Periodic Reporting for period 1 - PHAGECONTROL (The evolution of host manipulation by bacteriophage.)
Reporting period: 2022-11-01 to 2024-04-30
Parasites that manipulate their host are among the most fascinating adaptations in biology, that till date have not been discovered in microbial world. This manipulation manifests in behavioral patterns, which benefit the parasite by faciliating its transmission, but which harm the host. Bacteriophages are viruses that target bacteria and the most abundant parasites on the planet. So called Phage RS (Regulatory Switches) can control survival, social interactions and evolution of their bacteria. PHAGECONTROL aims to understand why phages modify bacterial phenotypes, hypothesizing that phage RS act as parasite manipulators. The three main research objetives and their associated research tasks are: 1) Find individual and social host behaviors which are controlled by RS phage; 2) Elucidate molecular mechanisms behind host control by RS phage; 3) Determine evolutionary forces behind host control by RS phage.
Expansion of knowledge on phage-host model system
Bioinformatics and experimental study was performed on the RS phage-host relationship. Two main clusters of SPbetaviruses, were found, with the well-known SPβ belonging to a minority cluster and showing tight linkage to host phylogeny and another mobile genetic element, skin. We also fund that most predicted SPbetaviruses do not exhibit horizontal transmission via lytic cycles, suggesting broader definitions of parasite manipulation, where phages modulate host phenotypes to facilitate gene exchange (Fig. 1).
Building phage and host collection
Collaborations provided new active RS-phages, which were used to lysogenize SPβ-free recipient strains. Additionally, medically relevant isolates such as NDmed were successfully lysogenized, showing robust biofilm formation. Efforts were also made to construct double lysogens, as phenotypic changes could depend on the presence of multiple phages. Some strains underwent whole-genome sequencing for further analysis.
Labeling strains with fluorescent reporters
To monitor phage-host interactions using microscopy and spectrofluorimetry, fluorescent reporters were introduced into selected strains, facilitating real-time observation of various cellular processes such as cell elongation, division, sporulation, and spore coat formation. These strains enable high-throughput phenotypic screening for host traits controlled by SPβ-like phages.
Screening for host phenotypes controlled by RS phage
Bioinformatics analysis of available transcriptomics data helped identify growth conditions and phage genes of interest, revealing variations in prophage gene expression based on different environmental factors (Fig. 2). The Synphage software was developed to visualize gene conservation within RS phages and to connect phenotype with genotype, aiding in the identification of genes shared among phages triggering similar host changes (Fig. 3). Pilot experiments focused on sporulation dynamics and macrocolony development, with findings indicating that RS phages affect these processes differently (Fig. 4). Effect of different RS phages on ecological competition was tested revealing that effect of these phages on host competitive fitness may be positive of negative (Fig. 6).
Optimization of high-throughput imaging
Efforts to optimize time-lapse imaging experiments using Elyra7 platform involved protocol adjustments for sample preparation, gas control, auto-focus, and multi-position imaging. This optimization is crucial for high-throughput screening experiments (Fig. 5).
Molecular mechanisms of host control by RS phage
Molecular tools were developed, including reporter strains and fluorescently labeled phages, to study host control strategies. For instance, reporters for prophage induction and fluorescently labeled phage particles were created to track phage dynamics within host cells. Fluorescence-independent methods such as PCR and qPCR were also used to track phage integration and excision (Fig. 7) and phage staining protocol was optimized (Fig. 8).
Host control strategy assessment
Studies on spore dispersibility and cell shape control provided insights into the strategies used by RS phages. For example, the excision of RS phage SPβ affects spore dispersibility, potentially benefiting horizontal transmission. The role of phi3T phage in altering cell shape was linked to its genetic cargo rather than att gene interruption (Fig. 9, Fig. 10).
Evolution of host control by RS phage
Tasks aimed to evaluate the costs and benefits of phage-mediated host control strategies on both phage and host fitness. For instance, removing sublancin cluster (bacteriocin encoded by phage, that benefits the host) negatively impacted phage fitness, demonstrating the intricate balance in phage-host interactions (Fig. 11).
Bioinformatics and experimental study was performed on the RS phage-host relationship. Two main clusters of SPbetaviruses, were found, with the well-known SPβ belonging to a minority cluster and showing tight linkage to host phylogeny and another mobile genetic element, skin. We also fund that most predicted SPbetaviruses do not exhibit horizontal transmission via lytic cycles, suggesting broader definitions of parasite manipulation, where phages modulate host phenotypes to facilitate gene exchange (Fig. 1).
Building phage and host collection
Collaborations provided new active RS-phages, which were used to lysogenize SPβ-free recipient strains. Additionally, medically relevant isolates such as NDmed were successfully lysogenized, showing robust biofilm formation. Efforts were also made to construct double lysogens, as phenotypic changes could depend on the presence of multiple phages. Some strains underwent whole-genome sequencing for further analysis.
Labeling strains with fluorescent reporters
To monitor phage-host interactions using microscopy and spectrofluorimetry, fluorescent reporters were introduced into selected strains, facilitating real-time observation of various cellular processes such as cell elongation, division, sporulation, and spore coat formation. These strains enable high-throughput phenotypic screening for host traits controlled by SPβ-like phages.
