Browsing by Author "Hynes, Michael F."

Now showing 1 - 17 of 17
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    AbiK: A Novel Polymerase That Confers Resistance to Phage Infection
    (2019-05-15) Soufi, Bahar; Zimmerly, Steven; Hynes, Michael F.; Hansen, David V.
    The abortive infection K (AbiK) system of Lactococcus lactis is a powerful antiviral defense mechanism that acts in the period after phage DNA enters the cell but before new progeny are released. The N-terminal domain of AbiK encodes a novel DNA polymerase that in vitro uses an internal amino acid to synthesize an un-templated DNA. The C-terminal domain of AbiK, on the other hand, has not been biochemically characterized. In the first of two projects presented in this thesis, strategies were devised to identify the amino acid priming site in AbiK. A close examination of data has narrowed down the priming site to two highly-conserved tyrosines in the N-terminal sequence of AbiK. In the second project, the characterization of the AbiK’s C terminal domain was undertaken to identify its function. This study uncovers a novel RNA modification activity, and provides evidence that AbiK’s C-terminal domain has ribonuclease activity.
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    Bacteriocin production in rhizobium leguminosarum bv. viciae strain 306
    (2000) Venter, Alexandra Patricia; Hynes, Michael F.
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    Conjugative Complexities: Defining the requirements for the transfer of Rhizobium leguminosarum plasmid pRleVF39b
    (2019-03-20) Wathugala, Nandun Dulmini; Hynes, Michael F.; Turner, Raymond Joseph; Harrison, Joe J.
    The genome of Rhizobium leguminosarum is compartmentalized into a chromosome and multiple large (> 100 kb) plasmids. Four conjugation systems have been identified that can mediate horizontal transfer of these plasmids, and type IVA-1 was identified on R. leguminosarum VF39SM plasmid pRleVF39b. This category of system is found among multiple Rhizobiaceae members from different geographical locations, on both non-symbiotic and symbiotic plasmids. The type IVA-1 conjugation system on pRleVF39b is distinctive due to the presence of a shorter relaxase gene (traA) compared to canonical relaxases, a repressor, TrbR, belonging to the xenobiotic response element (Xre)-family, and the lack of some common genes found in the other conjugation systems. There are 15 annotated Orfs adjacent to known transfer genes in the type IV conjugation locus that might have important functions in plasmid transfer. A series of deletions in each of the hypothetical genes was created to identify essential and facilitating genes for the transfer of pRleVF39b. The effect of these mutations on transfer of pRleVF39b to plasmid-free Agrobacterium recipients was analyzed. orf23, orf24, orf26, orf27, orf28 and orf29 mutants reduced the plasmid transfer frequency by 10-fold or more compared to wild type, suggesting a key role in conjugation. To define the TrbR regulon of pRleVF39b, putative promoter (P) regions of orf17, trbR, orf24, orf25, orf26 and orf29 were fused to a reporter gene gusA. The expression of promoters in WT VF39, VF39a-, VF39b-, trbR mutant and an orf29 mutant was analyzed using β-glucuronidase assays. PtrbR expression was not reduced due to trbR mutation. Thus, TrbR does not autoregulate itself. P24 showed ~30-fold increase and P25 showed ~10-fold increase in expression in VF39b- and trbR backgrounds, indicating that the operons of orf24 and orf25 are part of the TrbR regulon. These results were validated using qRT-PCR experiments. It was also identified that orf16 and orf17 are not part of the TrbR regulon. The absence of conjugative plasmid pRleVF39a in donors negatively affected plasmid transfer but this result was contradicted by qRT-PCR experiments that showed higher levels of mRNA transcripts for orf23, orf24, orf26, orf27 and orf28 in a pRleVF39a cured background.
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    Developing an in cellulo Carrier-Driven Crystallization System using Cry1Ac from Bacillus thuringiensis
    (2019-09-20) McDade, Kyle Harrison; Fraser, Marie Elizabeth; Turner, Raymond Joseph; Hynes, Michael F.
    X-ray crystallography is the dominant technique for determining the structures of proteins but suffers from two major issues, the crystallization bottleneck and the phase problem. To address these problems, a solution was proposed in which the Cry1Ac protein, which crystallizes spontaneously in cellulo upon sporulation of Bacillus thuringiensis, is used as a crystallization-carrier – to enable the crystallization of auxiliary proteins. Production of endogenous Cry1Ac was optimized through the characterization of sporulation-inducing media: in comparison to C2 and nutrient sporulation media, G-Tris induced the largest inclusions. Cry1Ac was purified using hexane and ultracentrifugation. Non-mechanical lysis methods were developed to aid purification, including the application of B. thuringiensis cell wall lyase, CwlC. Three fusions of cry1ac with the gene of a reporter, green fluorescent protein (gfp) from Aequorea victoria, were created and integrated within Escherichia coli and B. thuringiensis expression systems. Fusions were designed with GFP at the amino-terminus, at the carboxy-terminus and substituting for the toxin domains of Cry1Ac. Production of the fusion proteins within E. coli generated polarizing and fluorescent inclusions that persisted after sonication. Protein fusions and unfused-Cry1Ac produced recombinantly in E. coli failed to generate bipyramidal inclusions characteristic of Cry1Ac. The carboxy-terminal Cry1Ac-GFP fusion and Cry1Ac exhibited similar solubilities when exposed to high pH and chaotropic agents. Recombinant production of protein fusions and unfused-Cry1Ac within the acrystalloferous B. thuringiensis system was poor: Cells rarely contained inclusions, but inclusions of protein fusions appeared bipyramidal and fluorescent. It is suspected that a reduced rate of sporulation caused by the loss of plasmids pHT8, pAW63, and pHT73 resulted in reduced expression. This issue prohibited inclusion purification and further characterization.
