oa Elucidating T7-like Bacteriophage Isolated from Qatar's Sand

A bacteriophage is a virus that infects a bacteria, it uses the bacteria as a host to further replicate by controlling the replication and protein synthesis machinery of the cell. They are composed of a protein capsule and DNA or RNA genome. Bacteriophages are used in the treatment of bacterial infections, known as the phage therapy. This results from phages invading the bacteria, as they undergo a lytic cycle, where the replication and protein synthesis machinery is used to produce virions, that later cause the cell to lyse, thus killing the bacteria. This technique has higher efficiency compared to the use of antibiotics as bacteria can develop a resistance against antibiotics, while remaining susceptible to the infection of the phage. In addition, phages can evolve and adapt to new mutations that might arise in the bacteria. Lastly, phages might be engineered to contain a survival gene needed by the bacteria to ensure that the bacteria will replicate and synthesize the new virons, leading to lysis and death.

This research explores the potential of the use of bacteriophages in the treatment of water, to disinfect the water from microbial bacterial and other bacteria that were used to detoxify the water. Phages were previously extracted from Qatar's sand and were used as the infecting agents. The model host chosen is Arthobacter bacteria, which is a denitrification bacteria, as it is similar to the bacteria used in water treatment. Denitrifying bacteria reduce nitrates to nitrogen-containing gases, allowing for the recycling of nitrogen back to the atmosphere.

The goal of my research is to analyze the genome of a phage extracted from Qatar's Sand. A culture of Arthobacter grown in Smeg media was used as a host. Unlike the normal optimal growth temperature of Arthobacter at 32 °C, it was shown that higher phage titer was produced when the culture was grown to saturation in smeg media at 37 °C. In addition, the culture was initially grown in luria Broth which is rich in nutrients including tryptone, yeast extract and NaCl. However, low phage titer was produced compared to switching the media to smeg media, which contains Middlebrook 7H9 broth base with supplements of albumin, dextrose and salts.

After manipulating the growth conditions of the host culture to obtain the highest titer, the optimum conditions were found to be a saturated culture of Arthobacter grown in Smeg media at 37 °C. Later, lysis conditions of the phage were optimized by varying several factors. First, the type of the top agar and plates used for pour plating were varied. Several top agars were used this includes LB top agar, LB top agar in 1 mM CaCl2 and PYCA top agar. It was concluded that the top agar that resulted in the highest titer was LB top agar in 1 mM CaCl2. Several plates have been used for pour plating this include LB plates and PYCA plates. Plaques formed using LB plates produced unclear plaques, this unclearness is an indication of persistence of the lysogenic state and not the lytic state. In the lysogenic state the phage genome gets incorportated into the host genome and replicates along with the cell cycle, thus remaining in a dormant state. On the other hand, in the lystic cycle, the phage uses the replication and protein synthesis machinery to produce more phages, that will later lead to lysis. In contrast, PYCA plates containing CaCl2 formed clearer plaques, indicating dominance of the lytic state. Therefore, addition of calcium in the top agar and the plates aided in the phages’ shift from the lysogenic cycle to the lytic cycle. This correlates with previous work, where calcium was shown to be essential for the penetration of the phage's genome into the host.

Finally, the incubation temperature of the plates containing the infected host Arthobacter by Qatar's sand's phage was varied. Initially the plates were incubated at 32 °C, however no plaques were formed. When the temperature was later increased to 37 °C, lysis was observed and a high phage titer was obtained.

After obtaining a high titer lysate, the DNA was isolated and a sample of the phage was sent to obtain an electron micrograph image of the phage. A restriction enzyme digest was preformed on the isolated DNA and the profile created was compared to the profile of lambda phage. Both the phages are lytic and lyse at similar temperatures, which allows for comparisons.

Lastly, motifs were identified between the Qatar's sand bacteriophage and T7 bacteriophage using blast tools in a previously extracted and sequenced Qatar's sand phage, primers will then be designed to verify if they exist in the actual phage.

Future work will focus on the analyzing the sequence of the phage to identify potential lysis genes, In addition, strengthening the lytic ability of the phage by cloning the gene of lysozyme into a T7-based vector system and test its adaptability to temperature using Arthrobacter as the host system.