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Abstract Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with high death rates that have remained substantially unaltered for decades. Therefore, new treatment approaches are urgently needed. Human papillomavirus-negative tumors harbor areas of terminally differentiated tissue that are characterized by cornification. Dissecting this intrinsic ability of HNSCC cells to irreversibly differentiate into non-malignant cells may have tumor-targeting potential. We modeled the cornification of HNSCC cells in a primary spheroid model and analyzed the mechanisms underlying differentiation by ATAC-seq and RNA-seq. Results were verified by immunofluorescence using human HNSCC tissue of distinct anatomical locations. HNSCC cell differentiation was accompanied by cell adhesion, proliferation stop, diminished tumor-initiating potential in immunodeficient mice, and activation of a wound-healing-associated signaling program. Small promoter accessibility increased despite overall chromatin closure. Differentiating cells upregulated KRT17 and cornification markers. Although KRT17 represents a basal stem cell marker in normal mucosa, we confirm KRT17 to represent an early differentiation marker in HNSCC tissue. Cornification was frequently found surrounding necrotic areas in human tumors, indicating an involvement of pro-inflammatory stimuli. Indeed, inflammatory mediators activated the differentiation program in primary HNSCC cells. In HNSCC tissue, distinct cell differentiation states were found to create a common tissue architecture in normal mucosa and HNSCCs. Our data demonstrate a loss of cell malignancy upon faithful HNSCC cell differentiation, indicating that targeted differentiation approaches may be therapeutically valuable. Moreover, we describe KRT17 to be a candidate biomarker for HNSCC cell differentiation and early tumor detection.

Abstract Background Industrial by-products accrue in most agricultural or food-related production processes, but additional value chains have already been established for many of them. Crude glycerol has a 60% lower market value than commercial glucose, as large quantities are produced in the biodiesel industry, but its valorisation is still underutilized. Due to its high carbon content and the natural ability of many microorganisms to metabolise it, microbial upcycling is a suitable option for this waste product. Results In this work, the use of crude glycerol for the production of the value-added compound itaconate is demonstrated using the smut fungus Ustilago maydis. Starting with a highly engineered strain, itaconate production from an industrial glycerol waste stream was quickly established on a small scale, and the resulting yields were already competitive with processes using commercial sugars. Adaptive laboratory evolution resulted in an evolved strain with a 72% increased growth rate on glycerol. In the subsequent development and optimisation of a fed-batch process on a 1.5-2 L scale, the use of molasses, a side stream of sugar beet processing, eliminated the need for other expensive media components such as nitrogen or vitamins for biomass growth. The optimised process was scaled up to 150 L, achieving an overall titre of 72 g L− 1, a yield of 0.34 g g− 1, and a productivity of 0.54 g L− 1 h− 1. Conclusions Pilot-scale itaconate production from the complementary waste streams molasses and glycerol has been successfully established. In addition to achieving competitive performance indicators, the proposed dual feedstock strategy offers lower process costs and carbon footprint for the production of bio-based itaconate.

Abstract Background Transforming waste and nonfood materials into bulk biofuels and chemicals represents a major stride in creating a sustainable bioindustry to optimize the use of resources while reducing environmental footprint. However, despite these advancements, the production of high-value natural products often continues to depend on the use of first-generation substrates, underscoring the intricate processes and specific requirements of their biosyntheses. This is also true for Streptomyces lividans, a renowned host organism celebrated for its capacity to produce a wide array of natural products, which is attributed to its genetic versatility and potent secondary metabolic activity. Given this context, it becomes imperative to assess and optimize this microorganism for the synthesis of natural products specifically from waste and nonfood substrates. Results We metabolically engineered S. lividans to heterologously produce the ribosomally synthesized and posttranslationally modified peptide bottromycin, as well as the polyketide pamamycin. The modified strains successfully produced these compounds using waste and nonfood model substrates such as protocatechuate (derived from lignin), 4-hydroxybenzoate (sourced from plastic waste), and mannitol (from seaweed). Comprehensive transcriptomic and metabolomic analyses offered insights into how these substrates influenced the cellular metabolism of S. lividans. In terms of production efficiency, S. lividans showed remarkable tolerance, especially in a fed-batch process using a mineral medium containing the toxic aromatic 4-hydroxybenzoate, which led to enhanced and highly selective bottromycin production. Additionally, the strain generated a unique spectrum of pamamycins when cultured in mannitol-rich seaweed extract with no additional nutrients. Conclusion Our study showcases the successful production of high-value natural products based on the use of varied waste and nonfood raw materials, circumventing the reliance on costly, food-competing resources. S. lividans exhibited remarkable adaptability and resilience when grown on these diverse substrates. When cultured on aromatic compounds, it displayed a distinct array of intracellular CoA esters, presenting promising avenues for polyketide production. Future research could be focused on enhancing S. lividans substrate utilization pathways to process the intricate mixtures commonly found in waste and nonfood sources more efficiently.

Abstract Background Pamamycins are a family of highly bioactive macrodiolide polyketides produced by Streptomyces alboniger as a complex mixture of derivatives with molecular weights ranging from 579 to 705 Daltons. The large derivatives are produced as a minor fraction, which has prevented their isolation and thus studies of chemical and biological properties. Results Herein, we describe the transcriptional engineering of the pamamycin biosynthetic gene cluster (pam BGC), which resulted in the shift in production profile toward high molecular weight derivatives. The pam BGC library was constructed by inserting randomized promoter sequences in front of key biosynthetic operons. The library was expressed in Streptomyces albus strain with improved resistance to pamamycins to overcome sensitivity-related host limitations. Clones with modified pamamycin profiles were selected and the properties of engineered pam BGC were studied in detail. The production level and composition of the mixture of pamamycins was found to depend on balance in expression of the corresponding biosynthetic genes. This approach enabled the isolation of known pamamycins and the discovery of three novel derivatives with molecular weights of 663 Da and higher. One of them, homopamamycin 677A, is the largest described representative of this family of natural products with an elucidated structure. The new pamamycin 663A shows extraordinary activity (IC50 2 nM) against hepatocyte cancer cells as well as strong activity (in the one-digit micromolar range) against a range of Gram-positive pathogenic bacteria. Conclusion By employing transcriptional gene cluster refactoring, we not only enhanced the production of known pamamycins but also discovered novel derivatives exhibiting promising biological activities. This approach has the potential for broader application in various biosynthetic gene clusters, creating a sustainable supply and discovery platform for bioactive natural products.

Abstract Background Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria and is industrially important. The underlying biosynthetic processes are complex and hinder rational engineering, so industrial manufacturing currently relies on classical mutants for production. While the biochemistry underlying oxytetracycline synthesis is known to involve polyketide synthase, hyperproducing strains of S. rimosus have not been extensively studied, limiting our knowledge on fundamental mechanisms that drive production. Results In this study, a multiomics analysis of S. rimosus is performed and wild-type and hyperproducing strains are compared. Insights into the metabolic and regulatory networks driving oxytetracycline formation were obtained. The overproducer exhibited increased acetyl-CoA and malonyl CoA supply, upregulated oxytetracycline biosynthesis, reduced competing byproduct formation, and streamlined morphology. These features were used to synthesize bhimamycin, an antibiotic, and a novel microbial chassis strain was created. A cluster deletion derivative showed enhanced bhimamycin production. Conclusions This study suggests that the precursor supply should be globally increased to further increase the expression of the oxytetracycline cluster while maintaining the natural cluster sequence. The mutagenized hyperproducer S. rimosus HP126 exhibited numerous mutations, including large genomic rearrangements, due to natural genetic instability, and single nucleotide changes. More complex mutations were found than those typically observed in mutagenized bacteria, impacting gene expression, and complicating rational engineering. Overall, the approach revealed key traits influencing oxytetracycline production in S. rimosus, suggesting that similar studies for other antibiotics could uncover general mechanisms to improve production.

