Rhizobiophage Functional Genomics

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dc.contributor.advisorHynes, Michael F.
dc.contributor.advisorYost, Christopher Karl
dc.contributor.authorKoswaththa Muhandiramlage, Damitha Gunathilake
dc.contributor.committeememberDunfield, Peter F.
dc.contributor.committeememberStrous, Marc
dc.date2020-11
dc.date.accessioned2020-08-26T20:12:55Z
dc.date.available2020-08-26T20:12:55Z
dc.date.issued2020-08-19
dc.description.abstractRhizobiophages are bacterial viruses that specifically infect nitrogen-fixing, legume-nodulating bacterial group rhizobia. In this study, the whole genome sequences of eight rhizobiophages were generated. These included four Rhizobium leguminosarum myophages (AF3, P9VFCI, RL2RES and RL38JI), two R. leguminosarum siphophages (P11VFA and B1VFA), one R. leguminosarum phage with unknown morphology (V1VFA-S) and one M. japonicum phage (Cp1R7A-A1). Characterization of Cp1R7A-A1 indicated a siphophage morphology with a prolate capsid, a distinct genome, and a close phylogenetic relationship to certain Caulobacter siphophages with similar morphology. The myophages AF3, P9VFCI, RL2RES and RL38JI belong in the ICTV family Ackermanviridae and in a T4-like group of viruses. A recombination dependent replication mechanism and circularly permuted genomes due to a pure headful packaging mechanism were postulated for these phages. B1VFA and V1VFA-S had genomes similar to each other. A theta replication mechanism and phage genome terimini with direct terminal repeats were suggested for these two. P11VFA was similar to the R. leguminosarum phage L338C genome available in databases. Quantitative reverse transcription PCR for selected genes of phages Cp1R7A-A1 and Lo5R7ANS, using RNA extracts from Mesorhizobium japonicum separately infected with these phages, confirmed an early-late type maximum gene expression in Lo5R7ANS. Selected replication genes showed a maximum expression 30 minutes after infection, but structural and packaging gene expression maximized after 90 minutes. Discrete timepoints of maximum expression for any selected gene in Cp1R7A-A1 were not observed during the given incubation periods, probably due to the slow rate of replication and assembly. RNAseq analysis of Cp1R7A-A1 infected M. japonicum indicated the expression of all 237 putative phage genes and gave some insights into host gene expression during phage infection. Attempts were made to engineer R. leguminosarum and M. japonicum to carry a plasmid with a functional CRISPR/Cas9 system. A CRISPR/Cas9 system derived from Streptococcus pyogenes was cloned into the broad-host range vector pRK415. The new vector pRK415Cas9 functioned successfully in E. coli. However, it was not functional in Rhizobium or Mesorhizobium species. Cas9 promoter expression studies and RT-PCR revealed that the Cas9 promoter was expressed in Mesorhizobium. However, whether correct translation occurs has yet to be tested.en_US
dc.identifier.citationKoswaththa Muhandiramlage, D. G. (2020). Rhizobiophage Functional Genomics (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/38112
dc.identifier.urihttp://hdl.handle.net/1880/112430
dc.language.isoengen_US
dc.publisher.facultyScienceen_US
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.en_US
dc.subjectRhizobiophages, genomics, transcriptomics, CRISPR-Cas9en_US
dc.subject.classificationMicrobiologyen_US
dc.titleRhizobiophage Functional Genomicsen_US
dc.typedoctoral thesisen_US
thesis.degree.disciplineBiological Sciencesen_US
thesis.degree.grantorUniversity of Calgaryen_US
thesis.degree.nameDoctor of Philosophy (PhD)en_US
ucalgary.item.requestcopytrueen_US
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