The bacterial cell wall is a nearly universal structure that offers protection and gives the cell its shape. However, environmental stressors, such as cell wall-targeting antibiotics and... Show moreThe bacterial cell wall is a nearly universal structure that offers protection and gives the cell its shape. However, environmental stressors, such as cell wall-targeting antibiotics and hyperosmotic conditions, can induce bacteria to shift to a wall-deficient state. It is unknown whether the lack of this cell wall ‘barrier’ can enable DNA exchange via horizontal gene transfer (HGT), a process that facilitates the spread of antibiotic resistance amongst bacteria. The research in this thesis confirms that wall-deficiency enables HGT in the actinobacterium Kitasatospora viridifaciens. Specifically, we show that wall-deficient L-forms take up extracellular material including DNA, sugar polymers and lipid nanoparticles via an endocytosis-like mechanism, which in case of DNA uptake leads to genetic transformation. Moreover, wall-less cells exchange genomic DNA in a DNAse-resistant manner, most likely via cell-cell fusion and/or production of extracellular vesicles. We also isolated vesicle-producing actinobacteria from a wastewater treatment plant, an environment that is considered a hotspot for the spread of antibiotic resistance genes. Lastly, growth of K. viridifaciens under hyperosmotic stress conditions leads to genetic instability, which together with HGT can contribute to enhanced genome plasticity. In summary, this thesis provides important insights into the consequences of bacterial cell wall-deficiency for HGT and evolution. Show less
Streptomycetes are soil-dwelling Gram-positive bacteria and can produce clinical drugs and adapt to environmental fluctuations through diverse morphological changes. The morphological development... Show moreStreptomycetes are soil-dwelling Gram-positive bacteria and can produce clinical drugs and adapt to environmental fluctuations through diverse morphological changes. The morphological development of streptomycetes features apical growth, which is determined by a tip-organizing center containing machinery for the synthesis of a cellulose-like glycan deposited at the cell surface. This thesis demonstrated that the cellulose-like glycan likely cellulose and the related synthesis machinery are unique in comparison with other Gram-negative cellulose synthesis systems. It showed that the synthesis of this glycan is performed by the glycosyltransferase ClsA and matured galactose-oxidase protein GlxA. Subsequently, the lytic polysaccharides monooxygenase LpmP and the glucanase ClsZ, adjacent to CslA/GlxA, cooperatively cleave peptidoglycan and create a passage to assist the exposure of this glycan on cell surface. In addition, this thesis also revealed coordination of polar growth determinants is performed by a stomatin-like protein StlP, which locates in the same gene cluster with CslA/GlxA/CslZ/LpmP. Oligomerization of StlP forms a fluid microdomain at hyphal tips, which spatially confines the whole tip organizing center. Furthermore, this thesis further showed that the cellulose-like glycan of Streptomyces coelicolor is responsible for the aggregation of clumps, which is distinct from Streptomyces lividans where this glycan mediates germlings’ aggregation. Show less
Abstract Division of labor can evolve when social groups benefit from the functional specialization of its members. Recently, a novel means of coordinating the division of labor was found in the... Show moreAbstract Division of labor can evolve when social groups benefit from the functional specialization of its members. Recently, a novel means of coordinating the division of labor was found in the antibiotic-producing bacterium Streptomyces coelicolor, where specialized cells are generated through large-scale genomic re-organization. We investigate how the evolution of a genome architecture enables such mutation-driven division of labor, using a multiscale computational model of bacterial evolution. In this model, bacterial behavior—antibiotic production or replication—is determined by the structure and composition of their genome, which encodes antibiotics, growth-promoting genes, and fragile genomic loci that can induce chromosomal deletions. We find that a genomic organization evolves, which partitions growth-promoting genes and antibiotic-coding genes into distinct parts of the genome, separated by fragile genomic loci. Mutations caused by these fragile sites mostly delete growth-promoting genes, generating sterile, and antibiotic-producing mutants from weakly-producing progenitors, in agreement with experimental observations. This division of labor enhances the competition between colonies by promoting antibiotic diversity. These results show that genomic organization can co-evolve with genomic instabilities to enable reproductive division of labor. Show less
