Planctomonas deserti gen. nov., sp. nov., a new member of the family Microbacteriaceae isolated from soil of the Taklamakan desert.

A Gram-staining-positive, aerobic, irregular coccoid- to ovoid-shaped, non-spore-forming and motile bacterium, designated strain 13S1-3T, was isolated from a soil sample from the rhizosphere of Tamarix collected in the Taklamakan desert in Xinjiang Uygur Autonomous Region, PR China. The strain was examined by a polyphasic approach to clarify its taxonomic position. Strain 13S1-3T grew optimally at 28-30 °C, pH 7.0 and with 0-1 % (w/v) NaCl. The cell-wall peptidoglycan was of the B2γ type and contained d-alanine, d-glutamic acid, glycine, d-2,4-diaminobutyric acid and l-2,4-diaminobutyric acid. Ribose, xylose, glucose and galactose were detected as cell-wall sugars. The acyl type of the muramic acid was acetyl. The predominant menaquinones were MK-12, MK-11, MK-13 and MK-10. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids and one unidentified phospholipid. The major whole-cell fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The DNA G+C content was 70.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that 13S1-3T represented a member of the family Microbacteriaceae and showed the highest level of 16S rRNA gene sequence similarity with Frondihabitans australicus E1HC-02T (97.11 %). Phylogenetic trees revealed that 13S1-3T formed a distinct lineage with respect to closely related genera within the family Microbacteriaceae. On the basis of the results of phylogenetic, phenotypic and chemotaxonomic analyses, 13S1-3T is distinguishable from phylogenetically related genera in the family Microbacteriaceae, and represents a novel species of a new genus, for which the name Planctomonas deserti gen. nov., sp. nov. is proposed. The type strain is 13S1-3T (=KCTC 49115T=CGMCC 1.16554T).

Nakamurella deserti sp. nov., isolated from rhizosphere soil of Reaumuria in the Taklamakan desert.

A Gram-stain-positive, aerobic, non-motile, non-spore-forming, coccus-shaped actinobacterium, designated strain 12Sc4-1T, was isolated from a soil sample collected in the Taklamakan desert in Xinjiang Uygur Autonomous Region, China. Strain 12Sc4-1T grew at 8‒35 °C (optimum, 28‒30 °C), pH 6.0‒9.0 (optimum, pH 7.0) and in the presence of 0‒3 % (w/v) NaCl (optimum, 0 %). Phylogenetic analysis based on 16S rRNA gene sequence suggested that strain 12Sc4-1T belonged to the genus Nakamurella and shared the highest 16S rRNA gene sequence similarity to Nakamurella silvestris S20-107T (96.94 %). Strain 12Sc4-1T showed <96.0 % 16S rRNA gene sequence similarities to all other recognized members of the genus Nakamurella. Chemotaxonomic analyses showed that the isolate possessed meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan, glucose, mannose and galactose as whole-cell sugars, and MK-8(H4) as the predominant menaquinone. The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, phosphatidylinositol, an unidentified phospholipid, an unidentified phosphoglycolipid and an unidentified glycolipid. The major fatty acids were C16 : 0 and anteiso-C15 : 0. The DNA G+C content was 72.1 mol% (draft genome sequence). On the basis of phylogenetic, phenotypic and chemotaxonomic analyses, strain 12Sc4-1T represents a novel species of the genus Nakamurella, for which the name Nakamurelladeserti sp. nov. is proposed. The type strain is 12Sc4-1T (=KCTC 49114T=CGMCC 1.16555T).

Structure-based manual screening and automatic networking for systematically exploring sansanmycin analogues using high performance liquid chromatography tandem mass spectroscopy.

