Author: P. Collins

Microbacteriophage Piperis: A new virus discovered by Mitchell CC researchers

Complete Genome Sequence of Cluster EC Microbacteriophage Piperis

Ryan Kistemaker, Annica Teasley, Lavender Cook, Alyssa Moliis, Anna Josey, Thomas Ray, Farrah Collins, Alisa Whitcomb, Ryan Swarts, Yazmen Roseboro, Shallah Glover, Ian Goodman, Kaitlyn Dishman, Kimberlyn Pickens, Justin Leonard, D. Parks Collins

Abstract
Microbacteriophage Piperis is a cluster EC bacteriophage which was isolated from soil collected in Mooresville, NC. Piperis has a 53241bp genome with 91 predicted genes.

Genome Announcement
Microbacteriophages are viruses which are known to infect microbacterial host species. Microbacteriophage Piperis was isolated from soil collected Mooresville, North Carolina, USA, by using Microbacterium foliorumNRRL B-24224 SEA as a host. Piperis was isolated using the enriched isolation procedure at 30ºC in the fall of 2018. Its plaques consisted of numerous small plaques with occasional big plaques and were usually clear. Electron microscopy showed that this bacteriophage has Siphoviridae morphology.

Piperis plaques were selected, purified, and amplified, and the DNA was extracted. The Piperis genome was sequenced with the Illumina Sequencing platform with an approximate shotgun coverage of 43x. The genome was then annotated using the following databases and software: DNA Master, Glimmer, GeneMark, Starterator, Phamerator, PhagesDB.org, NCBI BLAST, and Conserved Domain Database at NCBI, HHPRED, ARAGORN, and PECAAN.

Piperis is a member of Actinobacteriophage cluster EC. The Piperis genome is 53241 base pairs with a circularly permuted genome end and a 68.9% GC content. After annotation, 91 genes were identified, with 32.9% of genes being assigned a function. Piperis was compared to other bacteriophage genomes by using NCBI Blast and was found to be most similar to bacteriophage KaiHaiDragon, with 97% identity over 95% coverage.

Piperis is arranged canonically. The left-hand side has structural genes, while the right-hand side has to do with structural DNA and replication of DNA. This right-hand side begins at gene 48, about halfway through Piperis. The genome ends are circularly permuted. A similar arrangement can be found in the EC cluster, along with KaiHaiDragon, the most similar phage to Piperis. Among the functions in this phage, lysin A can be found upstream from a membrane protein, which could indicate that this is a holin. 

When Piperis was compared to other genes in the EC cluster, the phage that is most similar to it is KaiHaiDragon. Most genes in both phages share similar synteny and have the same functions. Neither of these phages has a tRNA gene within their genome. Furthermore, none of the genes in this cluster are read in reverse.

Acknowledgements
This work was supported by the Howard Hughes Medical Institute (HHMI) Science Education Alliance–Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program. We thank Graham F. Hatfull, Welkin H. Pope, Deborah Jacobs-Sera, Daniel A. Russell, and Rebecca A. Garlena for their technical support during the sequencing and annotation of this genome.

References

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. JMol Biol 215:403–410. doi:10.1016/S0022-2836(05)80360-2.

Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res 33:W451–W454. doi:10.1093/nar/gki487.

Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12:395. doi:10.1186/1471-2105-12-395.

Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679. doi:10.1093/bioinformatics/btm009. 

Hatfull, G., Russell, D., & Pope, W. (2019). The Actinobacteriophage Database | Phage Piperis. Retrieved from https://phagesdb.org/phages/Piperis/

PECAAN. (2019). Retrieved fromhttps://discover.kbrinsgd.org/evidence/genes

Remmert M, Biegert A, Hauser A, Söding J. 2011. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods 9:173–175. doi:10.1038/nmeth.1818.

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Microbacteriophage LaviMo: A new virus discovered by Mitchell CC researchers

Complete Genome Sequence of Cluster EE Microbacteriophage LaviMo

Lavender Cook, Alyssa Moliis, Anna Josey, Thomas Ray, Ryan Kistemaker, Annica Teasley, Farrah Collins, Alisa Whitcomb, Ryan Swarts, Yazmen Roseboro, Shallah Glover, Ian Goodman, Kaitlyn Dishman, Kimberlyn Pickens, Justin Leonard, D. Parks Collins

Abstract

Microbacteriophage LaviMo is a cluster EE bacteriophage which was isolated from soil collected in Statesville, NC. LaviMo has a 17,453-bp genome with 27 predicted genes, including 3 minor tail genes.

Genome Announcement 

Microbacteriophages are viruses which are known to infect microbacterial host species. Microbacteriophage LaviMo was isolated from soil collected from Statesville, NC, USA, by using Microbacterium foliorumNRRL B-24224 SEA. LaviMo was isolated using the direct isolation procedure at 30°C in September 2018. Its plaques were clear. Electron microscopy showed that this bacteriophage has morphology consistent with Siphovirida, a family of double-stranded DNA viruses in the order Caudovirales.

LaviMo plaques were selected, purified, and amplified, and the DNA was extracted. The LaviMo genome was sequenced with the Illumina platform using 3-bp single-end reads and had an average coverage of 5179x. The genome was then annotated using the following databases and software: DNA Master (http://cobamide2.bio.pitt.edu), Glimmer, GeneMark, Starterator, Phamerator, PhagesDB.org, NCBI BLAST and Conserved Domain Database at NCBI, HHPRED, ARAGORN , tRNAscan-SE, and PECAAN (http://pecaan.kbrinsgd.org).