Streptomyces bacteria are a valuable source of natural products, many of which are used in the clinic or in biotechnology. In our search for novel antibiotics we discovered lugdunomycin, a natural... Show moreStreptomyces bacteria are a valuable source of natural products, many of which are used in the clinic or in biotechnology. In our search for novel antibiotics we discovered lugdunomycin, a natural product with a highly complex chemical architecture that is produced by Streptomyces sp. QL37. It is derived from the angucyclines, a well-known class of molecules known for their antibacterial and anticancer activities. Though angucyclines are produced in high quantities under most conditions, lugdunomycin is produced in minimal amounts. This thesis describes novel insights into the transcriptional control of the lugdunomycin biosynthetic gene cluster and into the lugdunomycin biosynthesis pathway. These insights may be applied to improve the yield of lugdunomycin and expand the chemical diversity of angucyclines. Using molecular biology, bioinformatic approaches and omics studies, such as metabolomics and transcriptomics, we have characterized the lugdunomycin biosynthetic gene cluster, the regulatory genes (lugRI–lugRV) required for transcriptional activation of the cluster, and the oxygenase genes (lugOI–lugOV) that play a key role in the different chemical rearrangements of the angucyclines. Furthermore, this thesis contains a detailed review of the regulatory network that controls antibiotic production in Actinobacteria. Show less
Filamentous Actinobacteria, such as Streptomyces, produce a plethora of chemically diverse bioactive metabolites that have found applications across medicine, agriculture and biotechnology. Yet,... Show moreFilamentous Actinobacteria, such as Streptomyces, produce a plethora of chemically diverse bioactive metabolites that have found applications across medicine, agriculture and biotechnology. Yet, the vast majority of the biosynthetic potential of Actinobacteria remains uncharacterised, largely because their biosynthetic gene clusters (BGCs) are poorly expressed in the laboratory, preventing the discovery of the cognate natural products. Additionally, only a narrow band of environments and a few taxonomic groups have been explored for gifted Actinobacteria. In this thesis different approaches are described, wherein we combined drug discovery with ecology, aimed at accessing the full potential of Actinobacteria. Bioactive Actinobacteria were isolated from a faecal sample of a 28,000-year-old-mammoth and their taxonomic and metabolic diversity was analysed. Furthermore, the effect of human stress hormones on the production of antibiotics by Streptomyces was investigated, resulting in the discovery of adrenaline as elicitor of siderophore production. This was later shown to be caused by the adrenaline analog catechol, which is ubiquitous in nature. Catechol also elicited the production of angucycline glycosides, well known for their therapeutic potential as anticancer and antibiotic compounds. Lastly, zebrafish were used as an in vivo model to explore the bioactive and functional potential of Actinobacteria within the animal microbiome. Show less
The ongoing increase in antimicrobial resistance combined with the low discovery of novel antibiotics is a serious threat to our health care. Genome mining has given new potential to the field of... Show moreThe ongoing increase in antimicrobial resistance combined with the low discovery of novel antibiotics is a serious threat to our health care. Genome mining has given new potential to the field of natural product discovery, as thousands of biosynthetic gene clusters (BGCs) are discovered for which the natural product is not known.Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a highly diverse class of natural products. The large number of different modifications that can be applied to a RiPP results in a large variety of chemical structures, but also stems from a large genetic variety in BGCs. As a result, no single method can effectively mine for all RiPP BGCs, making it an interesting source for new molecules.In this thesis, new methods are explored to mine genomes for the BGCs of novel RiPP variants, with a focus on discovering RiPPs that have new modifications. RRE-Finder is a new tool for the detection of RiPP Recognition Elements, domains that are often found in RiPP BGCs. DecRiPPter is another tool that employs machine learning models to discover new RiPP precursor genes encoded in the genomes. Both tools can be used to prioritize novel RiPP BGCs. Two candidate BGCs are characterized, one of which could be shown to specify a new RiPP, validating the approach. Show less
Actinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High... Show moreActinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High throughput screening has failed to deliver the new antibiotics we so desperately need to combat multidrug-resistant pathogens. Therefore, new systematic approaches are needed to further explore the rich potential of Actinobacteria. The work described in this thesis entails systems biology approaches consisting of technologies such as proteomics, genomics, metabolomics and DNA binding studies. These were then applied to identify the biosynthetic gene clusters (BGCs) that are responsible for the production of novel antibiotics. Small molecules were thereby used as elicitors to activate the expression of cryptic BGCs in Streptomyces roseifaciens. Furthermore, S. coelicolor M1152 that was optimized for heterologous expression of antibiotics, was analysed for changes in protein expression, to understand which changes correlate to optimal antibiotic production. Finally, the role of the nucleoid associated protein SCO1839 in development and antibiotic production was studied. Chip-seq technology showed that it binds to thousands of DNA sequences on the S. coelicolor chromosome, which contain the motif GATC. I hope that this thesis contributes to utilizing multi-dimensional ‘omics approaches to answer major biological questions. Show less
Streptomyces present a valuable platform for natural product discovery. Lugdunomycin is a novel angucycline-derived polyketide from Streptomyces sp QL37, with unprecedented skeleton and... Show moreStreptomyces present a valuable platform for natural product discovery. Lugdunomycin is a novel angucycline-derived polyketide from Streptomyces sp QL37, with unprecedented skeleton and antimicrobial activity. This dissertation covers the research on the biosynthesis of this novel antibiotic and the developmental biology of Streptomyces. The data in this thesis have provided important new insights into the puzzle of lugdunomycin biosynthesis and the sporulation-specific cell division of Streptomyces. By means of molecular biology, structural biology, biochemical, chemical and bioinformatics approaches, we have uncovered the potential functions of the key enzymes, especially those encoded oxygenases (LugOI-LugOV) in lugdunomycin biosynthesis. Furthermore, we extensively studied the role of SsgB in Streptomyces development, that led us to the discovery of longitudinal cell division that support the predominant role of SsgB in the accurate positioning of the division site and the placement of the Z-ring. Show less
Streptomyces are bacteria abundant in soil that participate in diverse and complex interactions. These bacteria are the main producers of the antibiotics we currently use in the clinic.... Show moreStreptomyces are bacteria abundant in soil that participate in diverse and complex interactions. These bacteria are the main producers of the antibiotics we currently use in the clinic. Streptomyces are also prolific producers of volatile compounds (VCs) that due to their low molecular weight and vapor pressure can easily travel and distribute through air, water and soil pores. The volatile character of such molecules could suggest that they participate in “long-distance” interactions. This dissertation studies the diversity and functions of VCs produced by Streptomyces. The experiments described here show that even very small VCs such as ammonia can act as antibiotics if they are produced in sufficiently large quantities. it also shows how the perceiver bacteria reacts and responds to antibiotic VCs. The role of other volatile compounds such as terpenes was also studied, in particular 2-methylisoborneol and geosmin. These terpene compounds are highly conserved among Streptomyces and hence may have an important role in the biology of these bacteria. This work includes some results on the possible role of these molecules and opens research questions about the possible regulatory mechanisms for the production of these compounds. Show less
Streptomyces are multicellular, Gram-positive bacteria in the phylum of actinobacteria which produce a high amount of bioactive natural products of which the expression is tightly coordinated with... Show moreStreptomyces are multicellular, Gram-positive bacteria in the phylum of actinobacteria which produce a high amount of bioactive natural products of which the expression is tightly coordinated with the life cycle. This thesis shows the identification of S. roseifaciens, a novel species with an uncommon, verticillate spore morphology and a unique household of SsgA-like proteins. Analyses of the peptidoglycan composition show that S. coelicolor show a pattern of 3-3 cross-linking befitting a tip-growing organism and change in composition between vegetative mycelium and spores. Kitasatosporae carry meso-DAP in the peptidoglycan of vegetative mycelium and LL-DAP in the peptidoglycan of spores. In line with this difference, the peptidoglycan architecture of these two growth stages undergoes such radical changes that they would seem to be from different species. S. coelicolor is naturally vancomycin resistant, but the addition of D-alanine and disruption in a single gene increases vancomycin sensitivity by a thousandfold. A knockout mutant of the alanine racemase, alr, requires exogenous addition of D-alanine. The Alr crystal structure of S. coelicolor and the D-cycloserine producer S. lavendulae were compared as to look for possible mechanisms for D-cycloserine resistance. Show less
