Genome Analysis of Burkholderia pseudomallei

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Genome Analysis of Burkholderia pseudomallei

• Xie Chao

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Introduction

• Burkholderia pseudomallei is a free-living
  gram-negative soil bacillus that causes
  melioidosis
• The genome was completely sequenced on May 2003
  by the Sanger Institute
• Two replicons - 4Mbp (chromosome 1)
  - 3Mbp (chromosome 2)
• No annotation yet

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Objectives

• To construct the genome annotation database
• To analyze the genome using the database
• General analysis
• Comparison of the two chromosomes
• Prediction of putative Pathogenicity Islands
  (clusters of virulence genes)

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Creation of B. pseudomallei Genome Database -
Annotation
Predict ORF by GeneMark
5679 ORFs predicted
ORFs gt BLAST
4987 ORFs have homologs
ORFs gt NCBI Conserved Domain Database
4161 ORFs contain Pfam domains 4332 ORFs contain
COG domains
ORFs gt Functional Categories
3553 ORFs in categories
ORFs gt Gene Ontology vocabulary to classify
genes In more detail
3066 ORFs in GO

• 30 Java or Perl programs have been created to
  carry out the tasks

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B. pseudomallei Genome Database

• 16 tables were created in the database to store
  the annotation results
• 19 Perl scripts were written for the web
  interface of the database
• http//origin.bic.nus.edu.sg/xiechao/query.html

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Analyses of B. pseudomallei Genome- General
features
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Analyses of B. pseudomallei Genome- Comparative
genomics

• The ORFs were compared with all the 155
  completely sequenced microbial genomes
• 37 of B. pseudomallei genes have best homology
  with R. solanacearum genes
• Percentage of B. pseudomallei ORFs with best
  homolog matches and their source organisms was
  shown

No Homolog 15
R. solanacearum 37
P. aeruginosa 7
C. violaceum 5
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Replicon 2 is more diverse than Replicon 1
Percentage of ORFs with best homolog matches and
their source organism for each B. pseudomallei
replicon.
No homolog
R. solanacearum 19
No homolog
R. solanacearum 49
P. aeruginosa 10
C. violaceum 6
C. violaceum 5
P. aeruginosa 5
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Chromosome vs Plasmid?

• No clear definition
• Origin of replication, DnaA vs RepA
• Chromosome generally housekeeping genes
• Plasmid - carry genes conferring growth
  advantage, virulence factors etc, but not
  essential.
• Therefore, examine the origin of replication and
  the genes encoded by the two replicons next

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Is Replicon 2 a megaplasmid?- analysis of
origins of replication

• A Java program ORIPredict was written to predict
  origin of replication
• GC-skew analysis (based on the bias toward G in
  the leading strand during replication) (Lobry,
  JR 1996)
• (C-G)/(CG) window size 20kbp sliding step
  5kbp

Replicon 2
Origin of replication
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Replicon 2 has plasmid-like ori

• Plasmid type origin of replication
• Plasmid replication initiator RepA
• Direct repeats in AT-rich region 15 (iteron)
• Low copy number plasmid partition protein ParA
  and ParB

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Housekeeping genes complete in Replicon 1
incomplete in Replicon 2

• Replicon 1 encodes a complete set of essential
  housekeeping genes required for
• (1) DNA replication, cell division
• (2) transcription and
• (3) translation.
• Replicon 1 other essential pathways
• purine and pyrimidine biosynthesis and salvage
• coenzyme biosynthesis
• amino acid biosynthesis
• electron transport and phosphorylation
• None of the above pathways is complete on
  replicon 2
• Encodes some genes in the pathways
• Keep the pathways work in various environment?
• More transcriptional regulators (p3.1e-6) more
  robust control?

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Conclusion I

• Chromosome 2 is a highly probable megaplasmid
• Possibly dispensable, as replicon 1 encodes a
  full set of housekeeping genes and other
  essential genes
• Size-3 Mbp plasmid?

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Prediction of Pathogenicity Islands in B.
pseudomallei

• Virulence genes are often clustered together,
  called Pathogenicity Island (PAI)
• Most PAIs are putative alien
• Different genomic properties compared to whole
  genome
• Karlin (2001) has reviewed 5 criteria to identify
  PAI
• GC frequency
• Dinucleotide bias
• Codon usage bias
• Amino acid usage bias
• Putative alien genes cluster

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Prediction of PAI in B. pseudomallei-
Implementation

• Java Program PAIPredict with graphical user
  interface was written
• The prediction of B. pseudomallei PAI was carried
  out using this program

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Prediction of PAI in B. pseudomalleiReplicon 1
Window size 100 kbp Sliding step 5 kbp
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Replicon 1 has 3 putative PAIs
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Replicon 2 results Window size 100 kbp Sliding
step 5 kbp
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Replicon 2 has 4 putative PAIs
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Conclusion

• The B. pseudomallei Genome Database (BpmDB) was
  constructed, and web interface of this database
  was designed.
• Many software programs were created
• PAI prediction package (PAIPredict)
• Origin of replication prediction (ORIPredict)
• Database interface Perl scripts
• 30 other Java or Perl program or scripts
• Chromosome 2 is probably a 3.2 Mbps megaplasmid
  (largest known so far is 2.1 Mbps)
• Experimental verification needed
• E.g. knock out replicon 2 ori
• Discovery of 5 new putative PAIs in B.
  pseudomallei genome 2 PAIs confirmed

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Acknowledgement

• I would like to express my sincere appreciation
  to my supervisors, Dr Chua Kim Lee and A/P Tan
  Tin Wee, for their guidance, patience, and frank
  advice throughout the entire project