Project description:Microbial drug discovery programs rely heavily on accessing bacterial diversity from the environment to acquire new specialized metabolite (SM) lead compounds for the therapeutic pipeline. Therefore, knowledge of how commonly culturable bacterial taxa are distributed in nature, in addition to the degree of variation of SM production within those taxa, is critical to informing these front-end discovery efforts and making the overall sample collection and bacterial library creation process more efficient. In the current study, we employed MALDI-TOF mass spectrometry and the bioinformatics pipeline IDBac to analyze diversity within phylotype groupings and SM profiles of hundreds of bacterial isolates from two Eunapius fragilis freshwater sponges, collected 1.5 km apart. We demonstrated that within two sponge samples of the same species, the culturable bacterial populations contained significant overlap in approximate genus-level phylotypes but mostly nonoverlapping populations of isolates when grouped lower than the level of genus. Further, correlations between bacterial phylotype and SM production varied at the species level and below, suggesting SM distribution within bacterial taxa must be analyzed on a case-by-case basis. Our results suggest that two E. fragilis freshwater sponges collected in similar environments can exhibit large culturable diversity on a species-level scale, thus researchers should scrutinize the isolates with analyses that take both phylogeny and SM production into account to optimize the chemical space entering into a downstream bacterial library.
Project description:Filamentous soil bacteria are known to produce diverse specialized metabolites. Despite having enormous potential as a source of pharmaceuticals, they often produce bioactive metabolites at low titers. Here, we show that inactivation of the pactamycin, NFAT-133, and conglobatin biosynthetic pathways in Streptomyces pactum ATCC 27456 significantly increases the production of the mitochondrial electron transport inhibitors piericidins. Similarly, inactivation of the pactamycin, NFAT-133, and piericidin pathways significantly increases the production of the heat-shock protein (Hsp) 90 inhibitor conglobatin. In addition, four new conglobatin analogues (B2, B3, F1, and F2) with altered polyketide backbones, together with the known analogue conglobatin B1, were identified in this mutant, indicating that the conglobatin biosynthetic machinery is promiscuous toward different substrates. Among the new conglobatin analogues, conglobatin F2 showed enhanced antitumor activity against HeLa and NCI-H460 cancer cell lines compared to conglobatin. Conglobatin F2 also inhibits colony formation of HeLa cells in a dose-dependent manner. Molecular modeling studies suggest that the new conglobatins bind to human Hsp90 and disrupt Hsp90/Cdc37 chaperone/co-chaperone interactions in the same manner as conglobatin. The study also showed that genes that are involved in piericidin biosynthesis are clustered in two different loci located distantly in the S. pactum genome.
Project description:Bacteria communities associated with plants have been given increasing consideration because they are arguably beneficial to their host plants. To understand the ecological and evolutionary impact of these mutualistic associations, it is important to explore the vast unknown territory of bacterial genomic diversity and their functional contributions associated with the major branches of the tree-of-life. Arguably, this aim can be achieved by profiling bacterial communities by applying high throughput sequencing approaches, besides establishing model plant organisms to test key predictions. This study utilized the Illumina Miseq reads of bacterial 16S rRNA sequences to determine the bacterial diversity associated with the endosphere of the leaves of the highly specialized rock spleenwort Asplenium delavayi (Aspleniaceae). By documenting the bacterial communities associated with ferns collected in natural occurrence and cultivation, this study discovered the most species-rich bacterial communities associated with terrestrial ferns reported until now. Despite the substantial variations of species diversity and composition among accessions, a set of 28 bacterial OTUs was found to be shared among all accessions. Functional analyses recovered evidence to support the predictions that changes in bacterial community compositions correspond to functional differentiation. Given the ease of cultivating this species, Asplenium delavayi is introduced here as a model organism to explore the ecological and evolutionary benefits created by mutualistic associations between bacteria and ferns.
Project description:The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.
Project description:Plants exude specialized metabolites from their roots, and these compounds are known to structure the root microbiome. However, the underlying mechanisms are poorly understood. We established a representative collection of maize root bacteria and tested their tolerance against benzoxazinoids (BXs), the dominant specialized and bioactive metabolites in the root exudates of maize plants. In vitro experiments revealed that BXs inhibited bacterial growth in a strain- and compound-dependent manner. Tolerance against these selective antimicrobial compounds depended on bacterial cell wall structure. Further, we found that native root bacteria isolated from maize tolerated the BXs better compared to nonhost Arabidopsis bacteria. This finding suggests the adaptation of the root bacteria to the specialized metabolites of their host plant. Bacterial tolerance to 6-methoxy-benzoxazolin-2-one (MBOA), the most abundant and selective antimicrobial metabolite in the maize rhizosphere, correlated significantly with the abundance of these bacteria on BX-exuding maize roots. Thus, strain-dependent tolerance to BXs largely explained the abundance pattern of bacteria on maize roots. Abundant bacteria generally tolerated MBOA, while low abundant root microbiome members were sensitive to this compound. Our findings reveal that tolerance to plant specialized metabolites is an important competence determinant for root colonization. We propose that bacterial tolerance to root-derived antimicrobial compounds is an underlying mechanism determining the structure of host-specific microbial communities.
