Impact and interaction of prophage elements in bacterial host strains of biotechnological relevance

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.

The fast-growing marine bacterium Vibrio natriegens represents an emerging model host for molecular biology and biotechnology, featuring a reported doubling time of less than 10 minutes. 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. Genome analysis revealed the presence of two prophage regions in the V. natriegens genome that were shown to undergo spontaneous induction under standard cultivation conditions. In a recent study, we generated a prophage-free variant of V. natriegens. Remarkably, the prophage-free strain exhibited a higher tolerance toward DNA damage and hypoosmotic stress. Moreover, it was shown to outcompete the wild-type strain in a competitive
growth experiment. Our study presents the prophage-free variant of V. natriegens as a promising platform strain for future 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.

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. However, established systems are often limited in terms of applications due to high background expression levels and inducer toxicity. In a recent study of this project, we successfully established a promoter library based on the principle of XS and counter-silencing. For selected candidates, background expression levels were confirmed to be significantly reduced in comparison to established heterologous expression systems. Finally, this principle was implemented in a metabolic toggle switch allowing the dynamic redirection of carbon flux between biomass and L-valine production in a C. glutamicum production strain.

From the beginning of the project to the end of the period covered by the report, we already gained important insights into the impact of prophage elements on bacterial production strains and on how the principle of XS facilitates evolutionary network expansion in the medically and biotechnologically relevant bacterial phylum of actinobacteria.
Further preliminary results of this project highlight bacteriophages and prophage elements as a rich source for the identification of novel antibacterial proteins as well as for the establishment of efficient molecular tools targeting key regulatory hubs of the host cell. Therefore, we expect further interesting results with medical and biotechnological relevance in the remaining time of the project.

Periodic Reporting for period 3 - PRO_PHAGE (Impact and interaction of prophage elements in bacterial host strains of biotechnological relevance)

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