The pseudotetrasaccharide acarbose, produced by Actinoplanes sp. SE50/110, is a relevant secondary metabolite used in diabetes type II medication. Although maltose plays a crucial role in acarbose biosynthesis, the understanding of the maltose/maltodextrin metabolism and its involvement in acarbose production is at an early stage. Here, we reconstructed the predicted maltose–maltodextrin pathway that involves four enzymes AmlE, MalZ, MalP, and MalQ. An investigation of enzyme activities was conducted through in vitro assays, leading to an expansion of previously postulated substrate spectra. The maltose-induced α-glucosidase AmlE is noteworthy for its high hydrolysis rate of linear α-1,4-glucans, and its capability to hydrolyze various glycosidic bonds. The predicted maltodextrin glucosidase MalZ showed slow hydrolysis activity on linear α-glucans, but it was resistant to acarbose and capable of releasing glucose from acarbose. AmlE compensates for the low activity of MalZ to ensure glucose supply. We determined the enzyme activity of MalP and its dual function as maltodextrin and glycogen phosphorylase. The 4-α-glucanotransferase MalQ plays a central role in the maltose/maltodextrin metabolism, alongside MalP. This study confirmed the simultaneous degradation and synthesis of long-chain α-glucans. The product distribution showed that with an increasing number of glycosidic bonds, less glucose is formed. We found that MalQ, like its sequence homolog AcbQ from the acarbose biosynthetic gene cluster, is involved in the formation of elongated acarviosyl metabolites. However, MalQ does not participate in the elongation of acarbose 7-phosphate, which is likely the more readily available acceptor molecule in vivo. Accordingly, MalQ is not involved in the formation of acarviosyl impurities in Actinoplanes sp. SE50/110.

Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation and stress response. Actinoplanes sp. SE50/110 is the natural producer of acarbose, an α-glucosidase inhibitor that is used in diabetes type 2 treatment. Acarbose biosynthesis is dependent on growth, making sigma factor engineering a promising tool for metabolic engineering. ACSP50_0507 is a homolog of the developmental and osmotic-stress-regulating Streptomyces coelicolor σHSc. Therefore, the protein encoded by ACSP50_0507 was named σHAs. Here, an Actinoplanes sp. SE50/110 expression strain for the alternative sigma factor gene ACSP50_0507 (sigHAs) achieved a two-fold increased acarbose yield with acarbose production extending into the stationary growth phase. Transcriptome sequencing revealed upregulation of acarbose biosynthesis genes during growth and at the late stationary growth phase. Genes that are transcriptionally activated by σHAs frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its S. coelicolor homolog. The DNA-binding motif of σHAs was determined based on transcriptome sequencing data and shows high motif similarity to that of its homolog. The motif was confirmed by in vitro binding of recombinantly expressed σHAs to the upstream sequence of a strongly upregulated gene. Autoregulation of σHAs was observed, and binding to its own gene promoter region was also confirmed.

Abstract Background Fatty acid-derived products such as fatty alcohols (FAL) find growing application in cosmetic products, lubricants, or biofuels. So far, FAL are primarily produced petrochemically or through chemical conversion of bio-based feedstock. Besides the well-known negative environmental impact of using fossil resources, utilization of bio-based first-generation feedstock such as palm oil is known to contribute to the loss of habitat and biodiversity. Thus, the microbial production of industrially relevant chemicals such as FAL from second-generation feedstock is desirable. Results To engineer Corynebacterium glutamicum for FAL production, we deregulated fatty acid biosynthesis by deleting the transcriptional regulator gene fasR, overexpressing a fatty acyl-CoA reductase (FAR) gene of Marinobacter hydrocarbonoclasticus VT8 and attenuating the native thioesterase expression by exchange of the ATG to a weaker TTG start codon. C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) produced in shaking flasks 0.54 ± 0.02 gFAL L−1 from 20 g glucose L−1 with a product yield of 0.054 ± 0.001 Cmol Cmol−1. To enable xylose utilization, we integrated xylA encoding the xylose isomerase from Xanthomonas campestris and xylB encoding the native xylulose kinase into the locus of actA. This approach enabled growth on xylose. However, adaptive laboratory evolution (ALE) was required to improve the growth rate threefold to 0.11 ± 0.00 h−1. The genome of the evolved strain C. glutamicum gX was re-sequenced, and the evolved genetic module was introduced into C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) which allowed efficient growth and FAL production on wheat straw hydrolysate. FAL biosynthesis was further optimized by overexpression of the pntAB genes encoding the membrane-bound transhydrogenase of E. coli. The best-performing strain C. glutamicum ∆fasR cg2692TTG CgLP12::(P tac -pntAB-T rrnB ) gX (pEKEx2-maqu2220) produced 2.45 ± 0.09 gFAL L−1 with a product yield of 0.054 ± 0.005 Cmol Cmol−1 and a volumetric productivity of 0.109 ± 0.005 gFAL L−1 h−1 in a pulsed fed-batch cultivation using wheat straw hydrolysate. Conclusion The combination of targeted metabolic engineering and ALE enabled efficient FAL production in C. glutamicum from wheat straw hydrolysate for the first time. Therefore, this study provides useful metabolic engineering principles to tailor this bacterium for other products from this second-generation feedstock.

Abstract Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Although these bacteria have naturally evolved strategies to cope with different kinds of stress, many biotechnological applications benefit from engineering of optimised chassis strains with specially adapted tolerance traits. Here, we explored the formation of outer membrane vesicles (OMV) of Pseudomonas putida KT2440. We found OMV production to correlate with the recombinant production of a natural compound with versatile beneficial properties, the tripyrrole prodigiosin. Further, several P. putida genes were identified, whose up‐ or down‐regulated expression allowed controlling OMV formation. Finally, genetically triggering vesiculation in production strains of the different alkaloids prodigiosin, violacein, and phenazine‐1‐carboxylic acid, as well as the carotenoid zeaxanthin, resulted in up to three‐fold increased product yields. Consequently, our findings suggest that the construction of robust strains by genetic manipulation of OMV formation might be developed into a useful tool which may contribute to improving limited biotechnological applications.

