Browsing by Author "Hynes, Michael"

Now showing 1 - 7 of 7
  • Thumbnail Image
    ItemOpen Access
    Biodegradation of Starch by Microbial Communities in Soil Collected from Olduvai Gorge in Northern Tanzania
    (2020-07-07) Akeju, Tolutope O.; Dunfield, Peter; Mercader, Julio; Dunfield, Peter; Mercader, Julio; Hynes, Michael; Snowdon, Lloyd; Samuel, Marcus
    Starch is the main component of most dietary intake by humans and it is synthesized as starch granules in different plant parts. Once exposed to the soil, starch is susceptible to biodegradation by enzymes produced by communities of soil bacteria and fungi. However, the preservation of starch granules in the archaeological record (thousands of years) has been documented. This preservation is poorly understood and there is a paucity of information in the scientific literature on the effects of oxygen and moisture on the enzymatic degradation of starches from different botanical sources in the soil. This study attempts to fill these knowledge gaps by examining soil–starch microcosms subjected to different moisture and oxygen conditions. These microcosms were sampled at regular intervals to observe the metabolic activity of the soil microbes using gas chromatography and also to identify the dominant microbial clades degrading the starch granules using 16S rRNA and 18S rRNA gene analyses. Commercial wheat, corn and potato starches were used in addition to a starch of ethnobotanical interest named Ipomoea longituba collected from Oldupai Gorge. Results indicated that wheat starch and Ipomoea longituba were the most susceptible to microbial degradation while potato starch was more recalcitrant. Starch degradation rates were significantly affected by starch type, soil moisture content, oxygen and calcium phosphate. 16S and 18S rRNA gene analyses showed that starch degradation involves both bacteria and fungi and important taxa that were involved in starch breakdown include Ascomycota, Alkalibacterium, Streptomyces and others, mostly Gram-positive bacteria. In addition, from the preliminary results on the microbial communities living in the rhizosphere of Ipomoea longituba and bulk soil, we can conclude that the microbiome of the rhizosphere of Ipomoea longituba is very different from the microbiome of the surrounding bulk soil that was collected from around 1 m2 of the plant.
  • Thumbnail Image
    ItemOpen Access
    Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins
    (BioMed Central, 2010-08-17) Tambalo, Dinah D.; Bustard, Denise E.; Del Bel, Kate L.; Koval, Susan F.; Khan, Morgan F.; Hynes, Michael
  • Thumbnail Image
    ItemOpen Access
    Comparing methods for recovering genomic RNA from SARS-CoV-2 in municipal wastewater for molecular analysis
    (2023-12-04) McCalder, Janine Alison; Hubert, Casey; Parkins, Michael; Corcoran, Jennifer; Gieg, Lisa; Hynes, Michael
    SARS-CoV-2, an enveloped virus with a single stranded positive sense RNA genome (Baltimore class 4), is shed in the excreta (e.g., feces, sputum, urine) of people infected with the virus, resulting in the presence of viral genetic fragments in municipal wastewater. This RNA is quantifiable using wastewater-based surveillance (WBS) molecular biology and genomic methods. RNA extracted from wastewater can be assessed using RT-qPCR to track disease trends within populations of various sizes (e.g., whole city, neighbourhoods, hospitals, etc) in a non-invasive, unbiased, and highly inclusive way. Despite accelerated development and utilisation of WBS during the COVID-19 pandemic, many unknowns and challenges still confront this burgeoning field. Fundamental structural differences between enveloped SARS-CoV-2 and the majority of viruses in wastewater (e.g., non-enveloped enteric viruses) raise important questions about how previously established methods perform for this novel coronavirus. Establishing a reliable workflow for wastewater-based monitoring of SARS-CoV-2 requires a series of connected methods, including viral recovery from wastewater and downstream molecular and/or genomic analyses. Selecting the best possible components of an overall workflow requires method comparison and optimisation. In this study, several wastewater processing methods were assessed for the recovery of a bovine coronavirus surrogate, which has a similar structure to that of SARS-CoV-2, to establish an effective method for viral recovery from wastewater. This comparison revealed that ultrafiltration and direct extraction performed better than electronegative membrane filtration, which exhibited RT-qPCR gene abundance estimates an order of magnitude lower than the other two methods. Consequently, two validated workflows implementing ultrafiltration and direct extraction were then compared during a 29-month longitudinal analysis of SARS-CoV-2 quantification using RT-qPCR. Despite each workflow representing a distinct method for viral recovery, trends were similar with both methods indicating that consistent application of a given workflow, and not necessarily which workflow is used, is paramount in WBS. RNA extraction methods were furthermore compared using an amplicon-based sequencing strategy to assess the SARS-CoV-2 variant profiles in municipal wastewater samples. This comparison did not reveal significant differences in sequencing results between the two extraction strategies, but rather underscored the importance of wastewater sample handling and storage prior to genomic assessment.