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    Genetic regulation of chemotaxis and motility in Rhizobium leguminosarum
    (2004) Del Bel, Kathryn L.; Hynes, Michael F.
    Rhizobium leguminosarum, a nitrogen-fixing root nodule bacterium, has over 20 genes coding MCP-type chemoreceptors, which could playa role in locating host legumes by chemotaxis. Transcriptional gusA fusions were made with the mcpC and mcpD genes, and were used to confirm that the mcp genes are down-regulated in the nodule. Transposon mutagenesis was completed with a mcpC fusion strain to identify possible genes which regulate mcp expression. A free-living mcp regulatory mutant was identified as a bacA mutation, while two mutants which showed less down-regulation in nodules than the wild-type were identified as hutC and a ribitiol transporter mutant. Changes in expression in these mutants in planta were confirmed by examining nodules histologically. The flagellin proteins from a R. leguminosarum strain were isolated and used to produce an anti-flagellar antibody. Immunoblots employing this antibody confirmed that flagella, like MCPs, are down-regulated in nodule bacteria.
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    Genetics of Ribitol Catabolism in Rhizobium leguminosarum
    (2019-09-20) Buhlers, Deborah; Hynes, Michael F.; Gieg, Lisa M.; Wong, Sui-Lam; Oresnik, Ivan; Niu, Dongyan
    Rhizobium leguminosarum bv. viciae strain VF39SM contains six plasmids; a strain cured of both pRleVF39c and pRleVF39d is unable to grow on ribitol, whereas strains cured of only one of the plasmids can grow on this substrate. BLAST database searches and previous work show that the pRleVF39d plasmid ribitol catabolic genes are widely distributed among many R. leguminosarum strains. The less common pRleVF39c plasmid locus contains genes encoding a DeoR transcriptional regulator (rlcR), an ATPase component of an ABC transporter (rlcB), a periplasmic binding protein of an ABC transporter (rlcA), two ABC transporter permease components (rlcC, rlcF), a glycerophosphoryl diester phosphodiesterase (rlcG), a hydrolase (rlcH), a ribitol dehydrogenase (rlcD) and a ribulokinase (rlcK). The pRleVF39d locus contains genes encoding a reductase (rldE), a ribitol-2-dehydrogenase (rldD), an ABC transporter permease (rldC), the ATPase of an ABC transporter (rldB), the periplasmic binding protein of an ABC transporter (rldA), and an AraC-like regulator (rldR). Mutagenesis of the genes of these two loci showed that rlcK, rlcD, rldD, rldA, and rldR are required for the catabolism of ribitol. Three c and d plasmid double mutants, rlcK-rldA, rlcK-rldD and rlcK-rldR, were also unable to grow in the presence of ribitol as sole carbon and energy source. The d plasmid ribitol catabolic genes comprise as single operon, while the c plasmid ribitol catabolic genes are in multiple operons, with rlcR and rlcB comprising a divergently transcribed operon, and the remaining c plasmid ribitol catabolic genes making up additional transcripts. The c plasmid ribitol catabolic genes are induced by ribitol and seed exudates of peas, beans and lentils. Nodulation competition assays using ribitol catabolic gene single and double mutants, demonstrated that ribitol catabolism is required for nodulation competitiveness on peas (cv. Little Marvel) and lentils (cv. Marble), but not for vetch or faba beans (cv. Windsor). Ribitol catabolic gene induction during interaction of R. leguminosarum VF39SM with lentils and vetch seedlings also showed that rldR, but not rlcA was induced during early stages of interaction with the roots of lentil seedlings, and that rlcA is induced within the nodules of vetch.
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    Isolation and characterization of rhizobium leguminosarum phages from western Canadian soils
    (2012) Restrepo, Marcela; Hynes, Michael F.
    Rhizobiophages are bacteriophages that infect members of the Rhizobia, a bacterial group that nodulates and fixes nitrogen inside the nodules of legume plants. Rhizobiophages have been used for phage typing of rhizobial populations and as tools for molecular genetics due to their transduction abilities, but their diversity remains largely uncharacterized. At the start of this study very little was known about Rhizobium leguminosarum rhizobiophages. Isolation of phages from soil samples in Alberta and Saskatchewan was performed. Thirty rhizobiophages were trapped from these soils using strains of R. leguminosarum (3841 or VF39SM) and R. gallicum. Optimal storage conditions, classification based on: host range, TEM and RAPD-PCR, and phage transduction abilities were determined. Two phages were selected for detailed study and genome sequencing: L338C, a Siphoviridae and Pl0VF, a Myoviridae. Finally qPCR was demonstrated to be a viable tool for the detection of Pl0VF in the soil.
