oa Analysis of Sand Bacteriophages Genome in Qatar

Abstract Objective: In this era of increasing antibiotic resistance (WHO)[1], we are running out of time as common bacterial infections are progressively rejecting drugs that would be standard for treatment. It is due to these reasons that research regarding bacteriophages, viruses that infect bacteria has seen a sudden revival. The Science Education Alliance- Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) headed by Dr. Graham Hatful from the Howard Hughes Medical Institute (HHMI)[2], University of Pittsburgh and Carnegie Mellon University has begun a nationwide initiative to discover, categorize and study the billions of bacteriophages that surround us. This initiative aims to create a thorough database containing genomic data for as many bacteriophages as possible. The study of these viruses is virtually new, and thus requires a significant effort; there is a novel, virtually untapped resource of genes and proteins that could greatly benefit our understanding of genomes and be used as treatments to bacterial infections that are resistant to antibiotics. Studying the bacteriophages is of great importance. The same protocols and procedures from the SEA-PHAGES program were applied to search for bacteriophages in Qatar within the sand and soil at Carnegie Mellon University Qatar. This is the first research of its kind to be carried out in Qatar and possibly at the Gulf region. Bacteriophages, which infect Arthrobacter.sp, were discovered in the sand from Al-Rayyan, Doha Qatar (Diversity of Bacteriophages in the ecology of Qatar). Following this, the viral plaques obtained were purified, isolated using a QIAamp MinElute Virus Kit[3] (Qiagen) and finally sequenced using Next Generation sequencing methods at Weil Cornell Medical University Qatar. Method: The sequenced DNA of phages were assembled using a Short Oligonucleotide Analysis Package- Denovo (SOAP-Denovo) software package. A program called Velvet has been also used in conjunction to ensure the best results were obtained using the optimal k-mer, providing the longest contig length. The longest length obtained was a contig of 12,380 at a k-mer of 59, along with a large number of smaller contigs. The largest 201 contigs have been annotated; gene nucleotide and protein sequences were collected, using the gene prediction software, GeneMarkS available online. Each of the genes from the contigs were then searched against the entire non redundant protein (NR) database of available annotated genes at the National Centre for Biotechnology Information (NCBI's) online database using Basic Local Alignment Search Tool (BLAST-p). They were also searched against non-submitted phage genomes collected at SEA-PHAGES. That result in about 558 annotated genes/proteins. All the proteins are being studied to understand better what the bacteriophage genome comprises of and how the proteins allow them to survive and infect bacteria in the sand collected from Al Rayyan in Doha. Results: So far three different bacteriophage contigs were found from the sequenced DNA, containing a total of 14 genes, most of which are derived from the Enterobacteriophage T7 cluster. However the individual genes displayed some variation from the original genes due to the small rate of mutations that naturally occurs with bacteriophages during replication. In addition to this, variations could be due to natural exchange of genetic information between viruses to form mosaic genomes. Thus each bacteriophage is unique due to this variation and each has proteins that can perform a slightly different function. One such gene was found had a 99% identity with Gene 3.5 from the Enterobacteriophage T7 family (phages that can infect Escherichia coli (E.coli) bacteria, which are commonly responsible for foodborne illnesses). This particular gene was searched against the PDB (protein database) from the same NCBI BLAST tool, to search for mutations. The query returned results of 100% identity with a (Chain L) T7 RNA Polymerase complexed with T7 lysozyme (interplay between an RNA polymerase and lysozyme). One particular bacteriophage contains a gene originating from a bacteriophage of the Yersinia species (phiA1122). This gene codes for a head-to-tail joining protein, which is a major component of a phage. This bacteriophage principally infects and neutralizes, Yersinia pestis, which is the causative agent of the Bubonic plague in humans[4] (Garcio.E). This particular gene must have been transferred during naturally occurring gene exchange between bacteriophages that infect the same organism simultaneously (Fleischmann, W) [5]. Discussion: The findings strongly suggest that Gene 3.5 has a function in breaking down the bacterial cell wall during infection. A further search on the Research Collaboratory for Structural Bioinformatics (RSCB) PDB was carried out to find a structural view of the protein. Upon studying the protein, it was found that the fragment containing the mutation is not seen in the crystal structure, which means it is a flexible loop. If this section of the protein moves in the crystal structure then it most probably moves in nature as well. This strongly suggests that the flexible mutated loop on the protein surface could affect of protein-protein interaction; and may result in slight changes in the mechanism (or temperature) by which the phage ligates or transfers genetic information from one phage to another. Future work: Future work aims to further study the Qatari bacteriophage genes and their proteins, their mutations and functions. Characterizing the remainder of the bacteriophage will allow us to better understand and annotate its genome. The relevance of these phages in a practical application could be in medical, industrial or agricultural uses. It could possibly have a role in treating E.coli infections or to prevent the contamination of such bacteria in the food industry. Further research could be done to sample more sand from various parts of Qatar (and various depths within the sand) to search for more bacteriophages. It is very likely that there are bacteriophages that could have more significant applications present within the sand. The same procedures could be followed to determine the functions and the nature of these phage genes.

References

[1] World Health Organization. 2015. Antimicrobial Resistance. Available at: http://www.who.int/mediacentre/factsheets/fs194/en/. Accessed on 13 November 2015.

[2] Howard Hughes Medical Institute. Science Education Alliance. Available at: http://www.hhmi.org/programs/science-education-alliance. Accessed on 13 November 2015.

[3] Qiagen.com. 2015. QIAamp MinElute Virus Kit. Available at: https://www.qiagen.com/us/shop/sample-technologies/combined-sample-technologies/preparation/qiaamp-minelute-virus-spin-kit/. Accessed on 13 November 2015.

[4] Garcio.E. 2003. The Genome Sequence of Yersinia pestis Bacteriophage φA1122 Reveals an Intimate History with the Coliphage T3 and T7 Genomes. Journal of Bacteriology. [Online]. Available at: http://jb.asm.org/content/185/17/5248.full. Accessed on 13 November 2015.

[5] Fleischmann. W.R. 1996. Chapter 43: Viral Genetics. Medical Microbiology 4th edition. Available at: http://www.ncbi.nlm.nih.gov/books/NBK8439/. Accessed on 13 November 2015.