Preliminary Bioinformatic Analysis of Deinococcus sonorensis

Introduction:

Deinococcus sonorensis is a species of the genus that has been intensively studied in this lab previously due to its abnormal and unique characteristics when it is grown on TGY media. A poster presentation to develop an optimal DNA extraction method even when it forms its plaque structure was completed about a year ago, and now it is time to revisit the advancements that have been made in the past six months. Fortunately Illumina sequencing became available and the mysteries of D. sonorensis has started to unravel, thanks to being able to bioinformatically process this species and the addition of biochemical testing that has shed light on some new ideas. Not only are we now able to grow D. sonorensis on media and prevent plaque formations, we can extract its DNA better than ever before, and have findings that allow us to infer what proteins or systems may be responsible for its odd behaviors that make this species so interesting in the genus. 

Methods:

D. sonorensis was put through various biochemical tests, including the observation of growth on different medias for at least four days to ensure results. An observation was made that it seemed to be lighter in color and less plaque-like when grown on a less nutrient dense media, such as R2A in comparison to what it is typically grown on, TGY. The colonies taken from R2A plates can actually be homogenized in to R2B, but special not was taken because they could not be easily homogenized in water. This indicated a potential hydrophobic property of the plaque that is not yet characterized. Thus, a DNA extraction was conducted using as many cells that could be taken from plate and divided between two tubes, and put through the QUIAGEN DNeasy kit. Originally the plan was to attempt a 5 minute bead beating for one tube and 2 minutes for the other in lieu of the now easier homogenization of the cells to cut down on DNA shearing, but the tubes were mixed up and combined so each became a mix of the two bead beating times. This sample was processed with others as mentioned in a previous post, and sequenced on Illumina. The data was retrieved in FastQ format and processed through Patrik, while an initial attempt at Galaxy processing was made. The FastQC check on Galaxy did come back favorable, but other technological roadblocks have prevented the ability to replicate the results of the Patrik processing on Galaxy which will need to be worked out at a later time. The new FASTA and annotated files were downloaded from Patrik and analyzed using SnapGene and UniProt BLAST for comparative analysis. 

Results:

The original sequencing data file was processed to be a total of 76 contigs, relatively very good for Illumina sequencing, with the largest contig being approximately a little over 1.2 Mbp. The total genome size was also a little over 4 Mbp. No CRISPR-Cas systems were detected, through initial programming or using CasTyper. What was identified was a complex system of Pil genes, that are responsible for the pili that most Deinococcus are likely known to have according to the consistent presence of these genes across many of their genomes. What is interesting about D, sonorensis is the presence of many that are not seen in other species such as D. aquaticus, and may play a role in making the pili into a system that allows it to form a plaque. Pili can be utilized by a cell for horizontal gene transfer, adhesion, motility, and biofilm formation. D. sonorensis is currently identified to have two PilA genes, PilB, PilC, PilM, PilQ. PilT, and PilN. Other genes that may associated with its biofilm formation may include an FnBP gene as well as PelA. FnBP and PelA genes are specifically tied to a biofilm formation, so the plaque is not being considered a biofilm of some sort rather than a distinct plaque. Other genes of interest that have been identified are ComF, a gene that is suspected to boost competence for horizontal gene transfer, and VanW, a part of the Vancomycin resistance pathway. 

Discussion:

The preliminary search for genes in the D. sonorensis genome has already yielded very interesting results that will be pursued in the next few weeks for clarity and potentially the creation of a model that can shed light on the newfound biofilm capability of the species. It is incredibly exciting to have been able to start putting pieces together as to why D. sonorensis does what it is, and knowing that this is likely a pili based biofilm formation will lead to the new experiments that can be used to test this hypothesis. Some of the next steps for this project will include imaging, hopefully SEM, TEM, Confocal, and atomic force, but that is yet to be determined. If so, we are hoping to see pili structures and even better attachments between cells that would be indicative of a biofilm formation, with the possibility of seeing other proteins or structures that are also involved in this biofilm. More bioinformatic research will also need to be conducted to further characterize the proteins and literary research will ideally lend a hand in determining the function of each of these proteins and how they may work together.