Screening for host phenotypes controlled by RS phage
Bioinformatics analysis of available transcriptomics data helped identify growth conditions and phage genes of interest, revealing variations in prophage gene expression based on different environmental factors (Fig. 2). The Synphage software was developed to visualize gene conservation within RS phages and to connect phenotype with genotype, aiding in the identification of genes shared among phages triggering similar host changes (Fig. 3). Pilot experiments focused on sporulation dynamics and macrocolony development, with findings indicating that RS phages affect these processes differently (Fig. 4). Effect of different RS phages on ecological competition was tested revealing that effect of these phages on host competitive fitness may be positive of negative (Fig. 6).
Optimization of high-throughput imaging
Efforts to optimize time-lapse imaging experiments using Elyra7 platform involved protocol adjustments for sample preparation, gas control, auto-focus, and multi-position imaging. This optimization is crucial for high-throughput screening experiments (Fig. 5).
Molecular mechanisms of host control by RS phage
Molecular tools were developed, including reporter strains and fluorescently labeled phages, to study host control strategies. For instance, reporters for prophage induction and fluorescently labeled phage particles were created to track phage dynamics within host cells. Fluorescence-independent methods such as PCR and qPCR were also used to track phage integration and excision (Fig. 7) and phage staining protocol was optimized (Fig. 8).
Host control strategy assessment
Studies on spore dispersibility and cell shape control provided insights into the strategies used by RS phages. For example, the excision of RS phage SPβ affects spore dispersibility, potentially benefiting horizontal transmission. The role of phi3T phage in altering cell shape was linked to its genetic cargo rather than att gene interruption (Fig. 9, Fig. 10).
Evolution of host control by RS phage
Tasks aimed to evaluate the costs and benefits of phage-mediated host control strategies on both phage and host fitness. For instance, removing sublancin cluster (bacteriocin encoded by phage, that benefits the host) negatively impacted phage fitness, demonstrating the intricate balance in phage-host interactions (Fig. 11).
- All prophage elements within model species have been extracted, clustered, and tested for potential antagonism or coupling. The results will be released into Zenodo along with the publication. Research community focusing on other mobile genetic elements of Bacillus will benefit from the results, multiple computational predictions will be laid out for experimental validation
- Synphage software is already available as github repository and website. It will benefit scientific community working not only of phages, but interested in gene conservation in general
- Verification of previously published research on role of spsM in biofilm formation. Our data showed that previous observation was due to additional mutations that spontaneously occur in ∆spsM genetic background. In collaboration with previous authors, we will release manuscript correcting previous conclusions.
- New microscopy platform Elyra7 is available, ability to track growing cells and fluorescently labelled phages will soon allow high-throughput screening experiment of host control by phage under various conditions and setups. It will also open possibilities for collaborations within and outside the department.
- New line of research on phage recombination – observed in poly-lysogens, became base for basic project application under national funding scheme. Exchange of accessory genes between prophage elements has a great potential to accelerate host evolution. The approach may be used as a strategy to evolve beneficial bacteria with desired traits.
- Internationalization: research team was recruited, that consists of international postdocs and PhD students. Postdoc researcher from Japan was hosted in frame of ERC-AMED collaboration. Few Erasmus students were already involved in the project (Group photo – Fig. 12). PI of the project, supported by ministry of higher education, received Slovenian citizenship by means of special naturalization for achievements in science, what takes away a large administrative burden and contributes to work and life quality and therefore leadership quality.
- Reintegration and attraction of additional funding: researcher was recruited back to Slovenia, currently supported by EMBO fellowship, still partly works on the action. PhD student received FEMS short-term fellowship, Erasmus+ short-term PhD fellowship. Postdoc researcher received FEMS travel grant.
- International collaborations were established: Bramkamp lab (University of Kiel, Germany), Middelboe lab (University of Copenhagen, Denmark), Krasny lab (Czech Academy of Science, Czech republic), Sagona lab (University of Warwick, UK), Briandet lab (INRAE, France), Hestbjerg Hansen lab (University of Copenhagen, Denmark)
- Synphage software is already available as github repository and website. It will benefit scientific community working not only of phages, but interested in gene conservation in general
- Verification of previously published research on role of spsM in biofilm formation. Our data showed that previous observation was due to additional mutations that spontaneously occur in ∆spsM genetic background. In collaboration with previous authors, we will release manuscript correcting previous conclusions.
- New microscopy platform Elyra7 is available, ability to track growing cells and fluorescently labelled phages will soon allow high-throughput screening experiment of host control by phage under various conditions and setups. It will also open possibilities for collaborations within and outside the department.
- New line of research on phage recombination – observed in poly-lysogens, became base for basic project application under national funding scheme. Exchange of accessory genes between prophage elements has a great potential to accelerate host evolution. The approach may be used as a strategy to evolve beneficial bacteria with desired traits.
- Internationalization: research team was recruited, that consists of international postdocs and PhD students. Postdoc researcher from Japan was hosted in frame of ERC-AMED collaboration. Few Erasmus students were already involved in the project (Group photo – Fig. 12). PI of the project, supported by ministry of higher education, received Slovenian citizenship by means of special naturalization for achievements in science, what takes away a large administrative burden and contributes to work and life quality and therefore leadership quality.
- Reintegration and attraction of additional funding: researcher was recruited back to Slovenia, currently supported by EMBO fellowship, still partly works on the action. PhD student received FEMS short-term fellowship, Erasmus+ short-term PhD fellowship. Postdoc researcher received FEMS travel grant.
- International collaborations were established: Bramkamp lab (University of Kiel, Germany), Middelboe lab (University of Copenhagen, Denmark), Krasny lab (Czech Academy of Science, Czech republic), Sagona lab (University of Warwick, UK), Briandet lab (INRAE, France), Hestbjerg Hansen lab (University of Copenhagen, Denmark)