an-optimized-enrichment-technique-for-isolation-arthrobacter

An Optimized Enrichment Technique for the Isolation of <em>Arthrobacter</em> Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

Cabrini College

Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribosomal subunit (SSU) processome is a large ribonucleoprotein (RNP) required for processing of precursors to the small subunit RNA, the 18S, of the ribosome. We have found that a subcomplex of SSU processome proteins, the t-Utps, is also required for optimal rRNA transcription in vivo in the yeast Saccharomyces cerevisiae. The t-Utps are ribosomal chromatin (r-chromatin)-associated, and they exist in a complex in the absence of the U3 snoRNA. Transcription is required neither for the formation of the subcomplex nor for its r-chromatin association. The t-Utps are associated with the pre-18S rRNAs independent of the presence of the U3 snoRNA. This association may thus represent an early step in the formation of the SSU processome. Our results indicate that rRNA transcription and pre-rRNA processing are coordinated via specific components of the SSU processome.

RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components.

EMS crews encounter implantable cardioverter defibrillators (ICDs) daily, but these encounters rarely involve ICDs firing repeatedly on an awake, alert and understandably frightened individual. But that's exactly what happened when an EMS crew from Cottage Grove, Minn., responded to a man with a known heart condition who reported that his implantable defibrillator was firing inappropriately.

Shock therapy. How donut magnets can suspend inappropriate ICD shocks.

Our study, focused on classroom-based research at the introductory level and using the Phage Genomics course as the model, shows evidence that first-year students doing research learn the process of science as well as how scientists practice science. A preliminary but notable outcome of our work, which is based on a small sample, is the change in student interest in considering different career choices such as graduate education and science in general. This is particularly notable, as previous research has described research internships as clarifying or confirming rather than changing undergraduates' decisions to pursue graduate education. We hypothesize that our results differ from previous studies of the impact of engaging in research because the students in our study are still in the early stages of their undergraduate careers. Our work builds upon the classroom-based research movement and should be viewed as encouraging to the Vision and Change in Undergraduate Biology Education movement advocated by the American Association for the Advancement of Science, the National Science Foundation, and other undergraduate education stakeholders.

Classroom-based science research at the introductory level: changes in career choices and attitude.

Mycobacteriophages represent a genetically diverse group of viruses that infect mycobacterial hosts. Although more than 80 genomes have been sequenced, these still poorly represent the likely diversity of the broader population of phages that can infect the host, Mycobacterium smegmatis mc(2)155. We describe here a newly discovered phage, Marvin, which is a singleton phage, having no previously identified close relatives. The 65,100-bp genome contains 107 predicted protein-coding genes arranged in a noncanonical genomic architecture in which a subset of the minor tail protein genes are displaced about 20 kbp from their typical location, situated among nonstructural genes anticipated to be expressed early in lytic growth. Marvin is not temperate, and stable lysogens cannot be recovered from infections, although the presence of a putative xis gene suggests that Marvin could be a relatively recent derivative of a temperate parent. The Marvin genome is replete with novel genes not present in other mycobacteriophage genomes, and although most are of unknown function, the presence of amidoligase and glutamine amidotransferase genes suggests intriguing possibilities for the interactions of Marvin with its mycobacterial hosts.

Mycobacteriophage Marvin: a new singleton phage with an unusual genome organization.

We propose an explicit formula for the n-point maximal-helicity-violating one-loop amplitude in a N=4 supergravity theory. This formula is derived from the soft and collinear factorizations of the amplitude.

Maximal-helicity-violating n-point one-loop amplitude in N=4 supergravity.

Development of a DNA Bar-coding Project as a Biology Laboratory Module.