LaviMo is the member of Actinobacteria cluster EE. The LaviMo genome is 17453 bps long, with a 68.9% GC content. After annotation, 25 genes were identified, with 80% of genes being assigned a function. LaviMo was compared to other bacteriophage genomes by using NCBI Blast (https://blast.ncbi.nlm.nih.gov/)and was found to be most similar to bacteriophage Scamander with 99.98% identity and over 97% coverage.

The LaviMo genome is arranged canonically, with the structural genes and viral assembly genes on the leftmost 15 kbp of the genome. At the rightmost 2 kbp of the genome, there are consecutive small reverse genes. A similar arrangement is found in other cluster EE phages. The LaviMo genome has a lysis cassette consisting of lysin A. Between the lysis cassette and immunity cassette there are 3 short reverse genes, which were not found to have any specific functions. Given their position in the genome it is possible that these genes are involved in DNA replication/phage reproduction systems. Other cluster EE bacteriophages such as Scamander have short reverse genes in this position; the number of genes present and the nucleotide sequences are the same for this region. The tape measure protein and minor tail proteins that follow it in Lavimo is identical to that sequence of genes found in the most closely related microbacteriophage, Scamander. The genome also contains the gene MerR-like helix-turn-helix DNA binding protein, which is shared by other cluster EE bacteriophages but is not found in other phage clusters.

When LaviMo was compared to the two cluster phages, it was found that all three genomes obtained a tape measure protein that was located in the same location within the genome. The terminase was also another gene found within all three cluster phages.

Acknowledgments

This work was supported by the Howard Hughes Medical Institute (HHMI) Science Education Alliance–Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program.

We thank Graham F. Hatfull, Welkin H. Pope, Deborah Jacobs-Sera, Daniel A. Russell, and Rebecca A. Garlena for their technical support during the sequencing and annotation of this genome.

References

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. JMol Biol 215:403–410. doi:10.1016/S0022-2836(05)80360-2.

Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res 33:W451–W454. doi:10.1093/nar/gki487.

Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12:395. doi:10.1186/1471-2105-12-395.

Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679. doi:10.1093/bioinformatics/btm009. 

Hatfull, G., Russell, D., & Pope, W. (2019). The Actinobacteriophage Database | Phage Piperis. Retrieved from https://phagesdb.org/phages/Piperis/

PECAAN. (2019). Retrieved fromhttps://discover.kbrinsgd.org/evidence/genes

Remmert M, Biegert A, Hauser A, Söding J. 2011. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods 9:173–175. doi:10.1038/nmeth.1818.

Discovering Ancient Viruses

In 2000, scientists drilled a hole into the Siberian permafrost to collect old soil and look for ancient predators. In fact, these soil samples were estimated to be around 30,000 years old. These viral predators may be even older. Ten years later, the researchers added portions of these samples to amoebae colonies to see if any of the viruses from the soil would infect the amoebae. They were basically “fishing” by using amoebae as bait. The scientists found a virus new to science and named it Pithovirus sibericum. It was giant, measuring about 1.5 micrometers long. After 30,000 years, P. sibericum had been thawed and brought back to life.

Viruses are a large part of the planet’s biomass. Bacteriophages, or phages, are specific viruses that infect bacteria. Phages are the most abundant organism on Earth, with an estimated number of ten million trillion trillion (10^31). If phages were the size of an average beetle, they would completely cover the entire surface of the Earth several miles deep. They are abundant, but we know so little. Groups of student researchers are exploring this unknown realm.

This past semester, scientists at Mitchell Community College collected soil samples from Iredell County, then extracted, purified, and amplified new phages. The research was done in partnership with Howard Hughes Medical Center’s SEA-PHAGES program. SEA-PHAGES is seeking to find, characterize, and sequence phages that infect bacteria from the Actinobacterial family. The goal is to better understand the diversity within and between these phages. Instead of amoebae, Mitchell researchers used a bacteria, Microbacterium foliorum, as bait to catch the viruses.

Mitchell scientists discovered seven new viruses (seen here), and chose two phages for DNA sequencing. Next semester, the students will learn more about these phages by exploring their genome. The diversity of phages that inhabit Iredell County soil will become a little less blurry.

Mitchell CC Phages 2018


Campus Ant Diversity 2018

Post by research students (Fall 2018)

During the fall 2017 semester, we conducted ant surveys on both our Statesville and Mooresville campuses. Once again we surveyed ants across six grass plots and six pavement plots. However, this time we only sampled from the Statesville campus. The figures below show these comparisons.

You will notice different compositions from 2017 to 2018. Why is this? Could it be because of temperature differences? On 8/21/17, the average temperature in Statesville was 78ºF (high- 89ºF, low- 68ºF) while on 8/27/18 the average temperature was 69ºF (high-73ºF, low-66ºF). Temperature is probably not the only factor involved. Hopefully, a deeper dive into data will allow us to figure this out.

Gray Fox

Gray foxes, Urocyon cinereoargenteus, are omnivorous members of the canine family. Their diet ranges from mammals and birds to invertebrates, fruits, and nuts depending on the season and what is available.

As you can see from the figure above, we didn’t catch any on camera from 7 a.m. to 6 p.m. There could be many factors that explain this. Gray foxes are really good tree climbers, and we didn’t have any cameras in trees. Also, this species of fox is primarily nocturnal. We have written about their tree-climbing ability as well as their avoidance behaviors here.

Above you can see that they didn’t overlap much with coyotes in space. It almost looks as though the coyotes have pushed the gray foxes closer to developed area. Gray foxes also tend to avoid coyotes in time, only overlapping 55% of the time. This strategy is used to avoid interactions with the much larger carnivore, the coyote.

We also found that gray foxes were:

  • Present at 6 trap stations
  • Found in 96 photos (30 independent observation)
  • Nocturnal 96.67% of the time

Gray fox climbing in Statesville