First commissioning results of the multicusp ion source at MIT (MIST-1) for H2+

IsoDAR is an experiment under development to search for sterile neutrinos using the isotope Decay-At-Rest (DAR) production mechanism, where protons impinging on 9Be create neutrons which capture on 7Li which then beta-decays producing ve. As this will be an isotropic source of ve, the primary driver current must be large (10 mA cw) for IsoDAR to have sufficient statistics to be conclusive within 5 years of running. H2+ was chosen as primary ion to overcome some of the space-charge limitations during low energy beam transport and injection into a compact cyclotron. The H2+ will be stripped into protons before the target. At MIT, a multicusp ion source (MIST-1) was designed and built to produce a high intensity beam with a high H2+ fraction. MIST-1 is now operational at the Plasma Science and Fusion Center (PSFC) at MIT and under commissioning.National Science Foundation (U.S.). (Grant PHY-1505858)Bose Foundatio

7-Deazaguanine modifications protect phage DNA from host restriction systems

Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G+), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2′-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2′-deoxy-7-amido-7-deazaguanine (dADG), 2′-deoxy-7-cyano-7-deazaguanine (dPreQ0) and 2′-deoxy-7- aminomethyl-7-deazaguanine (dPreQ1). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2′-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes

Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications

To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: Pseudomonas phage M6 with 5-(2-aminoethyl)deoxyuridine (5-NedU), Salmonella phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-NeOmdU) and Delftia phage phi W-14 (a.k.a. ΦW-14) with α-putrescinylthymidine (putT). Among >200 commercially available restriction endonucleases (REases) tested, phage M6, ViI, and phi W-14 genomic DNAs (gDNA) show resistance against 48.4, 71.0, and 68.8% of Type II restrictions, respectively. Inspection of the resistant sites indicates the presence of conserved dinucleotide TG or TC (TS, S=C, or G), implicating the specificity of TS sequence as the target that is converted to modified base in the genomes. We also tested a number of DNA methyltransferases (MTases) on these phage DNAs and found some MTases can fully or partially modify the DNA to confer more resistance to cleavage by REases. Phage M6 restriction fragments can be efficiently ligated by T4 DNA ligase. Phi W-14 restriction fragments show apparent reduced rate in E. coli exonuclease III degradation. This work extends previous studies that hypermodified T derived from 5hmdU provides additional resistance to host-encoded restrictions, in parallel to modified cytosines, guanine, and adenine in phage genomes. The results reported here provide a general guidance to use REases to map and clone phage DNA with hypermodified thymidine

Diverse Durham collection phages demonstrate complex BREX defence responses

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria–phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility

A new family of globally distributed lytic roseophages with unusual deoxythymidine to deoxyuridine substitution

Marine bacterial viruses (bacteriophages) are abundant biological entities that are vital for shaping microbial diversity, impacting marine ecosystem function, and driving host evolution.1, 2, 3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria[4],[5] that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6, 7, 8, 9, 10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11, 12, 13 the majority of which belong to the N4-like group (Podoviridae family) or the Chi-like group (Siphoviridae family), although ssDNA-containing roseophages are also known.[14] In our attempts to isolate lytic roseophages, we obtained two new phages (DSS3_VP1 and DSS3_PM1) infecting the model MRC strain Ruegeria pomeroyi DSS-3. Here, we show that not only do these phages have unusual substitution of deoxythymidine with deoxyuridine (dU) in their DNA, but they are also phylogenetically distinct from any currently known double-stranded DNA bacteriophages, supporting the establishment of a novel family (“Naomiviridae”). These dU-containing phages possess DNA that is resistant to the commonly used library preparation method for metagenome sequencing, which may have caused significant underestimation of their presence in the environment. Nevertheless, our analysis of Tara Ocean metagenome datasets suggests that these unusual bacteriophages are of global importance and more diverse than other well-known bacteriophages, e.g., the Podoviridae in the oceans, pointing to an overlooked role for these novel phages in the environment