ABSTRACTThe highly conserved eukaryotic histone chaperone ASF1 is involved in the assembly and disassembly of nucleosomes during transcription, DNA replication, and repair. It was the first chaperone discovered to be involved in all three of these processes. The filamentous fungus Sordaria macrospora is one of only two multicellular organisms where asf1 deletions are viable, which makes it useful for in vivo analysis of this central regulator of eukaryotic chromatin structure. Deletion of asf1 in S. macrospora leads to sterility, a reduction of DNA methylation, and upregulation of genes that are usually weakly expressed in the wild type. Here, we focused on the functions of the highly conserved core and the divergent C-terminal tail of ASF1, studied the effects of ASF1 on histone modifications, and tested its relevance for genomic stability. By co-immunoprecipitation and complementation analysis, we showed that substitutions of amino acid V94 or truncations of the C-terminal tail abolish histone binding and do not complement the sterile mutant phenotype. ∆asf1 is sensitive to the DNA-damaging agent methyl methanesulfonate, while complementation strains, even those with non-histone-binding variants, regain wild type-like resistance. The histone marks H3K27me3 and H3K56ac were shown to be influenced by the histone-binding capability of ASF1. To aid in subsequent analyses, we generated a chromosome-resolved genome assembly of S. macrospora. By using Hi-C, we detected a tandem duplication of around 600 kb on chromosome 2 in the mutant. Crossing experiments indicated linkage between the viability of Δasf1 strains and the presence of the duplication.IMPORTANCEHistone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication, and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone-binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the Δasf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the Δasf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.

ABSTRACT During infections, Staphylococcus aureus is exposed to hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), which are produced by the neutrophil myeloperoxidase as potent antimicrobial killing agents. In this work, we applied RNAseq transcriptomics, Brx-roGFP2 biosensor measurements, and phenotype analyses to investigate the stress responses and defense mechanisms of S. aureus COL toward HOSCN stress. Based on the RNAseq transcriptome profile, HOSCN exerts strong thiol-specific oxidative, electrophile, and metal stress responses as well as protein damage in S. aureus, which is indicated by the strong induction of the HypR, TetR1, PerR, QsrR, MhqR, CstR, CsoR, CzrA, AgrA, HrcA, and CtsR regulons. Phenotype analyses of various mutants in HOSCN-responsive genes revealed that the HOSCN reductase MerA conferred the highest resistance toward HOSCN stress in S. aureus COL, whereas the QsrR and MhqR electrophile stress regulons do not contribute to protection. Brx-roGFP2 biosensor measurements and bacillithiol (BSH)-specific Western blot analyses revealed a strong oxidative shift of the bacillithiol redox potential (E BSH) and increased S-bacillithiolations in S. aureus, indicating that BSH is oxidized to bacillithiol disulfide (BSSB) under HOSCN stress. While the ΔmerA mutant was delayed in recovery of the reduced E BSH, overproduction of MerA in the ΔhypR mutant enabled faster recovery of E BSH due to efficient HOSCN detoxification. Moreover, both MerA and BSH were shown to contribute to HOSCN resistance in growth assays. In summary, HOSCN provokes a thiol-specific oxidative, electrophile, and metal stress response, an oxidative shift in E BSH and increased S-bacillithiolation in S. aureus. IMPORTANCE Staphylococcus aureus colonizes the skin and the airways but can also lead to life-threatening systemic and chronic infections. During colonization and phagocytosis by immune cells, S. aureus encounters the thiol-reactive oxidant HOSCN. The understanding of the adaptation mechanisms of S. aureus toward HOSCN stress is important to identify novel drug targets to combat multi-resistant S. aureus isolates. As a defense mechanism, S. aureus uses the flavin disulfide reductase MerA, which functions as HOSCN reductase and protects against HOSCN stress. Moreover, MerA homologs have conserved functions in HOSCN detoxification in other bacteria, including intestinal and respiratory pathogens. In this work, we studied the comprehensive thiol-reactive mode of action of HOSCN and its effect on the reversible shift of the E BSH to discover new defense mechanisms against the neutrophil oxidant. These findings provide new leads for future drug design to fight the pathogen at the sites of colonization and infections.

Modern biotechnology benefits from the introduction of novel chassis organisms in remedying the limitations of already-established strains. For this, Paracoccus pantotrophus was chosen for in-depth assessment. Its unique broad metabolism and robustness against abiotic stressors make this strain a well-suited chassis candidate. This study set out to comprehensively overview abiotic influences on the growth performance of five P. pantotrophus strains. These data can aid in assessing the suitability of this genus for chassis development by using the type strain as a preliminary model organism. The five P. pantotrophus strains DSM 2944T, DSM 11072, DSM 11073, DSM 11104, and DSM 65 were investigated regarding their growth on various carbon sources and other nutrients. Our data show a high tolerance against osmotic pressure for the type strain with both salts and organic osmolytes. It was further observed that P. pantotrophus prefers organic acids over sugars. All of the tested strains were able to grow on short-chain alkanes, which would make P. pantotrophus a candidate for bioremediation and the upcycling of plastics. In conclusion, we were able to gain insights into several P. pantotrophus strains, which will aid in further introducing this species, or even another species from this genus, as a candidate for future biotechnological processes.

Abstract Background New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. Methods We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. Results Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. Conclusions In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Abstract Background The establishment of the gut microbiota in early life is a critical process that influences the development and fitness of vertebrates. However, the relative influence of transmission from the early social environment and host selection throughout host ontogeny remains understudied, particularly in avian species. We conducted conspecific and heterospecific cross-fostering experiments in zebra finches (Taeniopygia guttata) and Bengalese finches (Lonchura striata domestica) under controlled conditions and repeatedly sampled the faecal microbiota of these birds over the first 3 months of life. We thus documented the development of the gut microbiota and characterised the relative impacts of the early social environment and host selection due to species-specific characteristics and individual genetic backgrounds across ontogeny by using 16S ribosomal RNA gene sequencing. Results The taxonomic composition and community structure of the gut microbiota changed across ontogenetic stages; juvenile zebra finches exhibited higher alpha diversity than adults at the post-breeding stage. Furthermore, in early development, the microbial communities of juveniles raised by conspecific and heterospecific foster parents resembled those of their foster family, emphasising the importance of the social environment. In later stages, the social environment continued to influence the gut microbiota, but host selection increased in importance. Conclusions We provided a baseline description of the developmental succession of gut microbiota in zebra finches and Bengalese finches, which is a necessary first step for understanding the impact of the early gut microbiota on host fitness. Furthermore, for the first time in avian species, we showed that the relative strengths of the two forces that shape the establishment and maintenance of the gut microbiota (i.e. host selection and dispersal from the social environment) change during development, with host selection increasing in importance. This finding should be considered when experimentally manipulating the early-life gut microbiota. Our findings also provide new insights into the mechanisms of host selection. Video Abstract

Abstract Background One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth’s magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe3O4) that under microoxic to anoxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of > 30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear. Results Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the > 4300 genes was significantly enhanced during magnetosome formation. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The mam and mms gene clusters, specifically controlling magnetosome biosynthesis, were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ70 dependent promoters in other Alphaproteobacteria. Conclusions Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource (GEO Series accession number GSE197098).

