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Funding: European Regional Development Fund (ERDF) “On Implementation of Activity “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”.

Project Title: Structural studies of a Borrelia burgdorferi bacteriophage

Project No.:

Implementation period: 1 January 2021 – 30 June 2023

Project costs: 111 505.00 EUR

Project implementer: Dr. biol. Jānis Rūmnieks

Borrelia burgdorferi-associated bacteriophages (phages) are a biologically important but under-investigated feature of the Lyme disease-causing spirochete. A group of ubiquitous B. burgdorferi plasmids cp32s are in fact dormant genomes of a highly unusual bacteriophage φBB1 which can be induced to form virus particles and lyse the bacterium, however, virtually nothing is known about the underlying mechanisms of how this is accomplished. The structural proteins of the φBB1 phage are particularly diverged and have very little or no sequence similarity to other viruses.

The objective of the current project is to use cryo-electron microscopy and X-ray crystallography to determine a high-resolution structure of the φBB1 bacteriophage which will map the function of more than a dozen currently obscure phage proteins and will reveal the molecular architecture for an evolutionary distinct lineage of bacterial viruses. As a result, the project aims to provide a comprehensive molecular-level understanding of how the Borrelia phage particles are built which not only will be of considerable value to the fields of fundamental borrelial and bacteriophage research but will additionally provide a foundation for further studies to develop novel approaches and tools relevant to treatment of Lyme disease.

Information published 04.01.2021.

Project progress

 1 January 2021 – 31 March 2021

To facilitate studies on the φBB1 structural proteins, at the beginning of the project the entire phage morphogenesis module was PCR-amplified from Borrelia burgdorferi plasmid cp32-1 and cloned into an Escherichia coli plasmid vector. Using this construct as a template, a series of further constructs were designed for production of the phage capsid (head) constituent proteins with the aim to obtain this phage component in E. coli. The respective coding sequences, both individually and in different subsets, were cloned in expression vectors and tested for expression. Preliminary results indicate that the respective proteins can be produced in high levels and there are indications that they can assemble into capsid-like structures in the E. coli system.

Besides the recombinant approach, experiments to obtain φBB1 phage particles from B. burgdorferi cells upon induction were also commenced by testing different methods for induction and phage concentration.

Information published 31.03.2021.

Project progress

 1 April 2021 – 30 June 2021

The structures formed by the recombinantly produced φBB1 capsid proteins in Escherichia coli were investigated using negative-stain electron microscopy which confirmed assembly of homogeneous procapsids. A purification scheme was developed to obtain these structures in high purity. Activities to recombinantly produce the φBB1 terminase complex were initiated with the aim to package DNA into the procapsids and hence produce mature capsids in vitro.

Experimental work to recombinantly produce the φBB1 baseplate in Escherichia coli was also commenced. The respective protein-coding sequences, both individually and in different subsets, were cloned in expression vectors and tested for expression. The expression level of the different baseplate proteins was variable, and work was done to optimize their production levels.

Information published 30.06.2021.

Project progress

 1 July 2021 – 30 September 2021

Efforts to obtain recombinant φBB1 terminase complex were underway. Different expression methods, affinity purification tags and purification protocols were tested, and conditions were found under which it was possible to obtain the terminase complex in a soluble form. The complex was purified on an analytical scale and first attempts to package DNA in φBB1 procapsids were commenced. Additionally, experimental efforts were launched to develop plasmid constructs for recombinantly expressing φBB1 tail proteins in the Escherichia coli system and test their expression levels. Experimental work also continued for developing and testing different plasmid constructs and expression protocols for producing the φBB1 baseplate complex in Escherichia coli.

Information published 30.09.2021.

Project progress

 1 October 2021 – 31 December 2021

Experimental work was ongoing to recombinantly express the φBB1 tail proteins in Escherichia coli. Additionally, two-plasmid systems for coexpressing both terminase subunits, as well as different baseplate proteins, were constructed with the aim to increase their production levels. Efforts to find suitable experimental conditions for in vitro packaging φBB1 procapsids with DNA were also underway.

Additionally, structural analysis of the φBB1 bacteriophage was commenced. φBB1 procapsid structures were purified and sent for examination in a cryo-electron microscope at the CEITEC facility in Czech Republic. The first results indicate that the sample is homogeneous and suitable for further high-resolution data collection.

Information published 30.12.2021.

Project progress

 1 January 2022 – 31 March 2022

Experiments were ongoing to develop an expression system in Escherichia coli for co-expressing the φBB1 tail and baseplate proteins. Additionally, plasmid constructs for expression of the φBB1 baseplate hub, tail spike, and tail fiber proteins were created. Experiments were done to assess their expression, and conditions were established under which these proteins were soluble, or, if necessary, additional plasmid constructs were created by fusing a solubility-enhancing domain to the target proteins. Purification protocols were developed for the baseplate hub and tail spike proteins. The proteins were purified to homogeneity in a preparative scale and subjected to initial crystallization trials.

Information published 31.03.2022.

Progress of the project

1 April 2022 – 30 June 2022

A purification scheme for the φBB1 tail fiber protein was developed and the protein was purified on a preparative scale. Screening and optimization of crystallization conditions for the tail spike and tail fiber proteins were carried out and crystals suitable for testing in synchrotron were obtained. Efforts were also put towards obtaining selenomethionine-labeled tail spike and tail fiber proteins and their purification and crystallization.

A high-resolution cryo-EM dataset of the φBB1 procapsids was obtained and processing of the data was started. The collected movies were motion-corrected, the contrast transfer functions of the obtained micrographs were estimated, particles were picked and extracted, and initial 3D reconstruction performed.

Information published 30.06.2022.