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Importance: This study reports the results of the largest analysis of genome sequences from phages that infect the Alphaproteobacteria class of bacterial hosts. We analyzed over 100 whole genome sequences of phages to construct dotplots, categorize them into genetically distinct clusters, generate a bootstrapped phylogenetic tree, compute protein orthologs, and predict packaging strategies. We determined that the phage sequences primarily cluster by the bacterial host family, phage morphotype, and genome size. We expect that the findings reported in this seminal study will facilitate future analyses that will improve our knowledge of the phages that infect these hosts., Competing Interests: The authors declare no conflict of interest.

The Enterobacteriales order is composed of Gram-negative bacteria that range from harmless symbionts to well-studied pathogens. We announce complete genome sequences of five related SO-1-like Enterobacteriales bacteriophages (also known as the Dhillonvirus genus) isolated from wastewater that infect Escherichia coli (Opt-212, Over9000, Pubbukkers, and Teewinot) or Shigella boydii (StarDew).

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most destructive bacterial infection in honeybees. Even antibiotic-sensitive strains of P. larvae can produce recurrent AFB months to weeks post-antibiotic treatment due to the survival of bacterial spores. Recently, phages that infect P. larvae have been shown to effectively combat AFB in the field. Here, we present evidence that phages not only bind to vegetative P. larvae but also bind to P. larvae spores. Spore binding was observed in the results of three specific experiments: (1) bacteria counted by flow cytometry generated quantitative data of FITC-labeled phages that were bound to vegetative bacteria as well as those bound to spores, (2) electron microscopy captured images of phages bound to the surface of spores in both horizontal and vertical positions, and (3) phages incubated with P. larvae spores bound to the spores and created plaques in vegetative bacteria under conditions not conducive to spore activation, indicating that binding to spores is reversible and that the phages are still active. Identification of phages with reversible spore-binding capability for use in phage therapy may improve treatment of sporulating bacterial infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Brady, Roll, Walker, Fajardo, Breakwell, Eggett and Hope.)

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. Here, we discuss the likely mischaracterization of three 16S rRNA sequences previously published as P. larvae and provide the phylogenetic evidence that supported the GenBank reassignment of the sequences as B. laterosporus We explore the issues that arise by using only 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., (Copyright © 2018 American Society for Microbiology.)

Erwinia amylovora is a plant pathogen belonging to the Enterobacteriaceae family, a family containing many plant and animal pathogens. Herein, we announce nine genome sequences of E. amylovora bacteriophages isolated from infected apple trees along the Wasatch Front in Utah.

We present here the complete genomes of 18 phages that infect Paenibacillus larvae, the causative agent of American foulbrood in honeybees. The phages were isolated between 2014 and 2016 as part of an undergraduate phage discovery course at Brigham Young University. The phages were isolated primarily from bee debris and lysogens.

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., (Copyright © 2018 Walker et al.)

A critical area of emphasis for science educators is the identification of effective means of teaching and engaging undergraduate students. Personal microbiome analysis is a means of identifying the microbial communities found on or in our body. We hypothesized the use of personal microbiome analysis in the classroom could improve science education by making courses more applied and engaging for undergraduate students. We determined to test this prediction in three Brigham Young University undergraduate courses: Immunology, Advanced Molecular Biology Laboratory, and Genomics. These three courses have a two-week microbiome unit and students during the 2016 semester students could submit their own personal microbiome kit or use the demo data, whereas during the 2017 semester students were given access to microbiome data from an anonymous individual. The students were surveyed before, during, and after the human microbiome unit to determine whether analyzing their own personal microbiome data, compared to analyzing demo microbiome data, impacted student engagement and interest. We found that personal microbiome analysis significantly enhanced the engagement and interest of students while completing microbiome assignments, the self-reported time students spent researching the microbiome during the two week microbiome unit, and the attitudes of students regarding the course overall. Thus, we found that integrating personal microbiome analysis in the classroom was a powerful means of improving student engagement and interest in undergraduate science courses.

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., (Copyright © 2017 Esplin et al.)

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.