Abstract It is currently assumed that around 100 million years ago, the common ancestor to the Fabales, Fagales, Rosales and Cucurbitales in Gondwana, developed a root nodule symbiosis with a nitrogen-fixing bacterium. The symbiotic trait evolved first in Frankia cluster-2; thus, strains belonging to this cluster are the best extant representatives of this original symbiont. Most cluster-2 strains could not be cultured to date, except for Frankia coriariae, and therefore many aspects of the symbiosis are still elusive. Based on phylogenetics of cluster-2 metagenome-assembled genomes (MAGs), it has been shown that the genomes of strains originating in Eurasia are highly conserved. These MAGs are more closely related to Frankia cluster-2 in North America than to the single genome available thus far from the southern hemisphere, i.e., from Papua New Guinea. To unravel more biodiversity within Frankia cluster-2 and predict routes of dispersal from Gondwana, we sequenced and analysed the MAGs of Frankia cluster-2 from Coriaria japonica and Coriaria intermedia growing in Japan, Taiwan and the Philippines. Phylogenetic analyses indicate there is a clear split within Frankia cluster-2, separating a continental from an island lineage. Presumably, these lineages already diverged in Gondwana. Based on fossil data on the host plants, we propose that these two lineages dispersed via at least two routes. While the continental lineage reached Eurasia together with their host plants via the Indian subcontinent, the island lineage spread towards Japan with an unknown host plant.

The sigma H (σΗ) and sigma E (σE) subunits of Corynebacterium glutamicum RNA polymerase belong to Group 4 of sigma factors, also called extracytoplasmic function (ECF) sigma factors. Genes of the C. glutamicum σΗ regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the σE regulon, which is involved in cell surface stress response, have not been explored until now. Using the C. glutamicum RES167 strain and its derivative C. glutamicum ΔcseE with a deletion in the anti-σΕ gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum ΔcseE compared to C. glutamicum RES167. To confirm the functional link between σΕ and the corresponding promoters, we tested selected promoters using the in vivo two-plasmid system with gfpuv as a reporter gene and by in vitro transcription. Analyses with RNAP+σΗ and RNAP+σΕ, which were previously shown to recognize similar promoters, proved that the σΗ and σE regulons significantly overlap. The σE-controlled genes were found to be involved for example in protein quality control (dnaK, dnaJ2, clpB, and clpC), the regulation of Clp proteases (clgR), and membrane integrity maintenance. The single-promoter analyses with σΗ and σΕ revealed that there are two groups of promoters: those which are exclusively σΗ-specific, and the other group of promoters, which are σΗ/σE-dependent. No exclusively σE-dependent promoter was detected. We defined the consensus sequences of exclusively σΗ-regulated promotors to be −35 GGAAt and − 10 GTT and σΗ/σE-regulated promoters to be −35 GGAAC and − 10 cGTT. Fifteen genes were found to belong to the σΗ/σΕ regulon. Homology modeling showed that there is a specific interaction between Met170 in σΗ and the nucleotides −31 and − 30 within the non-coding strand (AT or CT) of the σΗ-dependent promoters. In σE, Arg185 was found to interact with the nucleotides GA at the same positions in the σE-dependent promoters.

Chinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and time-intensive long-term stability studies must be performed. Site-specific integration into safe harbors is a strategy to overcome these limitations of conventional cell line design. Recent publications predict safe harbors in CHO cells based on omics data sets or by learning from random integrations, but those predictions remain theory. In this study, we established a CRISPR/Cas9-mediated site-specific integration strategy based on ChIP-seq data to improve stability of recombinant CHO cells. Therefore, a ChIP experiment from the exponential and stationary growth phase of a fed-batch cultivation of CHO-K1 cells yielded 709 potentially stable integration sites. The reporter gene eGFP was integrated into three regions harboring specific modifications by CRISPR/Cas9. Targeted Cas9 nanopore sequencing showed site-specific integration in all 3 cell pools with a specificity between 23 and 73%. Subsequently, the cells with the three different integration sites were compared with the randomly integrated donor vector in terms of transcript level, productivity, gene copy numbers and stability. All site-specific integrations showed an increase in productivity and transcript levels of up to 7.4-fold. In a long-term cultivation over 70 generations, two of the site-specific integrations showed a stable productivity (>70%) independent of selection pressure.

Bacteria of the genus Streptomyces produce various specialized metabolites. Single biosynthetic gene clusters (BGCs) can give rise to different products that can vary in terms of their biological activities. For example, for alnumycin and the shunt product K115, antimicrobial activity was described, while no antimicrobial activity was detected for the shunt product 1,6-dihydro 8-propylanthraquinone. To investigate the antibacterial activity of 1,6-dihydro 8-propylanthraquinone, we produced alnumycin and 1,6-dihydro 8-propylanthraquinone from a Streptomyces isolate containing the alnumycin BGC. The strain was cultivated in liquid glycerol–nitrate–casein medium (GN), and both compounds were isolated using an activity and mass spectrometry-guided purification. The structures were validated via nuclear magnetic resonance (NMR) spectroscopy. A minimal inhibitory concentration (MIC) test revealed that 1,6-dihydro 8-propylanthraquinone exhibits antimicrobial activity against E. coli ΔtolC, B. subtilis, an S. aureus type strain, and a vancomycin intermediate-resistance S. aureus strain (VISA). Activity of 1,6-dihydro 8-propylanthraquinone against E. coli ΔtolC was approximately 10-fold higher than that of alnumycin. We were unable to confirm gyrase inhibition for either compound and believe that the modes of action of both compounds are worth reinvestigating.

Abstract Background Beneficial associations between plants and microbes are widespread in nature and have been studied extensively in the microbial-dominant environment of the rhizosphere. Such associations are highly advantageous for the organisms involved, benefiting soil microbes by providing them access to plant metabolites, while plant growth and development are enhanced through the promotion of nutrient uptake and/or protection against (a)biotic stresses. While the establishment and maintenance of mutualistic associations have been shown to require genetic and epigenetic reprogramming, as well as an exchange of effector molecules between microbes and plants, whether short RNAs are able to effect such changes is currently unknown. Here, we established an interaction between the model grass species Brachypodium distachyon (Bd, Pooideae) and the beneficial fungal root endophyte Serendipita indica (Si, syn. Piriformospora indica, Sebacinales) to elucidate RNA interference-based regulatory changes in gene expression and small (s)RNA profiles that occurred during establishment of a Sebacinalean symbiosis. Results Colonization of Bd roots with Si resulted in higher grain yield, confirming the mutualistic character of this interaction. Resequencing of the Si genome using the Oxford Nanopore technique, followed by de novo assembly yielded in 57 contigs and 9441 predicted genes, including putative members of several families involved in sRNA production. Transcriptome analysis at an early stage of the mutualistic interaction identified 2963 differentially expressed genes (DEG) in Si and 317 in Bd line 21-3. The fungal DEGs were largely associated with carbohydrate metabolism, cell wall degradation, and nutrient uptake, while plant DEGs indicated modulation of (a)biotic stress responses and defense pathways. Additionally, 10% of the upregulated fungal DEGs encode candidate protein effectors, including six DELD proteins typical for Sebacinales. Analysis of the global changes in the sRNA profiles of both associated organisms revealed several putative endogenous plant sRNAs expressed during colonization belonging to known micro (mi)RNA families involved in growth and developmental regulation. Among Bd- and Si-generated sRNAs with putative functions in the interacting organism, we identified transcripts for proteins involved in circadian clock and flowering regulation as well as immunity as potential targets of fungal sRNAs, reflecting the beneficial activity of Si. Conclusions We detected beneficial effects of Si colonization on Bd growth and development, and established a novel plant-mutualist interaction model between these organisms. Together, the changes in gene expression and identification of interaction-induced sRNAs in both organisms support sRNA-based regulation of defense responses and plant development in Bd, as well as nutrient acquisition and cell growth in Si. Our data suggests that a Sebacinalean symbiosis involves reciprocal sRNA targeting of genes during the interaction.

