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

UNLABELLED: 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. IMPORTANCE: The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.

Complete genome sequence of the Escherichia coli phage vB_Ec_Tarrare.

We report the isolation, sequencing, and annotation of the novel bacteriophage vB_Ec_Tarrare, which infects the Escherichia coli K-12 strain. It was isolated from a bat guano sample collected in Rindge, NH, USA. Its genome is 40,953 base pairs long with 49 putative protein-coding genes and no transfer RNAs.

Complete Genome Sequence of Bacteriophage vB_Hercules_Set, Which Infects Enteric Pathogen Salmonella enterica Serovar Typhimurium.

We report the isolation, sequencing, and annotation of bacteriophage vB_Hercules_Set, a kuttervirus infecting human pathogen Salmonella enterica. vB_Hercules_Set was isolated from a slurry of soil and deli meat collected in New Hampshire in 2021. The genome length is 157,338 nucleotides, containing 210 protein-coding genes and five tRNAs.

An undergraduate laboratory experiment with real-world applications: Utilizing templateless polymerase chain reaction and real-time polymerase chain reaction to test for SARS-CoV-2 RNA.

We present a course-embedded undergraduate research module that involves real-time polymerase chain reaction testing for the presence of SARS-CoV-2 in environmental samples. A positive control RNA was constructed and two RNA extraction methods were compared and a range of primers were available to compare. Using a combination of published protocols, we assembled a successful project that illustrated a topical exercise similar to real-world assay development. The exercise is aimed at upper-level undergraduates and requires 3 weeks of laboratory periods. The students were able to design and test experimental protocols, while learning about RNA detection. This project could be utilized in upper-level classes including molecular biology, biochemistry, biotechnology, or for independent research projects.

An undergraduate genome research course using "big data".

The ability to analyze large data sets ("Big Data") is an increasingly important skill in modern science. In Biochemistry, the increased volume and velocity of data is particularly evident in the rapid expansion of biological databases. We present a modular bioinformatics course to survey the analysis of genomic data for advanced undergraduates. Research activities include genome scanning for endogenous retroviruses, annotating genomic sequences and a brief exploration of programming in R. A summative poster session was used to disseminate their work. This course is amenable to remote or online instruction. Supplemental materials provided include a schedule and outline. This article reports a session from the virtual international 2021 IUBMB/ASBMB workshop, "Teaching Science on Big Data."

Prophage-mediated defence against viral attack and viral counter-defence.

Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defence promotes phage co-evolution.

Complete mitochondrial genome of the soft-shell clam Mya arenaria.

We have sequenced and characterized the complete mitochondrial genome of the soft-shell clam, Mya arenaria, an important organism for environmental toxicology and aquaculture. Mya arenaria is located in the taxonomic order Myoida, which lacks any member with a completely annotated mitogenome. The M. arenaria mitochondrial genome is 17 947 bp in length. Like most marine bivalves, the circular mitogenome codes entirely on the heavy strand, with no introns. As with other bivalves, the gene order of the mitochondrion is highly rearranged. The mitogenome contains 12 protein-coding genes but ATP8 is missing, consistent with about half of all bivalve genera. Twenty-three tRNAs were identified. Phylogenetic analysis shows that M. arenaria is related most closely with the bivalves Sinonovacula constricta, and Moerella iridescens, of the infraclass Euheterodonta (unassigned). This, along with the close grouping of the phylogenetic trees, confirms a close tie between Myoida and Euheterodonta (unassigned).

Analysis of Gene Expression in an Inbred Line of Soft-Shell Clams (Mya arenaria) Displaying Growth Heterosis: Regulation of Structural Genes and the NOD2 Pathway.

Mya arenaria is a bivalve mollusk of commercial and economic importance, currently impacted by ocean warming, acidification, and invasive species. In order to inform studies on the growth of M. arenaria, we selected and inbred a population of soft-shell clams for a fast-growth phenotype. This population displayed significantly faster growth (p < 0.0001), as measured by 35.4% greater shell size. To assess the biological basis of this growth heterosis, we characterized the complete transcriptomes of six individuals and identified differentially expressed genes by RNAseq. Pathways differentially expressed included structural gene pathways. Also differentially expressed was the nucleotide-binding oligomerization domain 2 (NOD2) receptor pathway that contributes to determination of growth, immunity, apoptosis, and proliferation. NOD2 pathway members that were upregulated included a subset of isoforms of RIPK2 (mean 3.3-fold increase in expression), ERK/MAPK14 (3.8-fold), JNK/MAPK8 (4.1-fold), and NFκB (4.08-fold). These transcriptomes will be useful resources for both the aquaculture community and researchers with an interest in mollusks and growth heterosis.

Genome Sequences of Newly Isolated Mycobacteriophages Forming Cluster S.

We describe the genomes of two mycobacteriophages, MosMoris and Gattaca, newly isolated on Mycobacterium smegmatis The two phages are very similar to each other, differing in 61 single nucleotide polymorphisms and six small insertion/deletions. Both have extensive nucleotide sequence similarity to mycobacteriophage Marvin and together form cluster S.

Community annotation and bioinformatics workforce development in concert--Little Skate Genome Annotation Workshops and Jamborees.

Recent advances in high-throughput DNA sequencing technologies have equipped biologists with a powerful new set of tools for advancing research goals. The resulting flood of sequence data has made it critically important to train the next generation of scientists to handle the inherent bioinformatic challenges. The North East Bioinformatics Collaborative (NEBC) is undertaking the genome sequencing and annotation of the little skate (Leucoraja erinacea) to promote advancement of bioinformatics infrastructure in our region, with an emphasis on practical education to create a critical mass of informatically savvy life scientists. In support of the Little Skate Genome Project, the NEBC members have developed several annotation workshops and jamborees to provide training in genome sequencing, annotation and analysis. Acting as a nexus for both curation activities and dissemination of project data, a project web portal, SkateBase (http://skatebase.org) has been developed. As a case study to illustrate effective coupling of community annotation with workforce development, we report the results of the Mitochondrial Genome Annotation Jamborees organized to annotate the first completely assembled element of the Little Skate Genome Project, as a culminating experience for participants from our three prior annotation workshops. We are applying the physical/virtual infrastructure and lessons learned from these activities to enhance and streamline the genome annotation workflow, as we look toward our continuing efforts for larger-scale functional and structural community annotation of the L. erinacea genome.