Bacteria employ a diverse array of strategies to survive under extreme environmental conditions but maintaining these adaptations comes at an energetic cost. If energy reserves drop too low, extremophiles may enter a dormant state to persist. We estimated bacterial dormancy and identified the environmental variables influencing our activity proxy in 10 hypersaline and freshwater lakes across the Western United States. Using ribosomal RNA:DNA ratios as an indicator for bacterial activity, we found that the proportion of the community exhibiting dormancy was 16% lower in hypersaline than freshwater lakes. Based on our indicator variable multiple regression results, saltier conditions in both freshwater and hypersaline lakes increased activity, suggesting that salinity was a robust environmental filter structuring bacterial activity in lake ecosystems. To a lesser degree, higher total phosphorus concentrations reduced dormancy in all lakes. Thus, even under extreme conditions, the competition for resources exerted pressure on activity. Within the compositionally distinct and less diverse hypersaline communities, abundant taxa were disproportionately active and localized in families Microbacteriaceae (Actinobacteria), Nitriliruptoraceae (Actinobacteria), and Rhodobacteraceae (Alphaproteobacteria). Our results are consistent with the view that hypersaline communities are able to capitalize on a seemingly more extreme, yet highly selective, set of conditions and finds that extremophiles may need dormancy less often to thrive and survive.

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., (Copyright © 2015 Merrill et al.)

Background: The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains., Results: Whole genome nucleotide and proteome comparison of the 93 extant Bacillus phages revealed 12 distinct clusters, 28 subclusters and 14 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member of the group. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,922 protein families (phams) of which only 951 (19%) had a predicted function. In addition, 3,058 (62%) of phams were orphams (phams containing a gene product from a single phage). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains., Conclusions: This analysis provides a basis for understanding and characterizing Bacillus phages and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: 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., Results: 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., Conclusions: 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.

Great Salt Lake (GSL) represents one of the world's most hypersaline environments. In this study, the archaeal and bacterial communities at the North and South arms of the lake were surveyed by cloning and sequencing the 16S rRNA gene. The sampling locations were chosen for high salt concentration and the presence of unique environmental gradients, such as petroleum seeps and high sulfur content. Molecular techniques have not been systematically applied to this extreme environment, and thus the composition and the genetic diversity of microbial communities at GSL remain mostly unknown. This study led to the identification of 58 archaeal and 42 bacterial operational taxonomic units. Our phylogenetic and statistical analyses displayed a high biodiversity of the microbial communities in this environment. In this survey, we also showed that the majority of the 16S rRNA gene sequences within the clone library were distantly related to previously described environmental halophilic archaeal and bacterial taxa and represent novel phylotypes.

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.

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.

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.

Background: Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it is typically the longest gene in mycobacteriophage genomes and because regions within the TMP gene are conserved., Results: A single gene, TMP, identified the known Mycobacteriophage clusters and subclusters using a Gepard dotplot comparison or a phylogenetic tree constructed from global alignment and maximum likelihood comparisons. Gepard analysis of 247 mycobacteriophage TMP sequences appropriately recovered 98.8% of the subcluster assignments that were made by whole-genome comparison. Subcluster-specific primers within TMP allow for PCR determination of the mycobacteriophage subcluster from DNA samples. Using the single-gene comparison approach for siphovirus coliphages, phage groupings by TMP comparison reflected relationships observed in a whole genome dotplot comparison and confirm the potential utility of this approach to another widely studied group of phages., Conclusions: TMP sequence comparison and PCR results support the hypothesis that a single gene can be used for distinguishing phage cluster and subcluster assignments. TMP single-gene analysis can quickly and accurately aid in mycobacteriophage classification.

Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1.

Serial dilution and plate counting is often taught in courses for both microbiology and allied health students. Lecture examples and examination questions addressing how the method is used can sometimes be contrived: artificial data sets may have little or no meaning other than to have students perform a calculation. Here we provide a set of activities employing data sets acquired from the primary literature. Our objective was to have the students think critically about a real scenario in which serial dilution and plate count was used. Each activity requires students to read a paragraph describing the study, predict the results, perform the appropriate calculations, and then evaluate the results in light of their predictions. To test the efficacy of these activities, a pretest quiz was given to approximately 100 students in an allied health/general microbiology course. After a lecture on how microbes are enumerated, students were given a different quiz. The class was then divided randomly into groups of three or four students and assigned one of the activities. A postactivity quiz was also administered. Approximately two weeks later, a serial dilution/plate count question was used on an examination and served as a final posttest. Standardized learning gains were calculated for the quiz administered after each learning activity. Even though learning gains were significantly higher after the lecture, there was also a significant improvement between the lecture and the activity. Using an exercise based on an authentic set of data significantly improved student learning gains, and is a useful practice for teaching microbiology.