Abstract Background Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. Results Supplemented with l-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L−1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from l-valine catabolism. Unfavorably, l-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post l-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with l-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. Conclusions Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.

The global characterization of transcriptional regulatory networks almost exclusively uses in vivo conditions, thereby providing a snapshot on multiple regulatory interactions at the same time. To complement these approaches, we developed and applied a method for characterizing bacterial promoters genome-wide by in vitro transcription coupled to transcriptome sequencing specific for native 5′-ends of transcripts. This method, called ROSE (run-off transcription/RNA-sequencing), only requires chromosomal DNA, ribonucleotides, RNA polymerase (RNAP) core enzyme, and a specific sigma factor, recognizing the corresponding promoters, which have to be analyzed. ROSE was performed on E. coli K-12 MG1655 genomic DNA using Escherichia coli RNAP holoenzyme (including σ70) and yielded 3226 transcription start sites, 2167 of which were also identified in in vivo studies, and 598 were new. Many new promoters not yet identified by in vivo experiments might be repressed under the tested conditions. Complementary in vivo experiments with E. coli K-12 strain BW25113 and isogenic transcription factor gene knockout mutants of fis, fur, and hns were used to test this hypothesis. Comparative transcriptome analysis demonstrated that ROSE could identify bona fide promoters that were apparently repressed in vivo. In this sense, ROSE is well-suited as a bottom-up approach for characterizing transcriptional networks in bacteria and ideally complementary to top-down in vivo transcriptome studies.

The pseudo-tetrasaccharide acarbose, produced by Actinoplanes sp. SE50/110, is a α-glucosidase inhibitor used for treatment of type 2 diabetes patients. In industrial production of acarbose, by-products play a relevant role that complicates the purification of the product and reduce yields. Here, we report that the acarbose 4-α-glucanotransferase AcbQ modifies acarbose and the phosphorylated version acarbose 7-phosphate. Elongated acarviosyl metabolites (α-acarviosyl-(1,4)-maltooligosaccharides) with one to four additional glucose molecules were identified performing in vitro assays with acarbose or acarbose 7-phosphate and short α-1,4-glucans (maltose, maltotriose and maltotetraose). High functional similarities to the 4-α-glucanotransferase MalQ, which is essential in the maltodextrin pathway, are revealed. However, maltotriose is a preferred donor and acarbose and acarbose 7-phosphate, respectively, serve as specific acceptors for AcbQ. This study displays the specific intracellular assembly of longer acarviosyl metabolites catalyzed by AcbQ, indicating that AcbQ is directly involved in the formation of acarbose by-products of Actinoplanes sp. SE50/110.

This study was performed to comparably assess two commercial real-time PCR assays for the identification of Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma rangeli were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for T. cruzi and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both T. cruzi and T. rangeli without further discrimination. To discriminate between T. cruzi- and T. rangeli-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94) T. cruzi-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite T. rangeli. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with T. rangeli in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of T. cruzi was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic T. rangeli according to the RealStar assay may be a disadvantage in areas of co-circulation with T. cruzi, while the test performance of the two compared assays will be quite similar in geographic settings where T. rangeli infections are unlikely.

Abstract Background Because of its tractability and straightforward cultivation, the magnetic bacterium Magnetospirillum gryphiswaldense has emerged as a model for the analysis of magnetosome biosynthesis and bioproduction. However, its future use as platform for synthetic biology and biotechnology will require methods for large-scale genome editing and streamlining. Results We established an approach for combinatory genome reduction and generated a library of strains in which up to 16 regions including large gene clusters, mobile genetic elements and phage-related genes were sequentially removed, equivalent to ~ 227.6 kb and nearly 5.5% of the genome. Finally, the fragmented genomic magnetosome island was replaced by a compact cassette comprising all key magnetosome biosynthetic gene clusters. The prospective 'chassis' revealed wild type-like cell growth and magnetosome biosynthesis under optimal conditions, as well as slightly improved resilience and increased genetic stability. Conclusion We provide first proof-of-principle for the feasibility of multiple genome reduction and large-scale engineering of magnetotactic bacteria. The library of deletions will be valuable for turning M. gryphiswaldense into a microbial cell factory for synthetic biology and production of magnetic nanoparticles.

Abstract Background Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the ‘magnetosome island’ (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain. Results Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content. Conclusions Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.

Abstract Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

Abstract Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host`s development, growth and health. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described at the phylogenetic level but rarely functionally. Multifaceted analysis of the microbial consortium of the ancient phytoplankton Botryococcus as an attractive model food web revealed that its all abundant bacterial members belong to a niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies seem adversely to affect the algal cell morphology. Synthetic rearrangement of a minimal community consisting of an alga, a mutualistic and a parasitic bacteria underpins the model of a eukaryote that maintains its own mutualistic microbial community to control its surrounding biosphere. This model of coexistence, potentially useful for defense against invaders by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction is an opportunity to unravel the relationships within the consortia and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.

Abstract The isolation and sequencing of new strains of Pseudomonas aeruginosa created an extensive dataset of closed genomes. Many of the publicly available genomes are only used in their original publication while additional in silico information, based on comparison to previously published genomes, is not being explored. In this study, we defined and investigated the genome of the environmental isolate P. aeruginosa KRP1 and compared it to more than 100 publicly available closed P. aeruginosa genomes. By using different genomic island prediction programs, we could identify a total of 17 genomic islands and 8 genomic islets, marking the majority of the accessory genome that covers ~ 12% of the total genome. Based on intra-strain comparisons, we are able to predict the pathogenic potential of this environmental isolate. It shares a substantial amount of genomic information with the highly virulent PSE9 and LESB58 strains. For both of these, the increased virulence has been directly linked to their accessory genome before. Hence, the integrated use of previously published data can help to minimize expensive and time consuming wetlab work to determine the pathogenetic potential.

Abstract Background So far, large numbers of studies investigating the microbiome have focused on gut microbiota and less have addressed the microbiome of the skin. Especially in avian taxa our understanding of the ecology and function of these bacteria remains incomplete. The involvement of skin bacteria in intra-specific communication has recently received attention, and has highlighted the need to understand what information is potentially being encoded in bacterial communities. Using next generation sequencing techniques, we characterised the skin microbiome of wild zebra finches, aiming to understand the impact of sex, age and group composition on skin bacteria communities. For this purpose, we sampled skin swabs from both sexes and two age classes (adults and nestlings) of 12 different zebra finch families and analysed the bacterial communities. Results Using 16S rRNA sequencing we found no effect of age, sex and family on bacterial diversity (alpha diversity). However, when comparing the composition (beta diversity), we found that animals of social groups (families) harbour highly similar bacterial communities on their skin with respect to community composition. Within families, closely related individuals shared significantly more bacterial taxa than non-related animals. In addition, we found that age (adults vs. nestlings) affected bacterial composition. Finally, we found that spatial proximity of nest sites, and therefore individuals, correlated with the skin microbiota similarity. Conclusions Birds harbour very diverse and complex bacterial assemblages on their skin. These bacterial communities are distinguishable and characteristic for intraspecific social groups. Our findings are indicative for a family-specific skin microbiome in wild zebra finches. Genetics and the (social) environment seem to be the influential factors shaping the complex bacterial communities. Bacterial communities associated with the skin have a potential to emit volatiles and therefore these communities may play a role in intraspecific social communication, e.g. via signalling social group membership.

