A Versatile Thioesterase Involved in Dimerization during Cinnamoyl Lipid Biosynthesis.

The cinnamoyl lipid compound youssoufene A1 (1), featuring a unique dearomatic carbon-bridged dimeric skeleton, exhibits increased inhibition against multidrug resistant Enterococcus faecalis as compared to monomeric youssoufenes. However, the formation process of this intriguing dearomatization/dimerization remains unknown. In this study, an unusual "gene-within-gene" thioesterase (TE) gene ysfF was functionally characterized. The gene was found to naturally encodes two proteins, an entire YsfF with α/ß-hydrolase and 4-hydroxybenzoyl-CoA thioesterase (4-HBT)-like enzyme domains, and a nested YsfFHBT (4-HBT-like enzyme). Using an intracellular tagged carrier-protein tracking (ITCT) strategy, in vitro reconstitution and in vivo experiments, we found that: i) both domains of YsfF displayed thioesterase activities; ii) YsfF/YsfFHBT could accomplish the 6π-electrocyclic ring closure for benzene ring formation; and iii) YsfF and cyclase YsfX together were responsible for the ACP-tethered dearomatization/dimerization process, possibly through an unprecedented Michael-type addition reaction. Moreover, site-directed mutagenesis experiments demonstrated that N301, E483 and H566 of YsfF are critical residues for both the 6π-electrocyclization and dimerization processes. This study enhances our understanding of the multifunctionality of the TE protein family.

Thermomonas mangrovi sp. nov., isolated from soil of a mangrove nature reserve.

A Gram-stain-negative, non-motile, aerobic and ovoid-rod-shaped bacterial strain, designated as 2C3345T, was isolated from soil of a mangrove nature reserve in Shenzhen, PR China and its taxonomic position was investigated. The 16S rRNA gene sequence of strain 2C3345T exhibited the highest similarity value to that of the type strains Thermomonas aquatica NBRC 113114T (98.4 %), Thermomonas carbonis KCTC 42013T (98.1 %), Thermomonas brevis DSM 15422T (98.0 %) and Thermomonas haemolytica DSM 13605T (97.9 %). Strain 2C3345T grew optimally at 28-30 °C, pH 7.0 and without NaCl. The predominant ubiquinone in 2C3345T was identified as Q-8. The major fatty acids of 2C3345T were iso-C15 : 0, iso-C16 : 0 and summed feature 9 (is0-C17:1ω9c and/or 10-methyl C16:0). The major polar lipids of 2C3345T were phosphatidylcholine (PC), phosphatidylglycerol (PG), diphosphatidglycerol (DPG), phosphatidylethanolamine (PE), an unidentified phospholipid, an unidentified aminolipid and two unidentified lipids. The DNA G+C content of strain 2C3345T was 69.9 %. The draft genome sequence of 2C3345T included 2 888 645 bp in total (N50=310 730 bp) with a medium read coverage of 388.6×and 21 scaffolds. In silico DNA-DNA hybridisation with type strains of species of the genus Thermomonas showed 24.4, 22.4, 24.1 and 23.2% relatedness to T. aquatica NBRC 113114T, T. carbonis KCTC 42013T, T. brevis DSM 15422T and T. haemolytica DSM 13605T, respectively. And the average nucleotide identity by blast (ANIb) values between 2C3345T and these four reference strains were 81.9, 80.0, 81.2 and 80.6 %, respectively, lower than the 95-96% threshold value for dividing prokaryotic species. On the basis of its phenotypic, chemotaxonomic, phylogenetic and phylogenomic properties, strain 2C3345T is considered to represent a novel species within the genus Thermomonas, for which the name Thermomonas mangrovi sp. nov. is proposed. The type strain is 2C3345T (=KCTC 92730T =MCCC 1K08335T).

Paracoccus onchidii sp. nov., a moderately halophilic bacterium isolated from a marine invertebrate from the South China Sea.