The filamentous bacteria Streptomyces are widespread inhabitants of terrestrial soils. Streptomycetes are not only among the most potent producers of valuable secondary metabolites (e.g.... Show moreThe filamentous bacteria Streptomyces are widespread inhabitants of terrestrial soils. Streptomycetes are not only among the most potent producers of valuable secondary metabolites (e.g. antibiotics), but also the source of various industrially relevant hydrolytic enzymes. The mode-of-growth of streptomycetes under industrial conditions is markedly different to that observed in their natural habitat. Most species form dense particles called pellets. Pellets are heterogeneous in size; more specifically at least two populations of differently-sized pellets exist in submerged cultures. Importantly, pellet size and production have been shown to be to be tightly correlated in streptomycetes. The study and control of pellet size heterogeneity in streptomycetes is the subject of the research presented in this thesis. Here, the various phenomena occurring throughout growth are characterized with the aim of understanding the factors underlying this phenomenon. Subsequently, the obtained knowledge is applied to obtain homogeneously-sized pellets of the industrial workhorse Streptomyces lividans. The work described in this thesis also addresses the fate of pellets at late stages of growth and a growth strategy representing a valuable alternative to conventional liquid cultures. Show less
Streptomyces are multicellular bacteria that grow as branched filaments and are best known for producing the majority of our antibiotics, many immunosuppressant and anticancer compounds.... Show moreStreptomyces are multicellular bacteria that grow as branched filaments and are best known for producing the majority of our antibiotics, many immunosuppressant and anticancer compounds. Unfortunately their multicellular life style creates many problems for efficient industrial production. In a bioreactor, depending on the environment and the genetics, it can grow quickly as dispersed mycelia or aggregate in slow growing pellets. Either morphology has advantages and disadvantages, which can be product specific. For my thesis I studied the mechanism by which these filaments can aggregate into dense pellets. I found a small gene cluster that produces poly-1,6-N-acetylglucosamine, a bacterial glue which binds neighboring cells and required for pellet formation in S. coelicolor. Subsequently we can use these genes to control the morphology of streptomycetes in a liquid environment, tailoring it for production. My work has given us new insights in the mechanims through which streptomycetes aggregate, but also has the potential to make streptomycetes a more favorable host for industial production. Show less
The filamentous soil bacteria of the genus Streptomyces are commercially exploited for the production of a wide range of natural products such as antibiotics, anticancer agents and... Show moreThe filamentous soil bacteria of the genus Streptomyces are commercially exploited for the production of a wide range of natural products such as antibiotics, anticancer agents and immunosuppressants. Additionally, there is a strong interest in the use of these bacteria for the production of industrial proteins. Optimal production of these secondary metabolites and enzymes is tightly coupled to morphology. However, relatively little is known about the genetic determinants influencing the morphology of streptomycetes in liquid-grown cultures. The work presented in this thesis focuses on the formation of extracellular macromolecules and their influence on morphogenesis, with a special emphasis on the role of the cellulose synthase-like protein CslA and the radical copper oxidase GlxA in the production of a tip-localized glycan. This research is of fundamental importance for understanding the determinants of growth and development of this multicellular model organism, and at the same time may help us to further optimize their exploitation for the industrial production of secondary metabolites and enzymes. Show less
