Gliding Motility Associated Lipo-Protein GldD in Cellulophaga Lytica

1. Gliding Motility Associated Lipo-Protein GldD in Cellulophaga Lytica Samuel Duberowski and Isaac Meredith Introduction Gliding motility is a type of cell motility that is poorly understood. However, many proteins have been found that are necessary for this motility to occur, specifically in the bacteria Cellulophaga lytica. The organism C. lytica  is the type species of the genus Cellulophaga, which belongs to the family Flavobacteriaceae within the phylum 'Bacteroidetes' and was isolated from marine beach mud in Limon, Costa Rica. (2) The protein GldD has been shown to be an essential aspect of cell motility in Flavobacterium Johnsoniae (1). Our research was performed on this gldD gene in C. lytica because it is not a highly researched organism that exhibits cell motility. • 3. Horizontal Gene Transfer • Our tree suggests that horizontal gene transfer has occurred • From ten sequence alignments we created a WebLogo (see figure 3.) to illustrate highly conserved regions between multiple organisms that appear to have this gene. These highly conserved regions are conserved in our gene and is evidence that the gldD is similar in other organisms and was correctly described. Figure 6. Phylogenetic Tree of C. lytica. This figure was produced by phylogeny.fr This test tells us whether or not Horizontal gene transfer has occurred by looking at the branches of the “tree” and determining if C. lytica has a similar gene to the organisms in its phylogenetic tree. Since C. lytica is on its own branch and has no close relative we can conclude that horizontal gene transfer has occurred. Our goal was to annotate the gldD gene. Annotating a gene is the process of looking at the DNA sequence to discover more about the function and location of a gene product. Our ultimate goal was to determine if the GldD protein had been correctly described by computer analysis and to trace the origins of the protein by comparing with similar organisms. Discussion Through further research, we concluded that our reading frame was correct by studying the computer readout of the triplet code. We found no evidence for an alternative start site. (Data not shown) The function of the gene product is not highly researched , and there is no known mechanism of how GldD is used in gliding motility. However, our results aligned with the results of Mark McBride and David Hunnicut whose research on Flavobacterium johnsoneia showed similar results to the GldD protein by concluding it was neccesary for gliding motility. Our results led us to believe that this protein GldD in C. lytica has been properly annotated. We also found evidence that expression of gldD may be regulated by an operon involving the gldE gene. Methods IMG-ACT laboratory notebook (3) was used to save and organize our research. The GldD protein is 185 amino acids in length. Below is the gene neighborhood of our gene gldD. Its proximity to gldE another gene that is very likely to be involved in gliding motility (1) suggests that our gene may be part of an operon along with the gldE gene. Figure 3. WebLogo diagram illustrates highly conserved regions in comparative analysis with 10 homolog genes. Letters represent amino acids, and size of letter indicates degree of conservation. 2. Cellular Localization Data We used TMMHMM and Signal P Cellular localization data searches to learn about the location of the protein in the cell. Figure 4. TMMHMM Diagram of Membrane localization. This illustrates that the location of the GldD protein is in the plasma membrane. This finding is supported by the paper by Mark McBride and David Hunnicutt. Figure 1. Gene neighborhood of gldD. gldD shown in red is directly left of gldE, and suggests that they may be expressed part of an operon and both utilized in gliding motility Future Direction Much more research needs to be done to discover the function of this protein in the process of gliding motility. This could be explored in the lab by using our fellow colleagues findings to learn how all these proteins interact with each other to better understand their role in gliding motility in C. lytica and other species. Further research could be done to determine the physical shape of the GldDprotein and the way it functions along with the other proteins to facilitate gliding motility. We decided that for our experiment we would knock out the gene gldD in C. lytica to see if it is also necessary for gliding motility in our organism. • 1. Sequence Based Similarity Data • By using a BLAST search with Swiss Prot database (2) we were able to compare our gene to several other genes. This is done by paralleling our protein with similar proteins in other organisms. Figure 5.Graph of a signal peptide. This tells us the location of the signal peptide is on 29. This is evidence that although GldD is localized in the membrane, its signal peptide is cleaved and therefore releases it from the membrane. This is consistent with the proposed naming of the protein by describing it as a lipo-protein. Acknowledgements Northwestern College Biology Department Dr. Klein Img-act Northwestern College Print Shop • References • McBride, Mark, and David W. Hunnicutt. “Cloning and Characterization of the Flavobacterium Johnsoniae Gliding Motility Genes gldD and gldE.” Journal of Bacteriology 183.14 (2001): 4167-4175 • http://www.ncbi.nlm.nih.gov. Web. 13 Nov. 2012. • http://img-act.jgi-psf.org Web. 13 Nov. 2012. Figure 2. Comparative blast search. This database compares the AA sequence of our target gene to its homolog in Muricauda Ruestringensis. This search had an e value of 2e-79, an alignment length of 184, and a score of 245 bits.