A novel moderately halophilic bacterial strain, designated Z330T, was isolated from the egg of a marine invertebrate of the genus Onchidium collected in the South China Sea. The 16S rRNA gene sequence of strain Z330T exhibited the highest similarity value to that of the type strain Paracoccus fistulariae KCTC 22803T (97.6%), Paracoccus seriniphilus NBRC 100798T (97.6%) and Paracoccus aestuarii DSM 19484T (97.6%). Phylogenomic and 16S rRNA phylogenetic analysis showed that strain Z330T was most closely related to P. seriniphilus NBRC 100798T and P. fistulariae KCTC 22803T. Strain Z330T grew optimally at 28-30 °C, pH 7.0-8.0 with the presence of 5.0-7.0% (w/v) NaCl. In addition, growth of strain Z330T occurred at 0.5-16% NaCl, indicated strain Z330T was a moderately halophilic and halotolerant bacterium of genus Paracoccus. The predominant respiratory quinone in strain Z330T was identified as ubiquinone-10. The major polar lipids of strain Z330T were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, glycolipid and six unidentified polar lipids. The major fatty acids of strain Z330T was summed feature 8 (C18:1 ω6c and/or C18:1 ω7c). The draft genome sequence of strain Z330T includes 4,084,570 bp in total (N50 = 174,985 bp) with a medium read coverage of 463.6 × and 83 scaffolds. The DNA G + C content of strain Z330T was 60.5%. In silico DNA-DNA hybridization with the four type strains showed 20.5, 22.3, 20.1 and 20.1% relatedness to Paracoccus fistulariae KCTC 22803T, Paracoccus seriniphilus NBRC 100798T, Paracoccus aestuarii DSM 19484T and Paracoccus denitrificans 1A10901T, respectively. And the average nucleotide identity (ANIb) values between strain Z330T and these four type strains were 76.2, 80.0, 75.8 and 73.8%, respectively, lower than the 95-96% threshold value for dividing prokaryotic species. On the basis of the phenotypic, phylogenetic, phylogenomic and chemotaxonomic properties, a novel species of the genus Paracoccus, Paracoccus onchidii sp. nov. is proposed with the type strain Z330T (= KCTC 92727T = MCCC 1K08325T).

Transcriptomic Analysis Reveals the Role of tmRNA on Biofilm Formation in Bacillus subtilis.

Bacillus strains are widely distributed in terrestrial and marine environments, and some of them are used as biocontrol organisms for their biofilm-formation ability. In Bacillus subtilis, biofilm formation is fine-tuned by a complex network, a clear understanding of which still requires study. In bacteria, tmRNA, encoded by the ssrA gene, catalyzes trans-translation that can rescue ribosomes stalled on mRNA transcripts lacking a functional stop codon. tmRNA also affects physiological bioprocesses in some bacteria. In this study, we constructed a ssrA mutant in B. subtilis and found that the biofilm formation in the ssrA mutant was largely impaired. Moreover, we isolated a biofilm-formation suppressor of ssrA, in which the biofilm formation was restored to a level even stronger than that in the wild type. We further performed RNAseq assays with the wild type, ssrA mutant, and suppressor of ssrA for comparisons of their transcriptomes. By analyzing the transcriptomic data, we predicted the possible functions of some differentially expressed genes (DEGs) in the tmRNA regulation of biofilm formation in B. subtilis. Finally, we found that the overexpression of two DEGs, acoA and yhjR, could restore the biofilm formation in the ssrA mutant, indicating that AcoA and YhjR were immediate regulators involved in the tmRNA regulatory web controlling biofilm formation in B. subtilis. Our data can improve the knowledge about the molecular network involved in Bacillus biofilm formation and provide new targets for manipulation of Bacillus biofilms for future investigation.

Youssoufenes A2 and A3, Antibiotic Dimeric Cinnamoyl Lipids from the ΔdtlA Mutant of a Marine-Derived Streptomyces Strain.

Two new dimeric cinnamoyl lipids (CL) featuring with an unusual dearomatic carbon-bridge, named youssoufenes A2 (1) and A3 (2), were isolated from the ΔdtlA mutant strain of marine-derived Streptomyces youssoufiensis OUC6819. Structures of the isolated compounds were elucidated based on extensive MS and NMR spectroscopic analyses, and their absolute configurations were determined by combination of the long-range NOE-based 1H-1H distance measurements and ECD calculations. Compounds 1 and 2 exhibited moderate growth inhibition against multi-drug-resistant Enterococcus faecalis CCARM 5172 with an MIC value of 22.2 µM.

Antibiotic Dixiamycins from a Cold-Seep-Derived Streptomyces olivaceus.

Two new (1 and 2) along with six known (3-8) dixiamycins were isolated from the culture broth of a cold-seep-derived actinomycete, Streptomyces olivaceus OUCLQ19-3. Structures of the isolated compounds were elucidated based on extensive MS and NMR spectroscopic analyses together with ECD calculations. In the antibacterial test, compounds 1-8 exhibited notable growth inhibitions against a panel of multi-drug-resistant (MDR) strains with MIC values of 0.78-6.25 µg/mL, among which 1, 2, and 5-7 were more potent than the positive control tetracycline.

Effective Generation of Glucosylpiericidins with Selective Cytotoxicities and Insights into Their Biosynthesis.

Exploring unknown glycosyltransferases (GTs) is important for compound structural glycodiversification during the search for drug candidates. Piericidin glycosides have been reported to have potent bioactivities; however, the GT responsible for piericidin glucosylation remains unknown. Herein, BmmGT1, a macrolide GT with broad substrate selectivity and isolated from Bacillus methylotrophicus B-9987, was found to be able to glucosylate piericidin A1 in vitro. Next, the codon-optimized GT gene sbmGT1, which was designed based on BmmGT1, was heterologously expressed in the piericidin producer Streptomyces youssoufiensis OUC6819. Piericidin glycosides thus significantly accumulated, leading to the identification of four new glucopiericidins (compounds 3, 4, 6, and 7). Furthermore, using BmmGT1 as the probe, GT1507 was identified in the genome of S. youssoufiensis OUC6819 and demonstrated to be associated with piericidin glucosylation; the overexpression of this gene led to the identification of another new piericidin glycoside, N-acetylglucosamine-piericidin (compound 8). Compounds 4, 7, and 8 displayed cytotoxic selectivity toward A549, A375, HCT-116, and HT-29 solid cancer cell lines compared to the THP-1 lymphoma cell line. Moreover, database mining of GT1507 homologs revealed their wide distribution in bacteria, mainly in those belonging to the high-GC Gram-positive and Firmicutes clades, thus representing the potential for identification of novel tool enzymes for compound glycodiversification. IMPORTANCE Numerous bioactive natural products are appended with sugar moieties and are often critical for their bioactivities. Glycosyltransferases (GTs) are powerful tools for the glycodiversification of natural products. Although piericidin glycosides display potent bioactivities, the GT involved in glucosylation is unclear. In this study, five new piericidin glycosides (compounds 3, 4, 6, 7, and 8) were generated following the overexpression of GT-coding genes in a piericidin producer. Three of them (compounds 4, 7, and 8) displayed cytotoxic selectivity. Notably, GT1507 was demonstrated to be related to piericidin glucosylation in vivo. Furthermore, mining of GT1507 homologs from the GenBank database revealed their wide distribution across numerous bacteria. Our findings would greatly facilitate the exploration of GTs to glycodiversify small molecules in the search for drug candidates.

An Unusual Type II Polyketide Synthase System Involved in Cinnamoyl Lipid Biosynthesis.

As a unique structural moiety in natural products, cinnamoyl lipids (CLs), are proposed to be assembled by unusual type II polyketide synthases (PKSs). Herein, we demonstrate that the assembly of the CL compounds youssoufenes is accomplished by a PKS system that uniquely harbors three phylogenetically different ketosynthase/chain length factor (KS/CLF) complexes (YsfB/C, YsfD/E, and YsfJ/K). Through in vivo gene inactivation and in vitro reconstitution, as well as an intracellular tagged carrier-protein tracking (ITCT) strategy developed in this study, we successfully elucidated the isomerase-dependent ACP-tethered polyunsaturated chain elongation process. The three KS/CLFs were revealed to modularly assemble different parts of the youssoufene skeleton, during which benzene ring closure happens right after the formation of an ACP-tethered C18 polyene. Of note, the ITCT strategy could significantly contribute to the elucidation of other carrier-protein-dependent biosynthetic machineries.

Expanding the Structural Diversity of Drimentines by Exploring the Promiscuity of Two N-methyltransferases.

Methylation is envisioned as a promising way to rationally improve key pharmacokinetic characteristics of lead compounds. Although diverse tailoring enzymes are found to be clustered with cyclodipeptide synthases (CDPSs) to perform further modification reactions on the diketopiperazine (DKP) rings generating complex DKP-containing compounds, so far, a limited number of methyltransferases (MTs) co-occurring with CDPS have been experimentally characterized. Herein, we deciphered the methylation steps during drimentines (DMTs) biosynthesis with identification and characterization of DmtMT2-1 (from Streptomyces sp. NRRL F-5123) and DmtMT1 (from Streptomyces youssoufiensis OUC6819). DmtMT2-1 catalyzes N4-methylation of both pre-DMTs and DMTs; conversely, DmtMT1 recognizes the DKP rings, functioning before the assembly of the terpene moiety. Notably, both MTs display broad substrate promiscuity. Their combinatorial expression with the dmt1 genes in different Streptomyces strains successfully generated eight unnatural DMT analogs. Our results enriched the MT tool-box, setting the stage for exploring the structural diversity of DKP derivatives for drug development.