Streptomyces are Gram-positive, soil dwelling bacteria that raised interest in the last 50 years for their high potential in antibiotic and protein production. Thanks to their saprophytic nature,... Show moreStreptomyces are Gram-positive, soil dwelling bacteria that raised interest in the last 50 years for their high potential in antibiotic and protein production. Thanks to their saprophytic nature, streptomycetes secrete a massive amount of industrial enzymes. They have a relatively low level of endogenous extracellular proteolytic activity when compared to other expression hosts (e.g. Bacillus), they are generally more suited to produce proteins encoded by high G+C actinomycete genes in their native form, coupled to efficient secretion so as to avoid that the proteins end up in inclusion bodies (often a problem when using e.g. E. coli) and making downstream processes easier. Despite their attractive potential, Streptomyces present several constraints which so far limit their application in industry. The first constraint is morphology: by growing as a network of hyphae, they produce dense pellets in liquid cultures that hold Streptomyces back from being one of the first choice cell factories in large scale fermentations. In addition, the limited availability of efficient expression systems for high-level transcription/translation and subsequent secretion is a further bottleneck. This thesis presents the work done to address these issues for the optimization of Streptomyces lividans for future industrial applications and enzyme production. Show less
Streptomycetes are Gram-positive multicellular soil-dwelling bacteria which are commercially used as natural producers of antibiotics, anticancer agents and immunosuppressants, as well as many... Show moreStreptomycetes are Gram-positive multicellular soil-dwelling bacteria which are commercially used as natural producers of antibiotics, anticancer agents and immunosuppressants, as well as many industrial enzymes (Hopwood 2007). Similarly to fungi, they carry out a complex developmental life cycle, forming highly structured multicellular colonies composed of physiologically distinct hyphae (Miguelez et al. 2000). A major scientific challenge lies in understanding how growth parameters are controlled in response to nutritional conditions, and how this affects the efficiency of production and secretion of proteins and antibiotics. The work presented in this thesis therefore aims to arrive at a better understanding of Streptomyces morphogenesis and development, and how these processes link to productivity. Studies performed include the analysis of several novel coiled-coil proteins and their effect on morphogenesis an d division, as well as fluorescent live imaging to better understand and localize dynamic secretion systems. Cryo-correlative light and electron microscopy, and specifically tomography, was performed to image vegetative cell division and its effect on development within a pellet. Combining the multi-scale information gained, a structured morphological model was created to provide a framework for rational design of Streptomyces sp. and provide a test drive for the fermentation process. Show less
The soil is a thriving ecosystem, composed of both living and non-living matter with a multitude of interactions between them. It is considered as the most diverse microbial habitat on earth. Soil... Show moreThe soil is a thriving ecosystem, composed of both living and non-living matter with a multitude of interactions between them. It is considered as the most diverse microbial habitat on earth. Soil microorganisms perform important functions in recycling nutrients. They act as nutrient scavengers, obtaining nutrition from dead, decaying matter of plants and animals. Actinomycetes (including Streptomyces) are a large group of filamentous bacteria and may account even 30% of the total microorganisms in the soil rhizosphere, depending on nutrient availability (Barreto et al., 2008; Kennedy, 1999).They are able to utilize broad range of naturally occurring polymers, including starch, cellulose, chitin and xylan. A sufficiency of utilisable organic material promotes the vegetative growth of these bacteria, thereby delaying morphological development and associated antibiotic production. My thesis focuses on understanding the molecular mechanisms by which Streptomyces link nutrient availability with antibiotic production and development. Show less
Streptomycetes are Gram-positive soil-dwelling bacteria, in appearance similar to filamentous fungi. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues... Show moreStreptomycetes are Gram-positive soil-dwelling bacteria, in appearance similar to filamentous fungi. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues, control specific steps of sporulation-specific cell division in streptomycetes. The expression level of SsgA, the best studied SALP, has a rather dramatic effect on septation and on hyphal morphology, which is not only of relevance for our understanding of (developmental) cell division but has also been succesfully applied in industrial fermentation, to improve growth and production of filamentous actinomycetes. The different regulation of ssgA transcription in different Streptomyces species, is at least one of the reasons why some strains are able to produce spores in liquid-grown mycelium, while others cannot. By far the most conserved SALP is SsgB, which is most likely is the archetypal SALP, with only SsgB orthologues occurring in all morphologically complex actinomycetes. SsgB is essential for sporulation of Streptomyces, and orthologues have a universally conserved function in actinomycete morphogenesis. Show less
