CGMCC 4.6864(T) = DSM 24617(T)).

Micromonospora sonneratiae sp. nov., isolated from a root of Sonneratia apetala.

A novel endophytic actinomycete, strain 274745(T), was isolated from a root of Sonneratia apetala collected in a mangrove forest in Sanya, Hainan province, China. The 16S rRNA gene sequence of strain 274745(T) showed the greatest similarity to Micromonospora pattaloongensis TJ2-2(T) (98.3 %). Phylogenetic analysis based on the gyrB gene also supported the close relationship of these two strains. The predominant menaquinone was MK-10(H8) and the major fatty acids were iso-C15 : 0, C17 : 0 and anteiso-C15 : 0. The characteristic whole-cell sugars were xylose and mannose. The cell wall contained meso-diaminopimelic acid and glycine. The polar lipid profile mainly comprised phosphatidylethanolamine, phosphatidylinositol and diphosphatidylglycerol. The DNA G+C content was 71.6 mol%. Furthermore, a combination of DNA-DNA relatedness and some physiological and biochemical properties indicated that the novel strain could be readily distinguished from the closest phylogenetic relatives. On the basis of these phenotypic and genotypic data, strain 274745(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora sonneratiae sp. nov. is proposed. The type strain is 274745(T) ( = CCTCC AA 2012003(T) = DSM 45704(T)).

Micromonospora wenchangensis sp. nov., isolated from mangrove soil.

An actinomycete, strain 2602GPT1-05(T), was isolated from a composite mangrove soil sample collected from Wenchang, Hainan province, China. Strain 2602GPT1-05(T) showed closest 16S rRNA gene sequence similarity to Micromonospora haikouensis 232617(T) (99.05 %), and phylogenetically clustered with Micromonospora haikouensis 232617(T), Micromonospora matsumotoense IMSNU 22003(T) (98.7 %) and Micromonospora rifamycinica AM105(T) (98.6 %) based on the 16S rRNA and gyrB gene sequence phylogenetic analysis. The strain harboured meso-DAP and glycine as major cell-wall amino acids, and MK-10(H6) and MK-9(H6) as predominant menaquinones. The characteristic whole-cell sugars were xylose, arabinose, glucose and galactose. The polar lipid profile comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, phosphatidylinositol mannosides, unknown phospholipid and an unknown phosphoglycolipid. The major cellular fatty acids were C18 : 1ω9c, iso-C15 : 0, 10-methyl C18 : 0 (tuberculostearic acid), C16 : 0, C18 : 0 and iso-C16 : 0. The DNA G+C content was 71.7 mol%. Furthermore, some physiological and biochemical properties and low DNA-DNA relatedness values enabled the strain to be differentiated from members of closely related species. On the basis of these phenotypic, genotypic and chemotaxonomic data, strain 2602GPT1-05(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora wenchangensis sp. nov. is proposed. The type strain is 2602GPT1-05(T) ( = CCTCC AA 2012002(T) = DSM 45709(T)).

Molecular characterization of Antarctic actinobacteria and screening for antimicrobial metabolite production.

The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231(T), Staphylococcus aurues ATCC 51650(T), methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44(T) and Pseudomonas aeruginosa ATCC 10145(T), respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.