Targeted isolation of new polycyclic tetramate macrolactams from the deepsea-derived Streptomyces somaliensis SCSIO ZH66.

With a combined strategy of bioinformatics analysis, gene manipulation coupled with variation of growth conditions, the targeted activation of polycyclic tetramate macrolactams (PTMs) in the deepsea-derived Streptomyces somaliensis SCSIO ZH66 was conducted, which afforded a new (1) PTM, named somamycin A, along with three enol-type tetramic acid tautomers (2-4, somamycins B-D) of 10-epi-hydroxymaltophilin, 10-epi-maltophilin and 10-epi-HSAF, respectively. The structures of compounds 1-4 were elucidated by extensive spectroscopic analyses together with ECD calculations. Compound 1 exhibited notable growth inhibition against plant pathogenic fungi Fusariumoxysporum MHKW and Alternariabrassicae BCHB with the MIC values of 1.6 and 3.1 µg/mL, respectively, which were more potent than those of the positive control nystatin; and compounds 3 and 4 displayed moderate antifungal activities. Moreover, compounds 1-4 exhibited moderate cytotoxicity against the human cancer cell lines of HCT116 and K562.

Genetic Manipulation of an Aminotransferase Family Gene dtlA Activates Youssoufenes in Marine-Derived Streptomyces youssoufiensis.

Disruption of an aminotransferase family gene dtlA activated the production of a novel dimeric benzoic polyene acids (BPAs), named youssoufene A1 (1), along with four new (2-5, youssoufenes B1-B4) and a known (6) monomeric BPA in the marine-derived Streptomyces youssoufiensis OUC6819. The structures of 1-5 were elucidated by extensive spectroscopic and computational approaches. Youssoufene A1 (1) exhibited notably increased growth inhibition (MIC = 12.5 µg/mL) against multidrug resistant Enterococcus faecalis compared to monomeric structures (2-6).

Fungal community analysis in seawater of the Mariana Trench as estimated by Illumina HiSeq.

This study assessed the diversity and distribution of fungal communities in thirteen marine seawater samples from four sites (L1, L3, L4 and L7) of the Mariana Trench, with a depth range of 1000-4000 meters, using Illumine Hiseq sequencing with fungal-specific primers targeting the internal transcribed spacer (ITS) region of the ribosomal rRNA gene. Sedimentary fungal communities showed high diversity with 209 880 reads belonging to 91 operational taxonomic units (OTUs). Of these OTUs, 45 belonged to the Ascomycota, 37 to Basidiomycota, 3 to Chytridiomycota, 1 to Glomeromycota, 1 to Cryptomycota, and 4 to unknown fungi. The major fungal orders included Saccharomycetales and Sporidiobolales. The commonly found fungal genera were Candida, Malassezia and Cryptococcus. These results suggest the existence of diverse fungal communities in the Mariana Trench marine seawater, which can be considered as a useful community model for further ecological and evolutionary study of fungi in the trench.

Genome mining of cyclodipeptide synthases unravels unusual tRNA-dependent diketopiperazine-terpene biosynthetic machinery.

Cyclodipeptide synthases (CDPSs) can catalyze the formation of two successive peptide bonds by hijacking aminoacyl-tRNAs from the ribosomal machinery resulting in diketopiperazines (DKPs). Here, three CDPS-containing loci (dmt1-3) are discovered by genome mining and comparative genome analysis of Streptomyces strains. Among them, CDPS DmtB1, encoded by the gene of dmt1 locus, can synthesize cyclo(L-Trp-L-Xaa) (with Xaa being Val, Pro, Leu, Ile, or Ala). Systematic mutagenesis experiments demonstrate the importance of the residues constituting substrate-binding pocket P1 for the incorporation of the second aa-tRNA in DmtB1. Characterization of dmt1-3 unravels that CDPS-dependent machinery is involved in CDPS-synthesized DKP formation followed by tailoring steps of prenylation and cyclization to afford terpenylated DKP compounds drimentines. A phytoene-synthase-like family prenyltransferase (DmtC1) and a membrane terpene cyclase (DmtA1) are required for drimentines biosynthesis. These results set the foundation for further increasing the natural diversity of complex DKP derivatives.