Project description:For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra recorded from single bacterial colonies picked from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis strains in <30 min solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin, caused by a single frameshift mutation. Next, we used IDBac to detect subtle intraspecies differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we used IDBac to simultaneously extract protein and specialized metabolite MS profiles from unidentified Lake Michigan sponge-associated bacteria isolated from an agar plate. In just 3 h, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 spectra) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on interspecies and intraspecies differences in specialized metabolite production. IDBac is an attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow for facile discrimination of closely related bacterial colonies.
Project description:UnlabelledSoil microbiome modification may alter system function, which may enhance processes like bioremediation. In this study, we filled microcosms with gamma-irradiated soil that was reinoculated with the initial soil or cultivated bacterial subsets obtained on regular media (REG-M) or media containing crude oil (CO-M). We allowed 8 weeks for microbiome stabilization, added crude oil and monoammonium phosphate, incubated the microcosms for another 6 weeks, and then measured the biodegradation of crude oil components, bacterial taxonomy, and functional gene composition. We hypothesized that the biodegradation of targeted crude oil components would be enhanced by limiting the microbial taxa competing for resources and by specifically selecting bacteria involved in crude oil biodegradation (i.e., CO-M). Postincubation, large differences in taxonomy and functional gene composition between the three microbiome types remained, indicating that purposeful soil microbiome structuring is feasible. Although phylum-level bacterial taxonomy was constrained, operational taxonomic unit composition varied between microbiome types. Contrary to our hypothesis, the biodegradation of C10 to C50 hydrocarbons was highest when the original microbiome was reinoculated, despite a higher relative abundance of alkane hydroxylase genes in the CO-M microbiomes and of carbon-processing genes in the REG-M microbiomes. Despite increases in the relative abundances of genes potentially linked to hydrocarbon processing in cultivated subsets of the microbiome, reinoculation of the initial microbiome led to maximum biodegradation.ImportanceIn this study, we show that it is possible to sustainably modify microbial assemblages in soil. This has implications for biotechnology, as modification of gut microbial assemblages has led to improved treatments for diseases like Clostridium difficile infection. Although the soil environment determined which major phylogenetic groups of bacteria would dominate the assemblage, we saw differences at lower levels of taxonomy and in functional gene composition (e.g., genes related to hydrocarbon degradation). Further studies are needed to determine the success of such an approach in nonsterile environments. Although the biodegradation of certain crude oil fractions was still the highest when we inoculated with the diverse initial microbiome, the possibility of discovering and establishing microbiomes that are more efficient in crude oil degradation is not precluded.
Project description:Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.
Project description:Marine and freshwater sponges harbor diverse communities of bacteria with vast potential to produce secondary metabolites that may play an important role in protecting the host from predators and infections. In this work, we initially used cultivation and metagenomics to investigate the microbial community of the freshwater sponge Spongilla lacustris collected in an Austrian lake. Representatives of 41 bacterial genera were isolated from the sponge sample and classified according to their 16S rRNA gene sequences. The genomes of 33 representative isolates and the 20 recovered metagenome-assembled genomes (MAGs) contained in total 306 secondary metabolite biosynthesis gene clusters (BGCs). Comparative 16S rRNA gene and genome analyses showed very little taxon overlap between the recovered isolates and the sponge community as revealed by cultivation-independent methods. Both culture-independent and -dependent analyses suggested high biosynthetic potential of the S. lacustris microbiome, which was confirmed experimentally even at the subspecies level for two Streptomyces isolates. To our knowledge, this is the most thorough description of the secondary metabolite production potential of a freshwater sponge microbiome to date. IMPORTANCE A large body of research is dedicated to marine sponges, filter-feeding animals harboring rich bacterial microbiomes believed to play an important role in protecting the host from predators and infections. Freshwater sponges have received so far much less attention with respect to their microbiomes, members of which may produce bioactive secondary metabolites with potential to be developed into drugs to treat a variety of diseases. In this work, we investigated the potential of bacteria associated with the freshwater sponge Spongilla lacustris to biosynthesize diverse secondary metabolites. Using culture-dependent and -independent methods, we discovered over 300 biosynthetic gene clusters in sponge-associated bacteria and proved production of several compounds by selected isolates using genome mining. Our results illustrate the importance of a complex approach when dealing with microbiomes of multicellular organisms that may contain producers of medically important secondary metabolites.
Project description:Chronic rhinosinusitis (CRS) is a clinical syndrome defined by symptoms including nasal congestion, facial pain and pressure, anosmia, and rhinorrhea lasting more than 12 weeks. Several mechanistically distinct processes lead to the development of clinical symptoms in CRS including innate immune dysfunction, dysregulated eicosanoid metabolism and perturbations in host-microbiome interactions [1]. We developed a database comprised of patient demographic information, lipid mediator metabolomic profiles, and 16S bacterial rRNA gene sequence data from 66 patients undergoing endoscopic sinus surgery. Briefly, ethmoid sinus tissue and middle meatal swabs were collected from patients, including non-CRS controls, CRS with polyps (CRSwNP), and CRS without polyps (CRSsNP). Lipid mediator pathways from arachidonic acid (AA) and docosahexanoic acid (DHA) were analyzed by liquid chromatography/tandem mass spectrometry. Bacterial taxa were profiled in parallel by 16S rRNA gene sequencing. This database provides a useful compendium of AA/DHA metabolomic profiles and associated bacterial microbiota in patients with varying disease subtypes, demographics, and risk factors/comorbidities.