Genes and structural proteins of the phage SYN5 of the marine cyanobacteria, Synechococcus

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2005.Includes bibliographical references (p. 157-171).Bacteriophage have been proposed to be the most abundant organisms on the planet, at an estimated 10³¹ particles globally (Hendrix et al., 1999). The majority of bacteriophage isolates (96%) are double-stranded DNA tailed phages (Caudovirales). These phages possess a distinctive icosehedral head, with a protein tail structure protruding from a single vertex. This organelle determines host specificity and provides the mechanism of passage of the phage genome into the host cell. Phages infecting differing microbial hosts may have access to a global pool of genes, albeit at different levels. Marine cyanobacteria of the genera Prochlorococcus and Synechococcus are numerically dominant photosynthetic cells in the large oligotrophic gyres of the open oceans, and contribute an estimated 30% to the oceanic photosynthetic budget. Cyanophages have been isolated which propagate on many strains of Synechococcus and Prochlorococcus. Cyanophages can effect community structure and succession through lytic infection of their hosts, and have implications in lateral gene transfer, mediated through lysogeny, mixed infections, pseudolysogeny, and transduction.(cont.) The broad host ranges (between genera) observed in some phages indicates that lateral gene transfer is not confined to cells of the same strain. These phage/host interactions begin by host recognition by the tail of the infecting phage. Few studies have examined the structural proteins of cyanophage, partially due to the lack of a robust protocol for the growth and purification of phage particles. Cyanophage Syn5 is a short-tailed phage isolated from the Sargasso Sea by Waterbury and Valois (1993) which infects Synechococcus strain WH8109. Methods of growing the host cells and the phage, and concentrating the phage by PEG precipitation were developed. These methods led to highly concentrated purified phage stocks, to titers of 1012 particles/ml. Preliminary characterization of the growth of Syn5 gave a burst size of approximately 30 phage/cell and a lytic period of approximately 10 hours when inoculated into exponentially growing host cells acclimated to a temperature of 26⁰C and a light intensity of 50[mu]E m⁻² s⁻¹. Isolation of the phage nucleic acid yielded dsDNA molecules of approximately 40kb. The Syn5 particles were comprised of twelve structural proteins, as determined by SDS-PAGE.(cont.) The most intense band on the gel was assigned to the capsid protein of Syn5 ([approx.] 35kDa). However, it was not possible to distinguish putative tail proteins via this method. Purified Syn5 particles were sent to the Pittsburgh Bacteriophage Institute for genome sequencing. The completed Syn5 genome was 46,214 bp long with a 237bp terminal repeat. Annotation of the completed Syn5 genome identified 61 putative ORFs, and revealed that Syn5 appeared closely related to the enteric phage T7 and cyanophages P-SSP7 and P60, as determined by gene similarity and synteny, although the genome was [approx.] 10kb longer than T7. Syn5 appeared to possess a more extensive DNA replisome that T7, containing copies of genes that encoded proteins of known T7 host co-factors, such as thioredoxin, utilized by the T7 DNAP. Several large ORFs were identified between the gene encoding the putative tail fiber and the gene encoding the putative terminase. These ORFs encoded proteins similar to some fibrous sequences within the NCBI non- redundant (nr) gene sequence database as of March, 2005; but had unknown functions within the phage. Unlike other recently sequenced cyanophages, SynS did not contain any photosynthetic genes.(cont.) The structural proteins of SynS, as visualized by SDS-PAGE, were characterized by mass-spectroscopy and N-terminal sequencing. This allowed the assignment of sequences to putative ORFs within the Syn5 genome. The Syn5 particle was comprised of eleven discreet protein chains of molecular weight 152kDa, 139kDa, 99kDa, 90kDa, 66kDa, 60kDa, 47kDa, 35kDa, 22kDa, 21kDa, and 16kDa. The identified proteins included the portal, capsid, two tail tube proteins, and three internal virion proteins. Each of the genes encoding these proteins were found in the same gene order in the Syn5 genome as the corresponding genes were ordered in the T7 genome. There were three unidentifiable proteins within the particle (66kDa, 47kDa, and 16kDa). These mapped to the area of the SynS genome between the gene encoding the putative tail fiber and the gene encoding the putative terminase. No minor capsid or decorative capsid proteins were detected. The copy numbers of the corresponding protein chains were similar to those known for T7, with the exception of the tail fiber, which was present at a number of three chains per particle in comparison to T7's eighteen per particle.(cont.) Polyclonal antibodies were raised against Syn5 particles. A Western blot with these antibodies showed that the tail fiber and the two unknown fibrous sequences were highly antigenic. This evidence implies that the unknown structures may act as host recognition proteins in addition to the tail fiber. Characterization of these novel proteins may provide insight to the host recognition abilities of cyanophages. An additional study was also carried out, investigating the high temperature limit of the growth of phage P22. The results revealed that the production of infectious particles was limited by the temperature sensitivity of the folding and assembly of the P22 tailspike protein. This work has been published and is included in the Appendix.by Welkin Hazel Pope.Ph.D

Discovery and Characterization of Bacteriophage LuckyBarnes

Here, we report the genome sequence of LuckyBarnes, a newly isolated singleton siphovirus that infects Brevibacterium iodinum ATCC 15728 and has a 50,774-bp genome with 67 predicted genes

Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes

Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous freestanding HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotopes

Genomic analysis of a large set of phages infecting the common hostMycobacterium smegmatis mc2155 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

Genomics and Proteomics of Mycobacteriophage Patience, an Accidental Tourist in the Mycobacterium Neighborhood

ABSTRACT Newly emerging human viruses such as Ebola virus, severe acute respiratory syndrome (SARS) virus, and HIV likely originate within an extant population of viruses in nonhuman hosts and acquire the ability to infect and cause disease in humans. Although several mechanisms preventing viral infection of particular hosts have been described, the mechanisms and constraints on viral host expansion are ill defined. We describe here mycobacteriophage Patience, a newly isolated phage recovered using Mycobacterium smegmatis mc2155 as a host. Patience has genomic features distinct from its M. smegmatis host, including a much lower GC content (50.3% versus 67.4%) and an abundance of codons that are rarely used in M. smegmatis. Nonetheless, it propagates well in M. smegmatis, and we demonstrate the use of mass spectrometry to show expression of over 75% of the predicted proteins, to identify new genes, to refine the genome annotation, and to estimate protein abundance. We propose that Patience evolved primarily among lower-GC hosts and that the disparities between its genomic profile and that of M. smegmatis presented only a minimal barrier to host expansion. Rapid adaptions to its new host include recent acquisition of higher-GC genes, expression of out-of-frame proteins within predicted genes, and codon selection among highly expressed genes toward the translational apparatus of its new host

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

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\u27 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

Comparative genomics of Cluster O mycobacteriophages

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

A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students

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

Comparative Genomics of Cluster O Mycobacteriophages

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

Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn

We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc2155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites