Genomics of IS994: Transposase Genes in Renibacterium salmoninarum

Gina Capri

Dr. Daniel Rockey, Department of Biomedical Sciences

Acknowledgements

• Howard Hughes Medical Institute• Undergraduate Research, Innovation, Scholarship, Creativity

(URISC)• Dr. Daniel Rockey, OSU Department of Biomedical Sciences• The Rockey Lab (Jae Dugan, Sara Weeks, Hency Chu, Damir

Alzhanov, Brendan Jeffrey)• Dr. Kevin Ahern• Dr. Mark Strom, Dr. Greg Wiens• USDA/NRI Genome Sequencing program

R. salmoninarum: background• Bacterium, causes bacterial kidney

disease (BKD) in salmonids

• Discovered in the 1930’s

• First isolated and cultured in 1956

• Affects both cultured and wild salmonids, forming focal abscesses in the kidney, spleen and liver. .

Bacterial Kidney Disease (BKD)

Normal vs. diseased juvenile salmon

Bacterial Kidney Disease

http://www.medvet.umontreal.ca/departements/patho_micro/ictyopathologie/images/reni3.jpg&imgrefurl=http://www.medvet.umontreal.ca/departements/patho_micro/ictyopathologie/traitements.htm&h=294&w=400&sz=93&hl=en&start=3&tbnid=oDiDHhdui2i8IM:&tbnh=88&tbnw=120&prev=/images%3Fq%3Drenibacterium%2Bsalmoninarum%26svnum%3D10%26hl%3Den%26lr%3D%26sa%3

Image courtesy of:

Focal abscess

Focal abscess

Renibacterium salmoninarum

• Gram-positive• Non-sporulating• Non-motile rod• Grows best at 15-18°C and

not at all at 25°C• Slow growing (2-3 weeks

on KDM2, can take up to 8 or 12 weeks)

• Survives inside eggs and phagocytes

R. salmoninarum genome

• 3607 identified open reading frames (ORFs)

• 3179312 base pair genome

• Bacterial genome: circular

• Encodes more than 50 transposases

= Open reading frame

A simplified view of a bacterial genome

Infected Kidney Samples

• Approximately 20 kidney samples were received for study from ODFW

• So far, I have extracted DNA from two samples

• “A” - 2002 CC00002 6/5/06

• “B” - 2003 CC00481 6/7/06

• Qiagen tissue protocol, followed by Qiagen genomic-tip protocol

IS994: Insertion Sequence

• IS3 family insertion sequence

• 60+ IS994 sequences in the genome

• First IS identified from R. salmoninarum

• Common element 1330, 1331 or 1345 bp in length

• Two open reading frames: orfA and orfB, indicated in ERGO as arrows.

Two Open Reading Frames (ORFs)

• orfA - 363 bp• orfB - 831-888 bp, variable• orfA and orfB separated by a single

stop codon

Transposase interrupts dipeptide-related chain

Related regions in other organisms:

PrimersOur region of interest

X X Y Y Z Z Z

1 4

2

• Expected: X - 535 nt, Y - 449 nt, Zwith-1783 nt or Zwithout- 456 nt• Possible contamination?

600500

400

300

200

100

~2000

1000

3

X = Primers 1 and 2Y = Primers 3 and 4Z = Primers 1 and 4

A B A B A B G

Xba-I restriction digest:

Where precisely are the restriction fragments <2kb located in the genome?

• The five segments smaller than 2.0 kb were identified.

Position/Enzyme

before site (size ) Function

after site(size) ERGO ORF#

109496 XbaI 14258 Transposase 1570 RRSA00337

472093 XbaI 348 Transposase 1854 RRSA02075

969223 XbaI 1921 Transposase 29263 RRSA03438

2353959 XbaI 21505 Transposase 1707 RRSA03708

3085404 XbaI 1340 Transposase 6143 RRSA01533

• These correspond closely to the five segments in the Xba-I restriction digest.

Xba-I restriction digest:

• Summary and questions?