Summary The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome‐reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High‐throughput phenotyping using robotics‐integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomass‐specific cutinase yield for strain GRS41_51_NprE was increased by ~ 200%, although the growth rate was reduced by ~ 60%. Importantly, the causative deletions of genomic clusters cg2801‐cg2828 and rrnC‐cg3298 could not have been inferred a priori. Strikingly, bioreactor fed‐batch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by ~ 25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a ‘high‐performance’ strain from batch screening into a ‘low‐performance’ strain in fed‐batch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C. glutamicum to both genomic deletions and different bioprocess conditions.

Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85°C, pH 6–7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using α- or β-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming α-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous β-glycosidase (EGIDFPOO_00532) with β-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles.

Mobile genetic elements (MGEs) contribute to instability of the host genome and plasmids. Previously, removal of the prophages in the industrial amino acid producer Corynebacterium glutamicum ATCC 13 032 resulted in strain MB001 which showed better survival under stress conditions and increased transformability. Still, eight families of Insertion Sequence (IS) elements with 27 potentially active members remain in MB001, two of which were demonstrated to be detrimental in biotechnological processes. In this study, systematical deletion of all complete IS elements in MB001 resulted in the MGE-free strain CR101. CR101 shows growth characteristics identical to the wildtype and the increased transformability of MB001. Due to its improved genome stability, we consider this strain to be an optimal host for basic research and biotechnology. As a “zero-background” host, it is also an ideal basis to study C. glutamicum IS elements. Re-sequencing of CR101 revealed that only five spontaneous point mutations had occurred during the construction process, highlighting the low mutation rate of C. glutamicum on the nucleotide level. In a second step, we developed an easily applicable ISCg1-based transposon mutagenesis system to randomly transpose a selectable marker. For optimal plasmid stability during cloning in Escherichia coli, the system utilizes a genetic switch based on the phage integrase Bxb1. Use of this integrase revealed the presence of a functional attB site in the C. glutamicum genome. To avoid cross-talk with our system and increase ease-of-use, we removed the attB site and also inserted the Bxb1 encoding gene into the chromosome of CR101. Successful insertion of single markers was verified by sequencing randomly selected mutants. Sequencing pooled mutant libraries revealed only a weak target site specificity, seemingly random distribution of insertion sites and no general strand bias. The resulting strain, ML103, together with plasmid pML10 provides a easily customizable system for random mutagenesis in an otherwise genomically stable C. glutamicum. Taken together, the MGE-free C. glutamicum strain CR101, the derivative ML103, and the plasmid pML10 provide a useful set of tools to study C. glutamicum in the future.

Bacterial symbionts of marine invertebrates are rich sources of novel, pharmaceutically relevant natural products that could become leads in combatting multidrug-resistant pathogens and treating disease. In this study, the bioactive potential of the marine invertebrate symbiont Thalassomonas actiniarum was investigated. Bioactivity screening of the strain revealed Gram-positive specific antibacterial activity as well as cytotoxic activity against a human melanoma cell line (A2058). The dereplication of the active fraction using HPLC-MS led to the isolation and structural elucidation of cholic acid and 3-oxo cholic acid. T. actiniarum is one of three type species belonging to the genus Thalassomonas. The ability to generate cholic acid was assessed for all three species using thin-layer chromatography and was confirmed by LC-MS. The re-sequencing of all three Thalassomonas type species using long-read Oxford Nanopore Technology (ONT) and Illumina data produced complete genomes, enabling the bioinformatic assessment of the ability of the strains to produce cholic acid. Although a complete biosynthetic pathway for cholic acid synthesis in this genus could not be determined based on sequence-based homology searches, the identification of putative penicillin or homoserine lactone acylases in all three species suggests a mechanism for the hydrolysis of conjugated bile acids present in the growth medium, resulting in the generation of cholic acid and 3-oxo cholic acid. With little known currently about the bioactivities of this genus, this study serves as the foundation for future investigations into their bioactive potential as well as the potential ecological role of bile acid transformation, sterol modification and quorum quenching by Thalassomonas sp. in the marine environment.

ABSTRACT Magnetosomes are complex membrane organelles synthesized by magnetotactic bacteria (MTB) for navigation in the Earth’s magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense, all steps of magnetosome formation are tightly controlled by >30 specific genes arranged in several gene clusters. However, the transcriptional organization of the magnetosome gene clusters has remained poorly understood. Here, by applying Cappable-seq and whole-transcriptome shotgun RNA sequencing, we show that mamGFDCop and feoAB1op are transcribed as single transcriptional units, whereas multiple transcription start sites (TSS) are present in mms6op, mamXYop, and the long (>16 kb) mamABop. Using a bioluminescence reporter assay and promoter knockouts, we demonstrate that most of the identified TSS originate from biologically meaningful promoters which mediate production of multiple transcripts and are functionally relevant for proper magnetosome biosynthesis. In addition, we identified a strong promoter in a large intergenic region within mamXYop, which likely drives transcription of a noncoding RNA important for gene expression in this operon. In summary, our data suggest a more complex transcriptional architecture of the magnetosome operons than previously recognized, which is largely conserved in other magnetotactic Magnetospirillum species and, thus, is likely fundamental for magnetosome biosynthesis in these organisms. IMPORTANCE Magnetosomes have emerged as a model system to study prokaryotic organelles and a source of biocompatible magnetic nanoparticles for various biomedical applications. However, the lack of knowledge about the transcriptional organization of magnetosome gene clusters has severely impeded the engineering, manipulation, and transfer of this highly complex biosynthetic pathway into other organisms. Here, we provide a high-resolution image of the previously unappreciated transcriptional landscape of the magnetosome operons. Our findings are important for further unraveling the complex genetic framework of magnetosome biosynthesis. In addition, they will facilitate the rational reengineering of magnetic bacteria for improved bioproduction of tunable magnetic nanoparticles, as well as transplantation of magnetosome biosynthesis into foreign hosts by synthetic biology approaches. Overall, our study exemplifies how a genetically complex pathway is orchestrated at the transcriptional level to ensure the balanced expression of the numerous constituents required for the proper assembly of one of the most intricate prokaryotic organelles.

This study represents one of the largest comparisons of biofilms from environmentally sampled plastic and nonplastic particles from aquatic environments. By including particles sampled through three separate campaigns in the Baltic, Sargasso, and Mediterranean seas, we were able to make cross-geographical comparisons and discovered common taxonomical signatures that define the plastic biofilm.

Abstract Background Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC. Results The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5′-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC–MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. Conclusion Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives.

