Welcome to the Enteropathogen Resource Integration Center. Using your web browser,

1. Welcome to the Enteropathogen Resource Integration Center. Using your web browser, #1) go to http://www.ericbrc.org/

2. The ERIC database houses all of the available genomes of the members of family Enterobacteriaceae Boxes, represent organisms with at least one genome sequence Human Pathogens -Calymmatobacterium -Cedecea -Citrobacter -Edwardsiella -Enterobacter -Escherichia -Ewingella -Hafnia -Klebsiella -Kluyvera -Leclercia -Leminorella -Moellerella -Morganella -Plesiomonas -Proteus -Providencia -Rahnella -Salmonella -Serratia -Shigella -Tatumella -Yersinia -Yokenella Phytopathogens/ Plant-associated Insect Pathogens /Endosymbionts -Arsenophonus -Buchnera -Sodalis -Wigglesworthia -Xenorhabdus -Brenneria -Dickeya -Erwinia -Pantoea -Pectobacterium -Phlomobacter -Sacchararobacter -Samsonia Environmental/ Animals/Industrial -Alterococcus -Budvicia -Buttiauxella -Obesumbacterium -Pragia -Trabulsiella

3. For each genome, ERIC has information for every gene with the most up to date annotations available #1) For example lets look at the annotation page for stx2A, the gene for shiga-toxin 2 (A subunit), type stx2A into Search All Genomes, and click submit #2) Your search results will look like this, every gene has a Feature ID which consists of 3 capitol letters a dash and 7 numbers (Ex. ABC-1234567) Click on the feature ID that corresponds to genome EDL933 version 2 (ABH-0025275)

4. On the left side of the page you will find some information, such as length of the gene/coding sequence and the length of the predicted protein There are also links to a variety of tools, you will be using the BLASTN and/or BLASTP against ASAP (ERIC) genomes to search in all 3 E. coli O157:H7 genomes

5. On the right side of the page are all of the annotations You can see the data for each annotation type, and can click on the evidence link to go where the information came from (for example click on Experimental for the first function annotation)

6. Mauve To work with the 3 O157:H7 alignment, you will need to follow a few steps #1) download progressive Mauve version 2.2.0 at http://gel.ahabs.wisc.edu/mauve/download.php #2) download the compressed folder called 3 O157 alignments.zip at http://gel.ahabs.wisc.edu/~baumler/ #3) extract (uncompress) all files from the folder

7. In your start menu under programs go to Mauve 2.2.0, start up Mauve, notice there is a users guide in pdf form in this folder, this will contain useful information and commands to navigate Note: your computer may need to update Java, since mauve uses a Java platform for the alignment. You should see a window for Mauve appear

8. Next double click on the uncompressed 3 O157H7 folder, it should contain the following 19 files, take the first one (3 O157 alignment), and drag and drop it into the mauve window It should start to say reading sequences here, and in a few seconds the alignment will appear, note computers with less than 512MB RAM may not be able to open the file

9. Your alignment should look like this Organism name notice the first is EDL933, the second is RIMD(Sakai), and the third is EC4042 (spinach) Using the up or down arrows, you can switch the position of the genomes

10. Your tool bar is at the top on the left, the tools you will use are in the View pulldown, and also the buttons Returns the viewer back to home Search for features Zoom in/out, you can also hold down the ctrl button and use the arrows on the keyboard Move left or right, you will find this useful to center a region of interest in the middle of the screen prior to zooming in

11. Top strand Bottom strand The colored blocks are called local colinear blocks (LCB’s), and represent regions of the genome that Mauve has identified as conserved, the lines connect the LCBS, notice that some are in different positions in the other genomes, some are inverted and appear on the bottom strand of the double stranded genome

12. When you move you mouse over a region of one genome it will show a black box and also show the corresponding region (boxes) in the other two genomes, try scrolling left to right on one genome

13. When viewing the LCB’s, mauve displays regions that are highly conserved/identical as full color. Areas that are unique/variable to one genome appear in white, and represent unique islands

14. Notice, that when you scroll (slowly) over a white region (island) the black boxes pause in the other genomes, then comes back once you have passed over the island and back into conserved regions

15. If you would like to look at all three LCB’s, even though one is in a different position, scroll over one LCB and click the mouse button

16. Lets use the zoom function, press the home button to restore the alignment to original view Now click on the white island in the top genome, and using the right button bring it to the center of the screen, now start to zoom in multiple times You will start to see the genes, scroll over one and pause, and a window will pop-up with the product annotation, so here you can view what genes are present in this EDL933 island, and not in the other two

17. Now place you mouse over one of the genes, in my example I have iha irgA homolog adhesion Click your mouse once on the gene, and a window will pop-up, scroll down and select View CDS iha in ERICdb This will open the page in the ERIC database for that gene, containing all of the annotations, you can look to see if it is involved in virulence

18. Lets use the search feature #1) Click on the search feature #2) Choose a genome (EDL933) #3) Type in a gene name (stx2A) #4) Click on search

19. Notice that it has found the stx2A gene (highlighted in blue), and also in the RIMD strain. Just because it isn't aligned in the EC4042 strain does not mean it isn't there, if you look to the right in the EC4042 genome, you will find it Stx2A

20. One last feature you can use in Mauve To find an island that is in 2 out of 3 strains you will use the backbone view Press the home button first Then go to the View pull down select color scheme then backbone color

21. Your alignment should look like this in backbone color, regions in all three appear in light purple color, there will be regions that are different colors that will correspond to 2 out of 3 genomes (you may have to zoom in a bit to see these regions Regions in only EDL933 and RIMD appear olive green Regions in only EDL933 and EC4042 appear maroon Regions in only RIMD and EC4042 appear tan/brown This is how you identify islands unique to 2/3 strains

22. Congratulations, you should be able to work on your individual assignments with your newfound knowledge using Mauve and the ERIC database Some other useful commands in mauve Export the current view as an image Ctrl+E Additional information is available in the Mauve users guide