| Antibiotic resistance is occurring more and more severe,and the new and effective antibiotics are urgently needed.Currently,new and good quality antibiotics are still lacking,and one of the main reasons is repeated screening of known microorganisms and antibiotics,which caused high cost and low efficiency.Actinobacteria are one of major source of antibiotics,and the actinobacterial diversity is the foundation for diversity of compounds.Therefore,exploitation of microbial source could enrich the pharmaceutical actinobacterial resource and increase the probability of discovery of new antibiotics from nature.Actinobacterial strains residing in special environments have the unique gene types and metabolic mechanisms,which is the basis of discovery of new antibiotics.Therefore,exploration of pharmaceutical actinobacterial resources have been become the hot research in the research field of new antibiotic discovery.Based on the research background above,two special environments including mangrove and desert were selected to explore pharmaceutical actinobacterial resource by using classical separation method.As we all know,the microorganisms cultured in the laboratory are less than 1%,to exploit effectively the remaining 99%of microbial resources,the in situ cultivation technology has been applied to capture microorganisms in mangrove soil that are uncultured and difficult to culture.Finally,novel strains discovered in the exploration were performed the polyphasic taxonomic identification.The mainly research contents are as follows:1.Endophytic actinobacteria were isolated and purified from the mangrove plants in Cotai Ecological Zones,Macao,China.Representative actinobacterial isolates were selected for antibacterial assay.A total of 192 endophytic actinobacterial strains were obtained from 12 mangrove plant samples,these strains distributed in 30 genera affiliated to 17 families of 8 orders.The dominant genus was Streptomyces,and 22 out of 192 actinobacterial strains were potential new species.Among them,4 new species have been identified and published.Antibacterial assay showed that 50 of the 82 fermentation strains exhibited antagonistic activity against "ESKAPE" bacterial strains,including 6 strains with strong antibacterial activity(diameter of inhibition zone>20 mm),21 rare actinobacterial strains with significant antibacterial activity(diameter of inhibition zone>12 mm)and 8 potenial novel strains with antibacterial activity.Among them,28 strains were chosen as candidate strains for chemical study,and 5 candidate strains were analyzed for biosynthetic gene clusters of secondary metabolites to evaluate their antibacterial potential.2.The in situ cultivation technology was applied to capture actinobacteria that are uncultured and difficult to culture from mangrove soil in Futian Mangrove National Nature Reserve and Mao Wei Sea Mangrove National Nature Reserve,and representative actinobacterial isolates were selected for tests of antibacterial activity and antibacterial mechanism of action.A total of 367 actinobacterial strains were obtained from 35 the in situ cultivation samples,and these strains distributed in 30 genera affiliated to 12 families of 7 orders.The dominant genus was Microbacterium,and 9 actinobacterial strains were potential new species.Antibacterial assay showed that 45 out of 85 fermentation strains exhibited antagonistic activity against "ESKAPE" bacterial strains,including 10 strains with strong antibacterial activity(diameter of inhibition zone>20 mm),13 rare actinobacteria strains with significant antibacterial activity(diameter of inhibition zone>12 mm)and 2 potenial novel strains with significant antibacterial activity.Eighty five of organic layer samples were screened for mechanism of action by double fluorescent protein reporter system,and 2 Streptomyces strains produced secondary metabolites with inhibitory activity against protein translation.According to the results obtained,25 strains were identified as candidate strains for chemical study.Among them,1 Streptomyces strain was analyzed for biosynthetic gene clusters of secondary metabolites,and it showed the biosynthetic potential of various antibacterial compounds.Currently,3 bioactive compounds have been isolated from this strain.3.Endophytic actinobacteria were isolated and purified from psammophytes collected in Taklamakan Desert,China.Representative actinobacterial isolates were selected for tests of antibacterial activity and antibacterial mechanism of action.A total of 320 endophytic actinobacterial strains were obtained from 15 plant samples,these strains distributed in 23 genera affiliated to 14 families of 9 orders.The dominant genus was Streptomyces,and 19 actinobacterial strains were potential new species.Among them,the taxonomic positions of 3 strains have been clarified.The results of antibacterial assay showed that 47 out of 75 fermentation strains exhibited antagonistic activity against"ESKAPE" bacterial strains,including 6 strains with strong antibacterial activity(diameter of inhibition zone>20 mm),10 rare actinobacteria strains with significant antibacterial activity(diameter of inhibition zone>12 mm)and 5 potenial novel strains with antibacterial activity.75 of organic layer samples were screened for mechanism of action by double fluorescent protein reporter system.Two Streptomyces strains produced secondary metabolites with inhibitory activity against DNA biosynthesis,and 2 Streptomyces strains produced secondary metabolites with inhibitory activity against protein translation.According to the results obtained,19 strains were identified as candidate strains for chemical study.4 candidate strains were analyzed for biosynthetic gene clusters of secondary metabolites to predict the biosynthetic potential of compounds with antibacterial activity.14 bioactive compounds including 2 new compounds have been isolated from 2 candidate strains under the guidance of genome mining.4.The taxonomic positions of 7 novel strains including 4 endophytic bacteria(1T4Z-3,4Q3S-7,5T4P-12-1 and 2T4P-2-4)isolated from mangrove plants in Macao and 3 endophytic actinobacteria(11W25H-1,8H24J-4-2 and 9W16Y-2)isolated from psammophytes collected in Taklamakan Desert,were clarified through the polyphase approach and the 7 novel strains were named as Amnibacterium endophyticum sp.nov.,Marmoricola mangrovicus sp.nov.,Aeromicrobium endophyticum sp.nov.,Aureimonas endophytica sp.nov.,Labedella phragmitis sp.nov.,Labedella populi sp.nov.and Aeromicrobium endophyticum sp.nov.,respectively.In conclusion,a total of 879 actinobacterial strains were isolated from mangrove and desert,which were distributed in 54 genera affiliated to 20 families of 9 orders.The results of antibacterial assay showed that 142 out of 242 fermentation strains exhibited antagonistic activity,including 22 strains with strong antibacterial activity(diameter of inhibition zone>20 mm),44 rare actinobacteria strains with significant antibacterial activity(diameter of inhibition zone>12 mm)and 15 potenial novel strains with antibacterial activity.One hundred sixty of organic layer samples were screened for antibacterial mechanism of action by double fluorescent protein reporter system.Four Streptomyces strains produced bioactive compounds with inhibitory activity against protein translation,and 2 Streptomyces strains produced bioactive compounds with inhibitory activity against DNA biosynthesis.According to the results above,35 strains were identified as candidate strains for chemical study,and 10 candidate strains were analyzed for biosynthetic gene clusters of secondary metabolites to predict the biosynthetic potential of compounds with antibacterial activity.Until now,17 bioactive compounds including 2 new compounds have been isolated from 3 candidate strains by our group.In addition,50 potential novel actinobacterial strains were obtained in this study,and the taxonomic positions of 7 strains have been clarified.Until March 2020,13 new species of endophytic actinobacteria were isolated from mangrove plants all over the world,among them,4 strains were from this study(mangrove plants in Macao).From 2017 to January 2020,5 new species of endophytic actinobacteria were isolated from desert plants all over the world,among them,3 strains were from in this study(Taklamakan Desert).The data above demonstrated that mangrove and desert harbor rich and novel actinobacterial resources,which are the treasure houses to discovery of new antibiotics.In addition,the in situ cultivation technology could provide more abundant microbial sources for discovery of natural products,which is worthy of further exploration. |
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