Education & Certifications
MS, Brigham Young University, Microbiology and Molecular Biology (2015)
BS, Brigham Young University, Molecular Biology (2014)
A PCR-BASED METHOD FOR DISTINGUISHING BETWEEN TWO COMMON BEEHIVE BACTERIA, PAENIBACILLUS LARVAE AND BREVIBACILLUS LATEROSPORUS.
Applied and environmental microbiology
Paenibacillus larvae and Brevibacillus laterosporus are two bacteria that are members of the Paenibacillaceae family. Both are commonly found in beehives and have historically been difficult to distinguish from each other due to related genetic and phenotypic characteristics and a shared ecological niche. Herein, we discuss the likely mischaracterization of three 16S rRNA sequences previously published as P. larvae and provide the phylogenetic evidence that supported the GenBank re-assignment of the sequences as B. laterosporus We explore the issues that arise by only using 16S rRNA or other single gene analyses to distinguish between these bacteria. We also present three sets of molecular markers, two sets that distinguish P. larvae from B. laterosporus and other closely related species within the Paenibacillus genus, and a third set that distinguishes B. laterosporus from P. larvae and other closely related species within the Brevibacillus genus. These molecular markers provide a tool for proper identification of these oft-mistaken species.Importance 16S rRNA gene sequencing in bacteria has long been held as the gold standard for typing bacteria and, for the most part, is an excellent method of taxonomically identifying different bacterial species. However, the high level of 16S rRNA sequence similarity of some published strains of P. larvae and B. laterosporus, as well as possible horizontal gene transfer events within their shared ecological niche complicates the use of 16S rRNA sequence as an effective molecular marker for differentiating these two species. Additionally, shared characteristics of these bacteria limit the effectiveness of using traditional phenotypic identification assays, such as the catalase test. The results from this study provide PCR methods to quickly differentiate between these two genera and will be useful when studying Brevibacillus, Paenibacillus, and other disease-relevant bacteria commonly found in beehives.
View details for DOI 10.1128/AEM.01886-18
View details for PubMedID 30217838
Transient Osmotic Perturbation Causes Long-Term Alteration to the Gut Microbiota.
2018; 173 (7): 1742
Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains invitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.
View details for DOI 10.1016/j.cell.2018.05.008
View details for PubMedID 29906449
Complete Genome Sequences of Paenibacillus larvae Phages BN12, Dragolir, Kiel007, Leyra, Likha, Pagassa, PBL1c, and Tadhana.
2018; 6 (24)
We present here the complete genomes of eight phages that infect Paenibacillus larvae, the causative agent of American foulbrood in honeybees. Phage PBL1c was originally isolated in 1984 from a P. larvae lysogen, while the remaining phages were isolated in 2014 from bee debris, honeycomb, and lysogens from three states in the USA.
View details for DOI 10.1128/genomeA.01602-17
View details for PubMedID 29903825
Genome Sequences of 19 Novel Erwinia amylovora Bacteriophages.
2017; 5 (46)
Erwinia amylovora is the causal agent of fire blight, a devastating disease affecting some plants of the Rosaceae family. We isolated bacteriophages from samples collected from infected apple and pear trees along the Wasatch Front in Utah. We announce 19 high-quality complete genome sequences of E.amylovora bacteriophages.
View details for DOI 10.1128/genomeA.00931-17
View details for PubMedID 29146842
Bacteriophages as an alternative to conventional antibiotic use for the prevention or treatment of Paenibacillus larvae in honeybee hives
JOURNAL OF INVERTEBRATE PATHOLOGY
2017; 150: 94–100
American Foulbrood (AFB) is an infectious disease caused by the bacteria, Paenibacillus larvae. P. larvae phages were isolated and tested to determine each phages' host range amongst 59 field isolate strains of P. larvae. Three phages were selected to create a phage cocktail for the treatment of AFB infections according to the combined phages' ability to lyse all tested strains of bacteria. Studies were performed to demonstrate the safety and efficacy of the phage cocktail treatment as a replacement for traditional antibiotics for the prevention of AFB and the treatment of active infections. Safety verification studies confirmed that the phage cocktail did not adversely affect the rate of bee death even when administered as an overdose. In a comparative study of healthy hives, traditional prophylactic antibiotic treatment experienced a 38±0.7% decrease in overall hive health, which was statistically lower than hive health observed in control hives. Hives treated with phage cocktail decreased 19±0.8%, which was not statistically different than control hives, which decreased by 10±1.0%. In a study of beehives at-risk for a natural infection, 100±0.5% of phage-treated hives were protected from AFB infection, while 80±0.5% of untreated controls became infected. AFB infected hives began with an average Hitchcock score of 2.25 out of 4 and 100±0.5% of the hives recovered completely within two weeks of treatment with phage cocktail. While the n numbers for the latter two studies are small, the results for both the phage protection rate and the phage cure rate were statistically significant (α=0.05). These studies demonstrate the powerful potential of using a phage cocktail against AFB and establish phage therapy as a feasible treatment.
View details for DOI 10.1016/j.jip.2017.09.010
View details for Web of Science ID 000416298200016
View details for PubMedID 28917651
A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites.