Identification of novel immunoreactive proteins and delineation of a specific epitope of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA), an increasingly recognized febrile tick-borne illness, is caused by a gram-negative obligate intracellular bacterium Anaplasma phagocytophilum. Because of nonspecific clinical manifestations, diagnosis of HGA highly depends on laboratory tests. Identification of immunoreactive proteins is prerequisite for development of specific and sensitive immunoassays for HGA. In this study, we identified novel immunoreactive proteins of A. phagocytophilum. Previous studies indicated that secreted proteins of A. phagocytophilum and other bacteria can be immunoreactive antigens. Here we in silico screened A. phagocytophilum genome for encoding proteins which bear features of type IV secretion system substrates. Among seventy seven predicted proteins, fourteen proteins were determined for antigenicity and nine proteins were showed to be immunoreactive antigens. In addition, an APH1384 peptide harboring a B cell epitope predicted by bioinformatics was found specifically reacting with anti-A. phagocytophilum sera. Hereby, we identified novel immunoreactive proteins and delineated a specific epitope of A. phagocytophilum, which might be employed for HGA diagnosis.

Characterization of the biosynthetic gene cluster of the polyene macrolide antibiotic reedsmycins from a marine-derived Streptomyces strain.

BACKGROUND: Polyene antibiotics are important as antifungal medicines albeit with serious side effects such as nephrotoxicity. Reedsmycin (RDM) A (1), produced by marine-derived Streptomyces youssoufiensis OUC6819, is a non-glycosylated polyene macrolide antibiotic with antifungal activity comparable to that of clinically used nystatin. To elucidate its biosynthetic machinery, herein, the rdm biosynthetic gene cluster was cloned and characterized. RESULTS: The rdm cluster is located within a 104 kb DNA region harboring 21 open reading frames (ORFs), among which 15 ORFs were designated as rdm genes. The assembly line for RDM A is proposed on the basis of module and domain analysis of the polyketide synthetases (PKSs) RdmGHIJ, which catalyze 16 rounds of decarboxylative condensation using malonyl-CoA as the starter unit (loading module), two methylmalonyl-CoA (module 1 and 2), and fourteen malonyl-CoA (module 3-16) as extender units successively. However, the predicted substrate specificity of AT0 in the loading module is methylmalonyl-CoA instead of malonyl-CoA. Interestingly, the rdm cluster contains a five-gene regulation system RdmACDEF, which is different from other reported polyene gene clusters. In vivo experiments demonstrated the XRE family regulator RdmA and the PAS/LuxR family regulator RdmF function in negative and positive manner, respectively. Notably, inactivation of rdmA and overexpression of rdmF led to increased production of RDM A by ~ 2.0-fold and ~ 2.5-fold, reaching yields of 155.3 ± 1.89 and 184.8 ± 9.93 mg/L, respectively. CONCLUSIONS: Biosynthesis of RDM A is accomplished on a linear assembly line catalyzed by Rdm PKSs harboring a unique AT0 under the control of a complex regulatory system. These findings enable generation of new biologically active RDM derivatives at high yield and with improved properties by engineered biosynthesis.

Discovery of an Unusual Fatty Acid Amide from the ndgRyo Gene Mutant of Marine-Derived Streptomyces youssoufiensis.

NdgRyo, an IclR-like regulator, was selected as the target gene to activate new secondary metabolites in the marine-derived Streptomyces youssoufiensis OUC6819. Inactivation of the ndgRyo gene in S. youssoufiensis OUC6819 led to the accumulation of a new fatty acid amide (1), with an unusual 3-amino-butyl acid as the amine component. Moreover, its parent fatty acid (2) was also discovered both in the wild-type and ΔndgRyo mutant strains, which was for the first time isolated from a natural source. The structures of compounds 1 and 2 were elucidated by combination of LC-MS and NMR spectroscopic analyses. This study demonstrated that the ndgRyo homologs might serve as a target for new compound activation in Streptomyces strains.

Geranylpyrrol A and Piericidin F from Streptomyces sp. CHQ-64 ΔrdmF.

Two new compounds, geranylpyrrol A (1) and piericidin F (2), were isolated from a reedsmycins nonproducing mutant strain of Streptomyces sp. CHQ-64. Their structures, including absolute configurations, were elucidated by extensive NMR, MS, NOESY, and ECD analyses. Geranylpyrrol A (1) is an unusual naturally occurring 2,3,4-trisubstituted pyrrole, and piericidin F (2) showed cytotoxicity against HeLa, NB4, A549, and H1975 cell lines with IC50 values of 0.003, 0.037, 0.56, and 0.49 µM, respectively.