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    Myoviridae as Part of Cattle Fecal Microbiome
    (2019-03-18) Rincón García, Mónica; van der Meer, Frank; Bachofen, Claudia; Orsel, Karin; Timsit, Édouard; Hynes, Michael F.
    Double-stranded deoxyribonucleic acid (dsDNA) phages from the order of Caudovirales are the most abundant viruses in the microbiome of the mammalian digestive tract. There is increasing awareness of the role of phages in modulating bacterial composition in digestive compartments. Mammalian digestive microbiomes are generally dominated by members of the Bacteroidetes and Firmicutes phyla. Moreover, phages from the Siphoviridae family (order Caudovirales) are the most abundant members in the rumen of cattle and large intestine of humans, horses and pigs. However, there is a knowledge gap regarding composition of the virome in the large intestine of cattle. To facilitate these studies, suitable methods to analyze bovine fecal sample for virus content are needed. Therefore, the objective of this thesis was to establish appropriate methodologies and evaluate fecal samples from various sources. Two virus particle purification (VPP) procedures, namely filtration and cesium chloride (CsCl) ultracentrifugation were compared, using electron microscopy (EM), polymerase chain reaction (PCR) and metagenomic analyses. In total, 39 cattle fecal samples were processed. Metagenomic analysis was the most suitable methodology for measuring relative abundance (RA) and diversity of Myoviridae in fecal samples from two beef and two dairy cattle. Overall, numbers of phage members of the order Caudovirales was higher compared to other dsDNA viruses, with Myoviridae being the most abundant family within this order. Furthermore, a functional procedure to estimate total viral dsDNA virome in feces was developed. In conclusion, this thesis includes methods to detect and characterize phages in cattle feces, with generation of novel data that improve understanding of viral diversity in fecal microbiomes.
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    Organisation and regulation of the fixG operon of Rhizobium leguminosarum
    (1995) Mitsch, Michael J.; Hynes, Michael F.
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    Plasmid biology of rhizobium leguminosarum: novel conjugation system and glycerol catabolic genes
    (2012) Ding, Hao; Hynes, Michael F.
    Large plasmids in rhizobia play important roles in rhizobia-legume symbioses and bacterial survival and competitiveness in the rhizosphere. In R. leguminosarum bv. viciae VF39SM, a novel conjugation system on plasmid pRleVF39b was isolated and found to be different from the rhizobial quorum sensing-regulated or the RctA-repressed conjugation systems. The entire transfer region on pRleVF39b encompasses a trb-like operon and traG, encoding the mating pore formation component and the coupling protein, and a relaxase gene traA located 9 kb downstream of the trb operon. Mutations in any of the above genes completely abolished the transfer of pRleVF39b. The transfer of pRleVF39b was affected mainly by its host genetic background. Under the conditions tested, pRle VF3 9b transferred at highest frequency at about 10-4 transconjugants per recipient from plasmid-free Agrobacterium tumefaciens UBAPF2 to another Agrobacterium recipient. The transfer frequency decreased about 10 to 100-fold when Rhizobium was the donor, and decreased at least 1000-fold when Rhizobium was the recipient. An xre-type transcriptional repressor gene trbR was identified in the region between traG and traA. Mutation in trbR led to SO-fold increases in trb operon expression and pRleVF39b transfer. A glycerol uptake and catabolic locus on pRleVF39c was found to comprise a glpR gene, encoding a transcriptional regulator, and the glp operon, including a glpD gene, encoding glycerol 3-phosphate dehydrogenase, glpSTPQUV encoding an ATP binding cassette transporter, and a glpK gene encoding glycerol kinase. All the genes within the operon were required for growth on glycerol, except for glpK whose function can be partially complemented by a glpKch gene on the chromosome. The glp operon was inducible by glycerol, glycerol 3-phosphate, and pea seed exudates. GlpR represses the expression of the glp operon in the absence of inducer. Glycerol uptake in Rhizobium was shown to be an active process mediated by an ABC transporter. Mutants that are unable to utilize glycerol as a sole carbon source were less competitive in nodulating peas than the wild-type, indicating that glycerol catabolism might confer advantages to the bacterium in the rhizosphere or in plants.
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    Rhizobiophage Functional Genomics
    (2020-08-19) Koswaththa Muhandiramlage, Damitha Gunathilake; Hynes, Michael F.; Yost, Christopher Karl; Dunfield, Peter F.; Strous, Marc
    Rhizobiophages 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.
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    The role of RpoN in gene regulation in rhizobium leguminosarum
    (2003) Clark, Scott Roy David; Hynes, Michael F.