2017; 551 (7682): 648–52
The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.
View details for DOI 10.1038/nature24661
View details for PubMedID 29168502
DDX6 Represses Aberrant Activation of Interferon-Stimulated Genes.
2017; 20 (4): 819–31
The innate immune system tightly regulates activation of interferon-stimulated genes (ISGs) to avoid inappropriate expression. Pathological ISG activation resulting from aberrant nucleic acid metabolism has been implicated in autoimmune disease; however, the mechanisms governing ISG suppression are unknown. Through a genome-wide genetic screen, we identified DEAD-box helicase 6 (DDX6) as a suppressor of ISGs. Genetic ablation of DDX6 induced global upregulation of ISGs and other immune genes. ISG upregulation proved cell intrinsic, imposing an antiviral state and making cells refractory to divergent families of RNA viruses. Epistatic analysis revealed that ISG activation could not be overcome by deletion of canonical RNA sensors. However, DDX6 deficiency was suppressed by disrupting LSM1, a core component of mRNA degradation machinery, suggesting that dysregulation of RNA processing underlies ISG activation in the DDX6 mutant. DDX6 is distinct among DExD/H helicases that regulate the antiviral response in its singular ability to negatively regulate immunity.
View details for DOI 10.1016/j.celrep.2017.06.085
View details for PubMedID 28746868
View details for PubMedCentralID PMC5551412
Software-based analysis of bacteriophage genomes, physical ends, and packaging strategies
Phage genome analysis is a rapidly growing field. Recurrent obstacles include software access and usability, as well as genome sequences that vary in sequence orientation and/or start position. Here we describe modifications to the phage comparative genomics software program, Phamerator, provide public access to the code, and include instructions for creating custom Phamerator databases. We further report genomic analysis techniques to determine phage packaging strategies and identification of the physical ends of phage genomes.The original Phamerator code can be successfully modified and custom databases can be generated using the instructions we provide. Results of genome map comparisons within a custom database reveal obstacles in performing the comparisons if a published genome has an incorrect complementarity or an incorrect location of the first base of the genome, which are common issues in GenBank-downloaded sequence files. To address these issues, we review phage packaging strategies and provide results that demonstrate identification of the genome start location and orientation using raw sequencing data and software programs such as PAUSE and Consed to establish the location of the physical ends of the genome. These results include determination of exact direct terminal repeats (DTRs) or cohesive ends, or whether phages may use a headful packaging strategy. Phylogenetic analysis using ClustalO and phamily circles in Phamerator demonstrate that the large terminase gene can be used to identify the phage packaging strategy and thereby aide in identifying the physical ends of the genome.Using available online code, the Phamerator program can be customized and utilized to generate databases with individually selected genomes. These databases can then provide fruitful information in the comparative analysis of phages. Researchers can identify packaging strategies and physical ends of phage genomes using raw data from high-throughput sequencing in conjunction with phylogenetic analyses of large terminase proteins and the use of custom Phamerator databases. We promote publication of phage genomes in an orientation consistent with the physical structure of the phage chromosome and provide guidance for determining this structure.
View details for DOI 10.1186/s12864-016-3018-2
View details for Web of Science ID 000384980300001
View details for PubMedID 27561606
View details for PubMedCentralID PMC5000459
Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages
2016; 11 (6)
Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared.
View details for DOI 10.1371/journal.pone.0156838
View details for Web of Science ID 000377824800025
View details for PubMedID 27304881
View details for PubMedCentralID PMC4909266
Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae
Spounavirinae viruses have received an increasing interest as tools for the control of harmful bacteria due to their relatively broad host range and strictly virulent phenotype.In this study, we collected and analyzed the complete genome sequences of 61 published phages, either ICTV-classified or candidate members of the Spounavirinae subfamily of the Myoviridae. A set of comparative analyses identified a distinct, recently proposed Bastille-like phage group within the Spounavirinae. More importantly, type 1 thymidylate synthase (TS1) and dihydrofolate reductase (DHFR) genes were shown to be unique for the members of the proposed Bastille-like phage group, and are suitable as molecular markers. We also show that the members of this group encode beta-lactamase and/or sporulation-related SpoIIIE homologs, possibly questioning their suitability as biocontrol agents.We confirm the creation of a new genus--the "Bastille-like group"--in Spounavirinae, and propose that the presence of TS1- and DHFR-encoding genes could serve as signatures for the new Bastille-like group. In addition, the presence of metallo-beta-lactamase and/or SpoIIIE homologs in all members of Bastille-like group phages makes questionable their suitability for use in biocontrol.
View details for DOI 10.1186/s12864-015-1757-0
View details for Web of Science ID 000359201700001
View details for PubMedID 26250905
View details for PubMedCentralID PMC4528723
Genome Sequences of Five Additional Brevibacillus laterosporus Bacteriophages.
2015; 3 (5)
Brevibacillus laterosporus has been isolated from many different environments, including beehives, and produces compounds that are toxic to many organisms. Five B. laterosporus phages have been isolated previously. Here, we announce five additional phages that infect this bacterium, including the first B. laterosporus siphoviruses to be discovered.
