Periodic Reporting for period 4 - PRO_PHAGE (Impact and interaction of prophage elements in bacterial host strains of biotechnological relevance)
Periodo di rendicontazione: 2022-07-01 al 2023-06-30
Phages, viruses that prey on bacteria, are the most abundant and diverse inhabitants of the Earth. Temperate bacteriophages are able to integrate into the host genome and maintain as prophages a long-term association with their host. Illustrated by the development of mutually beneficial traits, this close interaction between host and virus has significantly shaped bacterial evolution. However, the immense genetic resources of phage genomes still remain almost unexplored. For the transition to a sustainable bioeconomy, we strongly depend on microbes as hosts for the production of value-added compounds. PRO_PHAGE is exploiting recent advances in next-generation sequencing (NGS), single-cell analysis, and high-throughput (HT) phenotyping to evaluate the impact of phage elements on host fitness and to use this knowledge for the improvement of future metabolic engineering approaches.
By combining an explorative approach with subsequent molecular analysis of selected targets, PRO_PHAGE will deliver novel insights into this genetic resource and will reveal the risks and potential for metabolic engineering by pursuing four major objectives. 1) Based on a comprehensive bioinformatic analysis, the impact of phage elements is studied by HT phenotyping of selected strains. 2) A second objective addresses the regulatory interaction of the phage and the host by focusing on host-encoded xenogeneic silencing proteins and their role in the integration of foreign DNA. 3) The spontaneous activation of phage elements is studied at the genomic scale to decipher molecular triggers and their impact on host gene expression. For this purpose, we already developed a novel workflow combining fluorescence-activated cell sorting and NGS enabling the analysis of microbial population dynamics at unprecedented resolution. 4) Finally, the insights obtained are benchmarked for metabolic engineering approaches in order to generate robust and flexible chassis strains for industrial production.
By combining an explorative approach with subsequent molecular analysis of selected targets, PRO_PHAGE will deliver novel insights into this genetic resource and will reveal the risks and potential for metabolic engineering by pursuing four major objectives. 1) Based on a comprehensive bioinformatic analysis, the impact of phage elements is studied by HT phenotyping of selected strains. 2) A second objective addresses the regulatory interaction of the phage and the host by focusing on host-encoded xenogeneic silencing proteins and their role in the integration of foreign DNA. 3) The spontaneous activation of phage elements is studied at the genomic scale to decipher molecular triggers and their impact on host gene expression. For this purpose, we already developed a novel workflow combining fluorescence-activated cell sorting and NGS enabling the analysis of microbial population dynamics at unprecedented resolution. 4) Finally, the insights obtained are benchmarked for metabolic engineering approaches in order to generate robust and flexible chassis strains for industrial production.
In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. In this project, we focused on Corynebacterium glutamicum as one of the most important biotechnological platform organisms and on the fast-growing marine bacterium Vibrio natriegens representing an emerging model host for molecular biology and biotechnology. In RRO_PHAGE we successfully constructed several prophage-free variants of these strains. Final prophage-free strains proved as suitable platform strains for the engineering of small molecule or protein production and are by now frequently used platform strains for biotechnological applications.
Following on the question of how ‘foreign’/viral genetic material is tolerated by the host and how it is integrated into host regulatory networks, we systematically addressed the function of xenogeneic silencing (XS) proteins. In actinobacteria, Lsr2-like nucleoid-associated proteins function as XS of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant for the use as a potential drug target and for the identification of novel bioactive compounds. In our study, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and countersilencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum. Our results demonstrated that CgpS distinguishes between self and foreign DNA by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks. We further developed targeted counter-silencing approaches by implementing dCas9-based countersilencing providing an unprecedented opportunity for the targeted activation of XS target genes. The principle of XS was further harnessed for the establishment of inducible expression systems representing key modules in regulatory circuit design and metabolic engineering approaches.
Actinobacteria are well known as producers of a variety of bioactive compounds, including molecules with antibacterial, anti-fungal or anti-cancer activity. During phage isolation efforts conducted in this project, we noticed that viral infectios trigger antibiotic production in Streptomyces species. This led to the hypothesis that bacteria produce and secrete small secondary metabolites involved in the defense against viral infections at the multicellular level. Following this hypotheis, we showed that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrated that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level.
Following on the question of how ‘foreign’/viral genetic material is tolerated by the host and how it is integrated into host regulatory networks, we systematically addressed the function of xenogeneic silencing (XS) proteins. In actinobacteria, Lsr2-like nucleoid-associated proteins function as XS of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant for the use as a potential drug target and for the identification of novel bioactive compounds. In our study, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and countersilencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum. Our results demonstrated that CgpS distinguishes between self and foreign DNA by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks. We further developed targeted counter-silencing approaches by implementing dCas9-based countersilencing providing an unprecedented opportunity for the targeted activation of XS target genes. The principle of XS was further harnessed for the establishment of inducible expression systems representing key modules in regulatory circuit design and metabolic engineering approaches.
Actinobacteria are well known as producers of a variety of bioactive compounds, including molecules with antibacterial, anti-fungal or anti-cancer activity. During phage isolation efforts conducted in this project, we noticed that viral infectios trigger antibiotic production in Streptomyces species. This led to the hypothesis that bacteria produce and secrete small secondary metabolites involved in the defense against viral infections at the multicellular level. Following this hypotheis, we showed that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrated that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level.
This periodic report already covers the final period of PRO_PHAGE (01/07/22-30/06/23). However, several projects are still ongoing and to be finalized and published within the next months. In particular the discovery of antiphage molecules produced by bacteria opened up new research lines and puts my group at the forefront of this the rapidly emerging research field. We further describe an important impact of phage infection on cellular development in bacteria and show that development of transiently resistance is crucial for the containment of viral infections at the community level.
We gained significant mechanistic understanding of xenogeneic silencing mediated by Lsr2-like proteins. Synthetic countersilencing approaches based on the introduction of regulatory circuits and dCas9 were successfully implemented and allow for the first time the targeted activation of silenced gene clusters. The project focusing on dCas9-based countersilencing is still ongoing and will be published withing the next months.
Bioinformatic analysis of actinobacterial genomes revealed Lsr2-like proteins as abunant regulators in actinobacteriophages. Mapping the dynamics of Lsr2 binding to prophage elements in the course of prophage induction using ChIP-Seq revealed an important role of these proteins in the phage life cylce and an impact on further cryptic prophages residing in the same genome. These mechanistic insights gained for Lsr2-like proteins go certainly beyond the state of the art.
We gained significant mechanistic understanding of xenogeneic silencing mediated by Lsr2-like proteins. Synthetic countersilencing approaches based on the introduction of regulatory circuits and dCas9 were successfully implemented and allow for the first time the targeted activation of silenced gene clusters. The project focusing on dCas9-based countersilencing is still ongoing and will be published withing the next months.
Bioinformatic analysis of actinobacterial genomes revealed Lsr2-like proteins as abunant regulators in actinobacteriophages. Mapping the dynamics of Lsr2 binding to prophage elements in the course of prophage induction using ChIP-Seq revealed an important role of these proteins in the phage life cylce and an impact on further cryptic prophages residing in the same genome. These mechanistic insights gained for Lsr2-like proteins go certainly beyond the state of the art.