Unusual Helicases
Found in Erwinia phage RAY�Emily Jacobs, Katrina Saam, Ryan Koch, Melissa Guereca�1Division of Biological Sciences, Section of Molecular Biology, University of California, San Diego, La Jolla, United States
Goal: To study the Erwinia phage RAY proteins gp131 and gp250 with the intent of forming a conclusion about the functions of these proteins.
What is RAY?
What is Erwinia amylovora?
What are jumbo phage?
What is the SWI/SNF complex?
What is a DEAD-box helicase?
Rationale/Background
Hypothesis
Localization
PSI-BLAST and Conserved Domain Process
iTOL Tree Process
Sequence Alignments Process
Protein Homologue Tree Process
Experimental Approach
Figure 5. Sequence alignment comparing the amino acid sequences of gp131(top) and gp250 homologs (bottom). Blue boxes indicate regions in which there is a large amount of conservation between the sequences. Amino acids that are identical or similar to the column consensus are colored in red, and if the entire column shares the exact same amino acid, the sequences are highlighted in red. The light yellow highlighted regions are the highest significance conserved domains of gp250: SSL 2 which functions as a general RNA or DNA helicase and SF2_C, a structure of C-terminal helicase of superfamily 2 DEAD/H-box helicases. The bright yellow highlighted region is the DEXDc conserved region of gp131 which indicates a DEAD-like helicase that includes chromatin remodeling factors and recombination proteins. The green highlighted region is the HELICc conserved domain of gp131 which indicates a function of a helicase with a c-terminal domain. The protein homolog sequences that appear to exhibit the most similarity to both gp131 and gp250 are helicases and hypothetical proteins from Salmonella phage vB SaIM SA002 and Proteus phage 10.
Inferences and Conclusions
PSI-BLAST Search for Protein Homologs (gp131): The two proteins with the most similarity to gp131 are the putative DEAD-like helicase from Erwinia phage DesertFox and the DEAD-like helicase from Erwinia phage Ea35-70, each with a 99% identity to gp131. The proteins with highest identities to gp131 after these two are the putative DEAD-like helicase from Salmonella phage vB_SalM_SA002 and a hypothetical protein from Proteus phage 10.
iTOL Phage Phylogenetic Tree (gp131): gp131 is found primarily in jumbo phages, indicating that it is probably more useful to jumbophage than to regular phage. This makes sense given that gp131 likely has something to do with packaging and organizing DNA, a function that is probably more important in a phage with more DNA.
PSI-BLAST Search for Protein Homologues (gp250): Out of the 36 hits, the proteins most similar to gp250 are Erwinia phage Desertfox and Erwinia phage Ea35-70 which each contain a 99.61% identity, which are both part of the Superfamily 2 DNA/RNA helicase. The phage most similar to gp250 outside the Erwinia family, is Salmonella phage SA002, with a identity of 69.11%, which according to the NCBI database states this phage has the conserved structure of a replicative helicase.
iTOL Phage Phylogenetic Tree (gp250): The phage tree demonstrates that gp250 is highly conserved across most jumbo phage. Erwinia phage Desertfox showed to be most common to gp250. Outside of the Erwinia family the phage most common to gp250 is Salmonella phage SA002. This demonstrates that gp250 is an important protein for jumbo phage because as these phage evolved they predominantly preserved homologs of gp250, likely to unpack the >200 kpb of viral genome.
CONCLUSIONS: From these results, we can conclude that gp131 is a DEAD-like SWI/SNF related helicase that functions in genome packaging and organization. Likewise, gp250 functions as a helicase in the process of chromatin remodeling, potentially as a RAD2/SF2 helicase.
Results, cont.
Figure 3C. Erwinia cell infected with RAY phage, tagged for gp131 localization. The first row shows images from the cells containing 0.5% arabinose 80 minutes post infection (mpi), and the second row shows images from cells containing no arabinose 80 mpi. gp131 (stained in green) localizes outside of the phage nucleus, which is defined by the presence of the DNA (stained in blue), and in the cytoplasm. This indicates that gp131 localizes exclusively within the cytoplasm during the viral replication process.
Figure 3A. Uninfected Erwinia cells with fluorescence. gp131’s cell ( left) exhibits overlap between the blue stain, a fluorescence protein called DAPI that stains DNA, and gp131’s green stain, indicating gp131’s presence throughout the entire cell pre-infection. gp250’s cell ( right) shows no overlap between the two.
Figure 3B. Erwinia cell infected with RAY phage, tagged for gp250 localization. The first row shows images from the cells containing 0.5% arabinose 70 minutes post infection (mpi), and the second row shows images from cells containing 0.1% arabinose 90 mpi. gp250 (stained in green) localizes inside of the phage nucleus, which is defined by the presence of the DNA (stained in blue). This indicates that gp250 localizes exclusively within the nucleus during the viral replication process.
We would like to thank Professors Rachel Dutton and Joseph Pogliano, Amy Prichard, and Tara Spencer for their assistance and guidance throughout the research process. We would also like to thank our BIMM 170 classmates for providing a supportive environment where we could complete our research, and a final thank you to Julianne H. Grose from Brigham Young University for providing us with the phage.
Future Directions
Bibliography
Acknowledgements
We believe that gp131 and gp250 are analogous to the SWI/SNF complex found in eukaryotes.
Figure 4. Phylogenetic tree showing the distribution of RAY_gp131 and RAY_gp250 homologs. A filled in square represents high confidence alignments and an empty square represents low confidence alignments. Our results show that both gp131 and gp250 homologs are highly conserved among jumbo phage, especially gp250, which suggests that most jumbo phages require these helicases for infection.
What is the conservation of gp131 and gp250 across relatives of Erwinia phage RAY?
How closely aligned are the sequences of gp131 and gp250 with those of related proteins?
Where do gp250 and gp131 localize in the cell before and during infection?
Figure 2. Results from PHYRE2 analysis of gp131 (top) and gp250 (bottom). Both gp131 and gp250 were tagged as transcription regulatory proteins within the SWI/SNF nucleosome complex, suggesting similar functions within RAY.
What is the putative function of gp131/gp250?
Results
Figure 1. Jumbo phage nucleus. This diagram depicts the life cycle of a jumbo phage, including the formation of the phage nucleus (teal circle). The orange filaments are Phuz-like filaments the move the phage nucleus to the center of the cell and keep it centered. The blue bits in the center of the phage nucleus are the phage DNA, and the hexagons on the outside of the phage nucleus in the forth image are the phage capsids being filled with DNA.