In this work, a method was developed to study the structural proteome of mycobacteriophage Marvin, a recent isolate from soil with 107 predicted coding sequences. This prototype method was applied for semi-quantitative analysis of the composition of this mycobacteriophage virion using ion mobility spectrometry and data-independent acquisition (MS(E)-IMS). MS(E)-IMS was compared to a more conventional proteomics technique employing mass spectrometry with a data-dependent acquisition strategy. MS(E)-IMS provided broad coverage of the virion proteome and high sequence coverage for individual proteins. This shotgun method does not depend on the limited sensitivity of visualization of protein bands by staining reagents inherent in gel-based methods. The method is comprehensive, provides high sequence coverage and is proposed as a particularly efficient method for the study of bacteriophage proteomes.

Data-independent acquisition (MSE) with ion mobility provides a systematic method for analysis of a bacteriophage structural proteome.

Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc(2)155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode.

Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes.

Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training.

A broadly implementable research course in phage discovery and genomics for first-year undergraduate students.

There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit.

A course-based research experience: how benefits change with increased investment in instructional time.

In their 2012 report, the President's Council of Advisors on Science and Technology advocated "replacing standard science laboratory courses with discovery-based research courses"-a challenging proposition that presents practical and pedagogical difficulties. In this paper, we describe our collective experiences working with the Genomics Education Partnership, a nationwide faculty consortium that aims to provide undergraduates with a research experience in genomics through a scheduled course (a classroom-based undergraduate research experience, or CURE). We examine the common barriers encountered in implementing a CURE, program elements of most value to faculty, ways in which a shared core support system can help, and the incentives for and rewards of establishing a CURE on our diverse campuses. While some of the barriers and rewards are specific to a research project utilizing a genomics approach, other lessons learned should be broadly applicable. We find that a central system that supports a shared investigation can mitigate some shortfalls in campus infrastructure (such as time for new curriculum development, availability of IT services) and provides collegial support for change. Our findings should be useful for designing similar supportive programs to facilitate change in the way we teach science for undergraduates.

A central support system can facilitate implementation and sustainability of a Classroom-based Undergraduate Research Experience (CURE) in Genomics.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have higher transposon density (25%-50%) than euchromatic reference regions (3%-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% versus 11%-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 versus 8.4-8.8 genes per block), indicating higher rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution.

Mycobacteriophages - viruses of mycobacterial hosts - are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages - Corndog, Catdawg, Dylan, Firecracker, and YungJamal - designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

Article	Year
RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components. Genes & development\| PubMed ID: 15489292	2004
Shock therapy. How donut magnets can suspend inappropriate ICD shocks. JEMS : a journal of emergency medical services\| PubMed ID: 18602595	2008
Classroom-based science research at the introductory level: changes in career choices and attitude. CBE life sciences education\| PubMed ID: 21885824	2011
Mycobacteriophage Marvin: a new singleton phage with an unusual genome organization. Journal of virology\| PubMed ID: 22357284	2012
Maximal-helicity-violating n-point one-loop amplitude in N=4 supergravity. Physical review letters\| PubMed ID: 22401057	2012
Development of a DNA Bar-coding Project as a Biology Laboratory Module. Journal of microbiology & biology education\| PubMed ID: 23653719	2010
Data-independent acquisition (MSE) with ion mobility provides a systematic method for analysis of a bacteriophage structural proteome. Journal of virological methods\| PubMed ID: 24129072	2014
Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes. Journal of virology\| PubMed ID: 24335314	2014
A broadly implementable research course in phage discovery and genomics for first-year undergraduate students. mBio\| PubMed ID: 24496795	2014
A course-based research experience: how benefits change with increased investment in instructional time. CBE life sciences education\| PubMed ID: 24591510	2014
A central support system can facilitate implementation and sustainability of a Classroom-based Undergraduate Research Experience (CURE) in Genomics. CBE life sciences education\| PubMed ID: 25452493	2014
Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution. G3 (Bethesda, Md.)\| PubMed ID: 25740935	2015
Comparative genomics of cluster o mycobacteriophages. PloS one\| PubMed ID: 25742016	2015

David Dunbar

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