Abstract Background 3-Hydroxypropionic acid (3-HP) is a promising platform chemical with various industrial applications. Several metabolic routes to produce 3-HP from organic substrates such as sugars or glycerol have been implemented in yeast, enterobacterial species and other microorganisms. In this study, the native 3-HP metabolism of Cupriavidus necator was investigated and manipulated as it represents a promising chassis for the production of 3-HP and other fatty acid derivatives from CO2 and H2. Results When testing C. necator for its tolerance towards 3-HP, it was noted that it could utilise the compound as the sole source of carbon and energy, a highly undesirable trait in the context of biological 3-HP production which required elimination. Inactivation of the methylcitrate pathway needed for propionate utilisation did not affect the organism’s ability to grow on 3-HP. Putative genes involved in 3-HP degradation were identified by bioinformatics means and confirmed by transcriptomic analyses, the latter revealing considerably increased expression in the presence of 3-HP. Genes identified in this manner encoded three putative (methyl)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) and two putative dehydrogenases (hpdH and hbdH). These genes, which are part of three separate mmsA operons, were inactivated through deletion of the entire coding region, either singly or in various combinations, to engineer strains unable to grow on 3-HP. Whilst inactivation of single genes or double deletions could only delay but not abolish growth, a triple ∆mmsA1∆mmsA2∆mmsA3 knock-out strain was unable utilise 3-HP as the sole source of carbon and energy. Under the used conditions this strain was also unable to co–metabolise 3-HP alongside other carbon and energy sources such as fructose and CO2/H2. Further analysis suggested primary roles for the different mmsA operons in the utilisation of β-alanine generating substrates (mmsA1), degradation of 3-HP (mmsA2), and breakdown of valine (mmsA3). Conclusions Three different (methyl)malonate semialdehyde dehydrogenases contribute to 3-HP breakdown in C. necator H16. The created triple ∆mmsA1∆mmsA2∆mmsA3 knock-out strain represents an ideal chassis for autotrophic 3-HP production.

Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress. Key words: Corynebacterium glutamicum, Mycothiol, Mycothiol redox potential, Mrx1-roGFP2

The gilled mushroom Clitopilus passeckerianus (Entolomataceae, Agaricales, Basidiomycota) is well known to produce the terpenoid pleuromutilin, which is the biotechnological basis for medically important antibiotics such as lefamulin and retapamulin. Their unique mode of action and good tolerance entails an increasing demand of pleuromutilin-derived antibiotics in veterinary and human health care. Surprisingly, despite their pharmaceutical importance, no genome sequence is available of any pleuromutilin-producing fungus. Here, we present the high-quality draft genome sequence of the pleuromutilin-producer C. passeckerianus DSM1602 including functional genome annotation. More precisely, we employed a hybrid assembly strategy combining Illumina sequencing and Nanopore sequencing to assemble the mitochondrial genome as well as the nuclear genome. In accordance with the dikaryotic state of the fungus, the nuclear genome has a diploid character. Interestingly, the mitochondrial genome appears duplicated. Bioinformatic analysis revealed a versatile secondary metabolism with an emphasis on terpenoid biosynthetic enzymes in C. passeckerianus and also in related strains. Two alleles of biosynthetic gene clusters for pleuromutilin were found in the genome of C. passeckerianus. The pleuromutilin genes were reassembled with yeast-specific elements for heterologous expression in Saccharomyces cerevisiae. Our work lays the foundation for metabolic strain engineering towards higher yields of the valuable compound pleuromutilin.

Abstract Background The Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins—which favors correct folding and facilitates downstream processing—as well as its acceptance of methylated DNA and its low endogeneous protease activity. However, current inconsistencies in protein yields urge for a deeper understanding of the burden of heterologous protein production on the cell. In the current study, transcriptomics and $$^{13}\hbox {C}$$ 13C -based fluxomics were exploited to uncover gene expression and metabolic flux changes associated with heterologous protein production. The Rhodothermus marinus thermostable cellulase A (CelA)—previously shown to be successfully overexpressed in S. lividans—was taken as an example protein. Results RNA-seq and $$^{13}\hbox {C}$$ 13C -based metabolic flux analysis were performed on a CelA-producing and an empty-plasmid strain under the same conditions. Differential gene expression, followed by cluster analysis based on co-expression and co-localization, identified transcriptomic responses related to secretion-induced stress and DNA damage. Furthermore, the OsdR regulon (previously associated with hypoxia, oxidative stress, intercellular signaling, and morphological development) was consistently upregulated in the CelA-producing strain and exhibited co-expression with isoenzymes from the pentose phosphate pathway linked to secondary metabolism. Increased expression of these isoenzymes matches to increased fluxes in the pentose phosphate pathway. Additionally, flux maps of the central carbon metabolism show increased flux through the tricarboxylic acid cycle in the CelA-producing strain. Redirection of fluxes in the CelA-producing strain leads to higher production of NADPH, which can only partly be attributed to increased secretion. Conclusions Transcriptomic and fluxomic changes uncover potential new leads for targeted strain improvement strategies which may ease the secretion stress and metabolic burden associated with heterologous protein synthesis and secretion, and may help create a more consistently performing S. lividans strain. Yet, links to secondary metabolism and redox balancing should be further investigated to fully understand the S. lividans metabolome under heterologous protein production.

Streptomyces lividans TK24 is a relevant Gram-positive soil inhabiting bacterium and one of the model organisms of the genus Streptomyces. It is known for its potential to produce secondary metabolites, antibiotics, and other industrially relevant products. S. lividans TK24 is the plasmid-free derivative of S. lividans 66 and a close genetic relative of the strain Streptomyces coelicolor A3(2). In this study, we used transcriptome and proteome data to improve the annotation of the S. lividans TK24 genome. The RNA-seq data of primary 5′-ends of transcripts were used to determine transcription start sites (TSS) in the genome. We identified 5,424 TSS, of which 4,664 were assigned to annotated CDS and ncRNAs, 687 to antisense transcripts distributed between 606 CDS and their UTRs, 67 to tRNAs, and 108 to novel transcripts and CDS. Using the TSS data, the promoter regions and their motifs were analyzed in detail, revealing a conserved -10 (TAnnnT) and a weakly conserved -35 region (nTGACn). The analysis of the 5′ untranslated region (UTRs) of S. lividans TK24 revealed 17% leaderless transcripts. Several cis-regulatory elements, like riboswitches or attenuator structures could be detected in the 5′-UTRs. The S. lividans TK24 transcriptome contains at least 929 operons. The genome harbors 27 secondary metabolite gene clusters of which 26 could be shown to be transcribed under at least one of the applied conditions. Comparison of the reannotated genome with that of the strain Streptomyces coelicolor A3(2) revealed a high degree of similarity. This study presents an extensive reannotation of the S. lividans TK24 genome based on transcriptome and proteome analyses. The analysis of TSS data revealed insights into the promoter structure, 5′-UTRs, cis-regulatory elements, attenuator structures and novel transcripts, like small RNAs. Finally, the repertoire of secondary metabolite gene clusters was examined. These data provide a basis for future studies regarding gene characterization, transcriptional regulatory networks, and usage as a secondary metabolite producing strain.