Sansanmycins (SS), one of several known uridyl peptide antibiotics (UPAs) possessing a unique chemical scaffold, showed a good inhibitory effect on the highly refractory pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis, especially on the multi-drug resistant M. tuberculosis. This study employed high performance liquid chromatography-mass spectrometry detector (HPLC-MSD) ion trap and LTQ orbitrap tandem mass spectrometry (MS/MS) to explore sansanmycin analogues manually and automatically by re-analysis of the Streptomyces sp. SS fermentation broth. The structure-based manual screening method, based on analysis of the fragmentation pathway of known UPAs and on comparisons of the MS/MS spectra with that of sansanmycin A (SS-A), resulted in identifying twenty sansanmycin analogues, including twelve new structures (1-12). Furthermore, to deeply explore sansanmycin analogues, we utilized a GNPS based molecular networking workflow to re-analyze the HPLC-MS/MS data automatically. As a result, eight more new sansanmycins (13-20) were discovered. Compound 1 was discovered to lose two amino acids of residue 1 (AA1) and (2S, 3S)-N3-methyl-2,3-diamino butyric acid (DABA) from the N-terminus, and compounds 6, 11 and 12 were found to contain a 2',3'-dehydrated 4',5'-enamine-3'-deoxyuridyl moiety, which have not been reported before. Interestingly, three trace components with novel 5,6-dihydro-5'-aminouridyl group (16-18) were detected for the first time in the sansanmycin-producing strain. Their structures were primarily determined by detail analysis of the data from MS/MS. Compounds 8 and 10 were further confirmed by nuclear magnetic resonance (NMR) data, which proved the efficiency and accuracy of the method of HPLC-MS/MS for exploration of novel UPAs. Comparing to manual screening, the networking method can provide systematic visualization results. Manual screening and networking method may complement with each other to facilitate the mining of novel UPAs.

Exploiting Substrate Diversity of NRPS Led to the Generation of New Sansanmycin Analogs.

Further exploration of substrate diversity of the sansaninycin biosynthetic pathway using available halogen- and methyl-phenylalanines led to the generation of diverse sansanmycin derivatives, either at the single C- or N-terminus alone or at both C- and N-termini. The structures of all of these derivatives were determined by MS/MS spectra, and amongst them, the structures of [2-Cl-Phe]-sansanmycin H (1) and [2-Cl-Phe]-sansanmycin A (2) were further identified by NMR. Both the C-terminal derivative I and the N-terminal derivative 2 were assayed for their antibacterial activitiesi and compound 1 exhibited moderate activity against P. aeruginosa and ΔtolC mutant E. coli.

Synthesis and in vitro antitubercular evaluation of novel sansanmycin derivatives.

Tuberculosis (TB) is a major health problem worldwide. A series of novel sansanmycin derivatives were designed, semi-synthesized and evaluated for their activity against drug-susceptible Mycobacterium tuberculosis strain H(37)Rv with sansanmycin A (SSA) as the lead. Among these analogs tested, compound 1d possessing an isopropyl group at the amino terminal afforded an increased antimycobacterial activity with a MIC value of 8 µg/mL in comparison with SSA. Importantly, it was active for rifampicin- and isoniazid-resistant M. tuberculosis strain isolated from patients in China. These promising results offer an opportunity for further exploration of this novel class of analogs as antitubercular agents.

Effects of combined treatment with sansanmycin and macrolides on Pseudomonas aeruginosa and formation of biofilm.

OBJECTIVE: To observe the effects of combined treatment with sansanmycin and macrolides on Pseudomonas aeruginosa and formation of biofilm. METHODS: Micro-dilution method was used to determine the minimal inhibitory concentrations (MICs) of sansanmycin, gentamycin, carbenicillin, polymyxin B, roxithromycin, piperacillin, and tazobactam. PA1 and PA27853 biofilms were observed under optical microscope after staining and under SEM after treatment with sansanmycin at different dosages and combined treatment with sansanmycin and roxithromycin. Viable bacteria in PA1 and PA27853 biofilms were counted after treatment with sansanmycin at different dosages or combined treatment with sansanmycin and roxithromycin. RESULTS: The MIC of sansanmycin was lower than that of gentamycin and polymyxin B, but was higher than that of carbenicillin. Roxithromycin enhanced the penetration of sansanmycin to PA1 and PA27853 strains through biofilms. PA1 and PA27853 biofilms were gradually cleared with the increased dosages of sansanmycin or with the combined sansanmycin and roxithromycin. CONCLUSION: Sub-MIC levels of roxithromycin and sansanmycin substantially inhibit the generation of biofilms and proliferation of bacteria. Therefore, combined antibiotics can be used in treatment of intractable bacterial infection.