Bacterial capsules are composed of high-molecular-weight polysaccharides and/or polypeptides, and are associated with virulence and biofilm formation. Unfortunately, capsules do not stain well with crystal violet, methylene blue, or other simple stains. This unit describes two methods of capsule staining. The first is a wet-mount method using india ink; the capsule is visualized as a refractile zone surrounding a cell. The second is a direct-staining dry-mount method that precipitates copper sulfate and leaves the capsule as a pale blue zone. Both methods are easily performed within approximately 5 min.

Bacterial flagella are appendages used for motility. Their presence is a useful tool for identification and differentiation of prokaryotes. Since flagella are too thin to be seen by compound light microscopy, staining methods employ the use of a mordant (often tannic acid) to make them thick enough to see using an oil immersion objective. Two protocols are described. Basic Protocol 1 is a modified Leifson method and is the one that many microbiologists have adapted. Basic Protocol 2 is a wet-mount stain using a Ryu stain and is included because the stain is stable at room temperature. Both of these methods are fairly time-consuming, taking from 15 to as long as 60 min to perform., ((c) 2009 by John Wiley & Sons, Inc.)

Acid-fastness is an uncommon characteristic shared by the genera Mycobacterium (Section 10A) and Nocardia. Because of this feature, this stain is extremely helpful in identification of these bacteria. Although Gram positive, acid-fast bacteria do not take the crystal violet into the wall well, appearing very light purple rather than the deep purple of normal Gram-positive bacteria., ((c) 2009 by John Wiley & Sons, Inc.)

Simple staining involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail, in contrast to negative staining where the bacteria remain unstained against a dark background., ((c) 2009 by John Wiley & Sons, Inc.)

Endospore production is a very important characteristic of some bacteria, allowing them to resist adverse environmental conditions such as desiccation, chemical exposure, extreme heat, radiation, etc. The identification of endospores is also very important for the clinical microbiologist who is analyzing a patient's body fluid or tissue-there are not that many spore-forming genera. In fact, there are two major pathogenic spore-forming genera, Bacillus and Clostridium, together causing a number of lethal diseases-botulism, gangrene, tetanus, and anthrax, to name a few., ((c) 2009 by John Wiley & Sons, Inc.)

In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures., ((c) 2009 by John Wiley & Sons, Inc.)

Negative staining is one of the many staining techniques that can be employed for viewing of bacterial cell morphology and size. The advantages of the negative stain include the use of only one stain and the absence of heat fixation of the sample. Negative staining employs the use of an acidic stain and, due to repulsion between the negative charges of the stain and the bacterial surface, the dye will not penetrate the cell. In negative staining, the results yield a clear cell with a dark background., ((c) 2009 by John Wiley & Sons, Inc.)

A strategy was adapted for using the primary literature to foster active learning in an allied health microbiology course. Recent journal articles were selected that underscored the fundamental microbiological principles to be learned in each course unit. At the beginning of the semester, students were taught the relationship between the layout of scientific articles and the scientific method. During the rest of the semester, students were oriented to the topic of each paper by viewing videos from Unseen Life on Earth: an Introduction to Microbiology, reading assigned pages from the text, and participating in mini-lectures and discussions. After all preparatory material was completed, a paper was read and discussed in small groups and as a class. Students were assessed using daily reading quizzes and end-of-unit concept quizzes. While reading quizzes averaged approximately 93%, concept quiz grades averaged approximately 82%. Student recognition of the terms used in each unit's scientific article was assessed with pre-read and post-read wordlists. For the self-assessment, the percent change between pre-read and post-read word cognition was, as expected, highly significant. Approximately 80% of students agreed that reading the scientific articles was a valuable part of the class and that it provided meaning to their study of microbiology. Using the primary scientific literature facilitated active learning in and out of the classroom. This study showed that introducing the scientific literature in an allied health microbiology class can be an effective way of teaching microbiology by providing meaning through the current literature and understanding of the scientific method.