  • Thumbnail Image
    ItemOpen Access
    Evaluation of Genes for Escherichia coli Envelope Proteins for their Participation with the Small Multidrug Resistance Protein, EmrE, Mediated pH/Osmotic Tolerance
    (2017) Pushpker, Rajnigandha; Turner, Raymond; Storey, Douglas; Hynes, Michael; Schryvers, Anthony
    The small multidrug resistance (SMR) protein, EmrE transports quaternary cationic compounds (QCC) across the cytoplasmic membrane in the Gram-negative bacterium Escherichia coli. EmrE over-expression results in the reduction of host pH and osmotic tolerance, likely through EmrE-mediated biological QCC based osmoprotectant efflux. It is not known how the substrates translocated by EmrE into the periplasmic space escape across the outer membrane (OM). The observation that an OM protein, OmpW, participates in EmrE-mediated substrate efflux provided the hypothesis that additional OM and periplasmic proteins participate in the process. By conducting alkaline pH based growth phenotype screens I identified 10 additional proteins that may contribute to the EmrE-mediated osmoprotectant efflux: GspD, HofQ, YccZ, AcrA, EmrA, EmrB, ProX, OsmF, DcrB and YggM. Further screening of these using a hyperosmotic stress ± betaine growth phenotype assay identified OmpW and two periplasmic proteins, DcrB and YggM, to have involvement in the EmrE-mediated export of betaine.
  • Thumbnail Image
    ItemOpen Access
    Genomic and Proteomic Approaches to Understanding Conjugation in Rhizobium leguminosarum VF39
    (2023-03-02) Hiniduma Gama Achchi, Kasuni Maheshika Hemananda; Hynes, Michael; Chua, Gordon; Fraser, Marie; Schryvers, Anthony; Torres Tejerizo, Gonzalo
    The root nodule bacterium Rhizobium leguminosarum strain VF39 contains six large single copy plasmids and a single circular chromosome. These six plasmids are named pRleVF39a to pRleVF39f in order of increasing size. pRleVF39a and pRleVF39b are self-transmissible plasmids. pRleVF39b has a type IVa rhizobial conjugation system and is characterized by presence of a shorter relaxase gene (traA) than other systems. It also has a gene encoding a negative transcriptional regulator (trbR) belonging to the Xre family and shows a lack of some common genes found in the other conjugation systems. The transfer region of pRleVF39b contains 15 hypothetical protein encoding genes with no known function. It was hypothesized that some of these proteins interact with TraA to form a relaxase complex. Thus, TraA protein-protein interactions were analysed by performing a pull-down assay followed by LC-MS/MS identification. The analysis has shown TraA interaction with the proteins encoded in trb region of pRleVF39b, and some other chromosomally encoded proteins with known TraA interaction. These interactions appear to be largely dependent on the presence of the coupling protein, TraG. Transposon sequencing experiments were performed to identify any additional genes potentially involved in pRleVF39a and pRleVF39b plasmid transfer. The results revealed 67 genes potentially involved in pRleVF39b plasmid transfer and 53 genes putatively involved in pRleVF39a plasmid transfer. The previously known but not well-studied repressor (traM) and activator (traR) in the pRleVF39a conjugation system and hyp1 and hyp2 genes located immediately downstream of traR were identified as potential regulators of pRleVF39a plasmid transfer. The pRleVF39b gene list contains 23 genes identified from previous studies and 44 genes newly identified from this work. The effect of the mutation of traG, encoding the coupling protein, on transfer of pRleVF39b to plasmid-free Agrobacterium recipients was analyzed. A traG marked mutant reduced the plasmid transfer frequency by ~200 fold compared to the wild type. Transfer frequency of the pRleVF39b plasmid was zero when the traG marked mutant was made in a pRleVF39a cured background, suggesting that residual transfer by traG mutants made in the wild type background might be a result of cointegrates between pRleVF39a and pRleVF39b.