View details for DOI 10.1128/genomeA.01146-15
View details for PubMedID 26494658
View details for PubMedCentralID PMC4616168
- Correction for Sheflo et al., Complete Genome Sequences of Five Brevibacillus laterosporus Bacteriophages. Genome announcements 2015; 3 (5)
The Genomes, Proteomes, and Structures of Three Novel Phages That Infect the Bacillus cereus Group and Carry Putative Virulence Factors
JOURNAL OF VIROLOGY
2014; 88 (20): 11846-11860
This article reports the results of studying three novel bacteriophages, JL, Shanette, and Basilisk, which infect the pathogen Bacillus cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 open reading frames (ORFs) and 220 ORFs, respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and 4, respectively, were predicted to be present by sequence analysis. Structural protein homology to well-characterized phages suggested that JL and Shanette were members of the family Myoviridae, which was confirmed by TEM. The third phage, Basilisk, was similar only to uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T=9 icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes, including TerC and TerD involved in tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus, B. anthracis, and B. thuringiensis. This study provides a basis for understanding these three phages and other related phages as well as their contributions to the pathogenicity of B. cereus group bacteria. Importance: The Bacillus cereus group of bacteria contains several human and plant pathogens, including B. cereus, B. anthracis, and B. thuringiensis. Phages are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding the evolution of pathogenic strains. Herein we provide the results of detailed study of three novel B. cereus phages, two highly related myoviruses (JL and Shanette) and an unrelated siphovirus (Basilisk). The detailed characterization of host range and superinfection, together with results of genomic, proteomic, and structural analyses, reveal several putative virulence factors as well as the ability of these phages to infect different pathogenic species.
View details for DOI 10.1128/JVI.01364-14
View details for Web of Science ID 000342688000020
View details for PubMedID 25100842
View details for PubMedCentralID PMC4178739
Characterization of Paenibacillus larvae bacteriophages and their genomic relationships to firmicute bacteriophages
Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages.P. larvae phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 are myoviruses with ~50 kbp genomes. The six P. larvae phages form three distinct groups by dotplot analysis. An annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. Sixty phage assembly or structural protein genes and 133 regulatory or other non-structural protein genes were identifiable among the six P. larvae phages. Jimmer1, Jimmer2, and Davies formed stable lysogens resistant to superinfection by genetically similar phages. The correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages Emery and Abouo in electron micrographs. A Phamerator database was assembled with the P. larvae phage genomes and 107 genomes of Firmicute-infecting phages, including 71 Bacillus phages. Phamerator identified conserved domains in 1,501 of 6,181 phamilies (only 24.3%) encoded by genes in the database and revealed that P. larvae phage genomes shared at least one phamily with 72 of the 107 other phages. The phamily relationship of large terminase proteins was used to indicate putative DNA packaging strategies. Analyses from CoreGenes, Phamerator, and electron micrograph measurements indicated Jimmer1, Jimmer2, Abouo and Davies were related to phages phiC2, EJ-1, KC5a, and AQ113, which are small-genome myoviruses that infect Streptococcus, Lactobacillus, and Clostridium, respectively.This paper represents the first comparison of phage genomes in the Paenibacillus genus and the first organization of P. larvae phages based on sequence and structure. This analysis provides an important contribution to the field of bacteriophage genomics by serving as a foundation on which to build an understanding of the natural predators of P. larvae.
View details for DOI 10.1186/1471-2164-15-745
View details for Web of Science ID 000341790000001
View details for PubMedID 25174730
View details for PubMedCentralID PMC4168068
Genome Sequences of Three Novel Bacillus cereus Bacteriophages.
2014; 2 (1)
The Bacillus cereus group is an assemblage of highly related firmicute bacteria that cause a variety of diseases in animals, including insects and humans. We announce three high-quality, complete genome sequences of bacteriophages we isolated from soil samples taken at the bases of fruit trees in Utah County, Utah. While two of the phages (Shanette and JL) are highly related myoviruses, the bacteriophage Basilisk is a siphovirus.
View details for DOI 10.1128/genomeA.01118-13
View details for PubMedID 24459255
View details for PubMedCentralID PMC3900887
Genome sequences of five b1 subcluster mycobacteriophages.
2013; 1 (6)
Mycobacteriophages infect members of the Mycobacterium genus in the phylum Actinobacteria and exhibit remarkable diversity. Genome analysis groups the thousands of known mycobacteriophages into clusters, of which the B1 subcluster is currently the third most populous. We report the complete genome sequences of five additional members of the B1 subcluster.
View details for DOI 10.1128/genomeA.00968-13
View details for PubMedID 24285667
View details for PubMedCentralID PMC3869329
Complete Genome Sequences of Five Paenibacillus larvae Bacteriophages.
2013; 1 (6)
Paenibacillus larvae is a pathogen of honeybees that causes American foulbrood (AFB). We isolated bacteriophages from soil containing bee debris collected near beehives in Utah. We announce five high-quality complete genome sequences, which represent the first completed genome sequences submitted to GenBank for any P. larvae bacteriophage.
View details for DOI 10.1128/genomeA.00668-13
View details for PubMedID 24233582
View details for PubMedCentralID PMC3828306