Microbial communities residing in the gastrointestinal tracts of animals have profound impacts on the physiological processes of their hosts. In humans, host-specific and environmental factors likely interact together to shape gut microbial communities, resulting in remarkable inter-individual differences. However, we still lack a full understanding of to what extent microbes are individual-specific and controlled by host-specific factors across different animal taxa. Here, we document the gut microbial characteristics in two estrildid finch species, the Bengalese finch (Lonchura striata domestica) and the zebra finch (Taeniopygia guttata) to investigate between-species and within-species differences. We collected fecal samples from breeding pairs that were housed under strictly controlled environmental and dietary conditions. All individuals were sampled at five different time points over a range of 120 days covering different stages of the reproductive cycle. We found significant species-specific differences in gut microbial assemblages. Over a period of 3 months, individuals exhibited unique, individual-specific microbial profiles. Although we found a strong individual signature in both sexes, within-individual variation in microbial communities was larger in males of both species. Furthermore, breeding pairs had more similar microbial profiles, compared to randomly chosen males and females. Our study conclusively shows that host-specific factors contribute structuring of gut microbiota.

Present in many industrial effluents and as common degradation product of organic matter, phenol is a widespread compound which may cause serious environmental problems, due to its toxicity to animals and humans. Degradation of phenol from the environment by mesophilic bacteria has been studied extensively over the past decades, but only little is known about phenol biodegradation at high temperatures or low pH. In this work we studied phenol degradation in the thermoacidophilic archaeon Saccharolobus solfataricus P2 (basonym: Sulfolobus solfataricus) under extreme conditions (80°C, pH 3.5). We combined metabolomics and transcriptomics together with metabolic modeling to elucidate the organism’s response to growth with phenol as sole carbon source. Although S. solfataricus is able to utilize phenol for biomass production, the carbon source induces profound stress reactions, including genome rearrangement as well as a strong intracellular accumulation of polyamines. Furthermore, computational modeling revealed a 40% higher oxygen demand for substrate oxidation, compared to growth on glucose. However, only 16.5% of oxygen is used for oxidation of phenol to catechol, resulting in a less efficient integration of carbon into the biomass. Finally, our data underlines the importance of the phenol meta-degradation pathway in S. solfataricus and enables us to predict enzyme candidates involved in the degradation processes downstream of 2-hydroxymucconic acid.

Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.

Summary: Heterologous expression of a biosynthesis gene cluster from Amycolatopsis sp. resulted in the discovery of two unique class IV lasso peptides, felipeptins A1 and A2. A mixture of felipeptins stimulated proliferation of cancer cells, while having no such effect on the normal cells. Detailed investigation revealed, that pre-treatment of cancer cells with a mixture of felipeptins resulted in downregulation of the tumor suppressor Rb, making the cancer cells to proliferate faster. Pre-treatment with felipeptins made cancer cells considerably more sensitive to the anticancer agent doxorubicin and re-sensitized doxorubicin-resistant cells to this drug. Structural characterization and binding experiments showed an interaction between felipeptins resulting in complex formation, which explains their synergistic effect. This discovery may open an alternative avenue in cancer treatment, helping to eliminate quiescent cells that often lead to cancer relapse.

The family of Ustilaginaceae belongs to the order of Basidiomycetes. Despite their plant pathogenicity causing, e.g., corn smut disease, they are also known as natural producers of value-added chemicals such as extracellular glycolipids, organic acids, and polyols. Here, we present 17 high-quality draft genome sequences (N50 > 1 Mb) combining third-generation nanopore and second-generation Illumina sequencing. The data were analyzed with taxonomical genome-based bioinformatics methods such as Percentage of Conserved Proteins (POCP), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) analyses indicating that a reclassification of the Ustilaginaceae family might be required. Further, conserved core genes were determined to calculate a phylogenomic core genome tree of the Ustilaginaceae that also supported the results of the other phylogenomic analysis. In addition, to genomic comparisons, secondary metabolite clusters (e.g., itaconic acid, mannosylerythritol lipids, and ustilagic acid) of biotechnological interest were analyzed, whereas the sheer number of clusters did not differ much between species.

Increasing the growth rate of the industrial host Corynebacterium glutamicum is a promising target to rise productivities of growth coupled product formation. As a prerequisite, detailed knowledge about the tight regulation network is necessary for identifying promising metabolic engineering goals. Here, we present comprehensive metabolic and transcriptional analysis of C. glutamicum ATCC 13032 growing under glucose limited chemostat conditions with μ = 0.2, 0.3, and 0.4 h–1. Intermediates of central metabolism mostly showed rising pool sizes with increasing growth. 13C-metabolic flux analysis (13C-MFA) underlined the fundamental role of central metabolism for the supply of precursors, redox, and energy equivalents. Global, growth-associated, concerted transcriptional patterns were not detected giving rise to the conclusion that glycolysis, pentose-phosphate pathway, and citric acid cycle are predominately metabolically controlled under glucose-limiting chemostat conditions. However, evidence is found that transcriptional regulation takes control over glycolysis once glucose-rich growth conditions are installed.

Species of the genus Shewanella are widespread in nature in various habitats, however, little is known about phages affecting Shewanella sp. Here, we report the isolation of phages from diverse freshwater environments that infect and lyse strains of Shewanella oneidensis and other Shewanella sp. Sequence analysis and microscopic imaging strongly indicate that these phages form a so far unclassified genus, now named Shewanella phage Thanatos, which can be positioned within the subfamily of Tevenvirinae (Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Caudovirales; Myoviridae; Tevenvirinae). We characterized one member of this group in more detail using S. oneidensis MR-1 as a host. Shewanella phage Thanatos-1 possesses a prolate icosahedral capsule of about 110 nm in height and 70 nm in width and a tail of about 95 nm in length. The dsDNA genome exhibits a GC content of about 34.5%, has a size of 160.6 kbp and encodes about 206 proteins (92 with an annotated putative function) and two tRNAs. Out of those 206, MS analyses identified about 155 phage proteins in PEG-precipitated samples of infected cells. Phage attachment likely requires the outer lipopolysaccharide of S. oneidensis, narrowing the phage’s host range. Under the applied conditions, about 20 novel phage particles per cell were produced after a latent period of approximately 40 min, which are stable at a pH range from 4 to 12 and resist temperatures up to 55°C for at least 24 h. Addition of Thanatos to S. oneidensis results in partial dissolution of established biofilms, however, early exposure of planktonic cells to Thanatos significantly enhances biofilm formation. Taken together, we identified a novel genus of Myophages affecting S. oneidensis communities in different ways.

Actinomycete bacteria from marine environments represent a potential source for new antibiotics and anti-tumor drugs. Ten strains belonging to the genus Streptomyces isolated from the marine sponge Antho dichotoma collected at the bottom of the Trondheim fjord (Norway) were screened for antibiotic activity. Since only few isolates proved to be bioactive in the conditions tested, we decided to gain an insight into their biosynthetic potential using genome sequencing and analysis. Draft genomes were analyzed for the presence of secondary metabolite biosynthesis gene clusters (BGCs) using antiSMASH software. BGCs specifying both known and potentially novel secondary metabolites were identified, suggesting that these isolates might be sources for new bioactive compounds. The results of this analysis also implied horizontal transfer of several gene clusters between the studied isolates, which was especially evident for the lantibiotic- and thiopeptide-encoding BGCs. The latter implies the significance of particular secondary metabolites for the adaptation of Streptomyces to the spatially enclosed marine environments such as marine sponges. Two bioactive isolates, one showing activity against both yeast and Bacillus subtilis, and one only against yeast were analyzed in details, leading to the identification of cycloheximide, linearmycins, and echinomycins that are presumably responsible for the observed bioactivities.