  • Thumbnail Image
    ItemOpen Access
    Isolation, characterization, and applications of rhizobiophages
    (2014-10-02) Halmillawewa, Anupama; Hynes, Michael
    Rhizobiophages are the group of bacteriophages that infect rhizobia. The rhizobia constitute a bacterial group that includes members of several different genera and are capable of nodulating legume plant roots, and providing a source of fixed nitrogen for the plant. Rhizobiophages can play a vital role in the Rhizobium-legume symbiosis by altering the population dynamics of the rhizobia present in the rhizosphere, which can be used in agriculture to improve the efficacy of commercial Rhizobium inoculants and mitigate the Rhizobium competition problem. As a prerequisite for the application of such technology, a thorough understanding of rhizobiophage biology is required. Isolation of rhizobiophages from soil samples obtained from Alberta, Saskatchewan, Ontario and British Columbia was performed using several different strains of rhizobia as trapping hosts. The isolated phages were characterized using host range, transmission electron microscopy, protein profiles and genomic characterization. All phage isolates were characterized as tailed phages belonging to the Order Caudovirales, while further classification allowed us to place them in families Siphoviridae, Myoviridae and Podoviridae. Depending on the host range, morphotype and trapping host, several phages were selected and detailed genome characterization was performed using whole genome sequencing. Five rhizobiophage genomes were sequenced to finished state (vB_RleM_P10VF, vB_RglS_P106B, vB_RleM_PPF1, vB_MloP_Lo5R7ANS, and vB_MloP_Cp1R7ANS-C2) and annotated. The complete genome sequences of vB_RglS_P106B (KF977490), vB_RleM_PPF1 (KJ746502), vB_RleM_P10VF (KM199770) and vB_MloP_Lo5R7ANS (KM199771) are publically available at the database of National Center of Biological Information (NCBI). The genome of vB_MloP_Cp1R7ANS-C2 will be submitted to the NCBI in the near future. The integration of temperate phage vB_RleM_PPF1 into its bacterial host R. leguminosarum F1 was also studied. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host. To develop a phage-based inoculant technology, the phages of Rhizobium leguminosarum with broadest possible host range were selected and tested in nodulation competition assays. The presence of phages altered the nodule occupancy by phage-sensitive and phage-resistant strains of rhizobia, increasing the efficacy of nodulation by phageresistant rhizobia under controlled environmental conditions.
  • Thumbnail Image
    ItemOpen Access
    Replication, stability, and incompatibility determinants of Rhizobium leguminosarum plasmids
    (2013-08-16) Yip, Cynthia; Hynes, Michael
    The large stable plasmids of rhizobia play an important role in survival, environmental competition, and plant symbiosis. The six plasmids of Rhizobium leguminosarum bv. viciae VF39SM comprise nearly 35% of the bacterium’s genome and are all repABC replicons, which are defined by the presence of a highly unusual operon containing both replication (repC) and partitioning functions (repA and repB) under the control of a single promoter. The six repABC operons were isolated and were found to confer the autonomous replication, incompatibility characteristics, and strong stability of the large repABC plasmid from which they were derived. The repABC operons of the three largest plasmids were found to have strong incompatibility determinants in the non-protein coding regions, and the location and number of these determinants were unique to each operon. The varying incompatibility determinants were predicted to be the centromere-like site of the plasmid, based on the presence of a 16 nucleotide consensus palindromic sequence. However, in all three repABC operons, the intergenic region between repB and repC was the strongest incompatibility factor, and is predicted to encode a counter-transcribed RNA (ctRNA). Mutations in the promoter region of the ctRNAs result in loss of incompatibility. Deletion of the repA and repB genes of the repABC operons of the three largest plasmids abolished replicon stability, whereas deletion of the intergenic region increased the stability significantly, such that the intergenic deletion replicons were more stable than the entire repABC operon. Depending on the rhizobial host, the intergenic deletion replicon had increased stability due either to an increase in copy number (Agrobacterium tumefaciens host), or a cointegration event with the chromosome or a resident plasmid (A. tumefaciens or R. leguminosarum host). Genomic rearrangements are common in the rhizobial species in this study, but appear to be especially prevalent in R. leguminosarum VF39SM. Cointegration events to mitigate plasmid incompatibility or loss of plasmid stability are common and result in consistent and reproducible patterns, even in a RecA-deficient background.