Three Prochlorococcus cyanophage genomes : signature features and ecological interpretations

© 2005 Sullivan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The definitive version was published in PLoS Biology 3 (2005): e144, doi:10.1371/journal.pbio.0030144.The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish ‘‘core’’ genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of ‘‘cyanobacterial’’ genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent ‘‘signature’’ cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages—T7 and T4—but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.This research was supported by the US DOE under grant numbers DEFG02– 99ER62814 and DE-FG02–02ER63445, and the National Science Foundation under grant number OCE-9820035 (to SWC)

PhD

dissertationThe scaffolding protein of the dsDNA bacteriophage P22 was studied in order to better understand the molecular basis of procapsid assembly. The procapsid is a structural precursor to the capsid, a protein shell that encloses and protects a viral chromosome. The phage P22 procapsid is composed of icosahedrally arranged coat protein subunits encasing a core of scaffolding protein and contains a ring of 12 portal protein subunits at one of its 12 icosahedral vertices. The scaffolding protein activates the assembly of coat protein subunits, directs the positioning of those subunits such that a closed shell of the correct dimension is formed, and is required for the inclusion of portal and injection proteins into the procapsid. DNA enters the procapsid through the portal vertex and the scaffolding protein exits the procapsid intact before DNA packaging is completed. How scaffolding protein accomplishes these processes is the focus of the work presented in this dissertation. The scaffolding protein was first examined by a functional domain analysis in vivo. Scaffolding protein truncation mutants were cloned and expressed in P22's host, Salmonella enterica serovar Typhimurium, during infection with a P22 strain defective in scaffolding protein synthesis. Mutants were tested for their ability to complement multiple rounds of infection, exit from the procapsid, recruit portal and injection proteins, and form procapsid-like particles. Successive N-terminal deletions defined the boundaries of amino acids necessary for these functions, including a 15 amino acid minimal coat protein binding domain. Scaffolding protein's ability to bind coat protein and promote assembly of procapsid-like particles both was also studied. Point mutations within the coat protein binding domain were generated and mutant scaffolding proteins were expressed in vivo during scaffolding deficient P22 infection. Fifteen different single amino acid substitutions failed to abrogate coat protein binding in vivo. Scaffolding protein mutants were subsequently overexpressed, purified, and assayed, in vitro, for the abilitiy bind coat protein as well as assemble procapsid-like particles from purified monomeric coat protein. Mutations that affected both processes were found

Photosynthesis: Life from Light

In this course, you will journey through the web of physical, chemical, and biological reactions that collectively constitute photosynthesis. We will begin with light harvesting and follow photons to the sites of primary photochemistry: the photoreaction centers. A molecular-scale view will show in atomic detail how these protein complexes capture and energize electrons. Then we will follow the multiple pathways electrons take as they carry out their work. Consequent reactions, such as the synthesis of ATP and the reduction of CO2 during the synthesis of carbohydrates, will also be discussed in structural detail. Lastly, we will delve into the evolution of these systems and also discuss other photosynthetic strategies, such as light-driven proton pumps and anoxygenic photosynthesis. The course will include a visit to an electron microscope to allow students to directly observe proteins involved in photosynthesis. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching

Nano-life: An Introduction to Virus Structure and Assembly

Watson and Crick noted that the size of a viral genome was insufficient to encode a protein large enough to encapsidate it and reasoned, therefore that a virus shell must be composed of multiple, but identical subunits. Today, high resolution structures of virus capsids reveal the basis of this genetic economy as a highly symmetrical structure, much like a geodesic dome composed of protein subunits. Crystallographic structures and cryo-electron microscopy reconstructions combined with molecular data are beginning to reveal how these nano-structures are built. Topics covered in the course will include basic principles of virus structure and symmetry, capsid assembly, strategies for enclosing nucleic acid, proteins involved in entry and exit, and the life cycles of well understood pathogens such as HIV, influenza, polio, and Herpes. A review of cutting edge structural methods is also covered. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting