Transcript Bio_db_presentation_on_Vibrio_cholera_rb

The DNA Sequencing of both
Chromosomes of the Cholera
pathogen Vibrio cholerae
John F. Heidelberg, Jonathan A. Eisen, William C. Nelson, Rebecca A. Clayton, Michelle L. Gwinn, Robert J. Dodson,
Daniel H. Haft, Erin K. Hickey, Jeremy D. Peterson, Lowell Umayam, Steven R. Gill, Karen E. Nelson, Timothy D. Read,
Hervé Tettelin, Delwood Richardson, Maria D. Ermolaeva, Jessica Vamathevan, Steven Bass, Haiying Qin, Ioana
Dragoi, Patrick Sellers, Lisa McDonald, Teresa Utterback, Robert D. Fleishmann, William C. Nierman, Owen White,
Steven L. Salzberg, Hamilton O. Smith, Rita R. Colwell, John J. Mekalanos, J. Craig Venter & Claire M. Fraser
Nature 406, 477-483 (3 August 2000) | doi:10.1038/35020000; Received 3 April 2000; Accepted 18 May 2000
Andrew Herman, Richard Brous, Jennifer Okonata
Biological Databases
10/18/2010
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Outline
• What is Vibrio cholerae?
• A genomic comparative analysis of VC
• Methods and results of genomic
comparisons.
• The biological significance of the data.
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Outline
• What is Vibrio cholerae?
• A genomic comparative analysis of VC
• Methods and results of genomic
comparisons.
• The biological significance of the data.
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What is Vibrio cholera?
• An aetiological agent of cholera
– highly infectious
– epidemic form
• Species includes both pathogenic and nonpathogenic strains
• Contains a wide variety of strains and biotypes
– Zooplankton in a sessile stage
– Planktonic state in the water column
– Capacity to act as a pathogen within the human gastrointestinal
tract.
– In aquatic environments, chitin is a source of both carbon and
nitrogen.
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What is Vibrio cholera? (cont.)
• Receives and transfers genes for toxin
– El Tor N16961 revealed a single copy of the cholera toxin (CT)
gene
• Colonization factors
• Antibiotic resistance
• Capsular polysaccharides that provide resistance to chlorine
• Surface antigens
• Represents the significant portion of the culturable heterotrophic
bacteria of oceans, coastal waters and estuaries
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Outline
• What is Vibrio cholerae?
• A genomic comparative analysis of VC
• Methods and results of genomic
comparisons.
• The biological significance of the data.
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Genomic Comparative Analysis of Vibrio
Cholera
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The genome of V. cholerae was sequenced by the whole genome random
sequencing method
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Two circular chromosomes
– 2,961,146 base pairs
– 1,072,314 (chromosome 2) base pairs
– average G+C content = 46.9% and 47.7%
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3,885 predicted open reading frames
•
792 predicted Rho-independent terminators
– 2,770 and 1,115 ORFs and 599 and 193 Rho-independent terminators
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Genomic Comparative Analysis of Vibrio
Cholera (cont.)
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Two-chromosome structure of V. cholerae allows for comparison:
– Between the two chromosomes of this organism
– Between either of the V. cholerae chromosomes
– The chromosomes of other microbial species.
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Chromosome 1
– Most genes that are required for growth and viability
– Asymmetry in the distribution of genes known to be essential for growth
and virulence
– Significantly more genes encoding:
• DNA replication and repair
• Transcription and translation
• Cell-wall biosynthesis
• Central catabolic and biosynthetic pathways
• Genes known to be essential in bacterial pathogenicity
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Genomic Comparative Analysis of Vibrio
Cholera (cont.)
• Chromosome 2:
– Larger fraction (59%) of hypothetical genes/genes of unknown function
(Figure 4)
– This partitioning is highly localized in the integron island
– Carries the 3-hydroxy-3-methylglutaryl CoA reductase
• A gene acquired from an archaea
– Evidence suggests megaplasmid origins
• Unclear why chromosome 2 has not been integrated into
chromosome 1
• Specialized function that provides the evolutionary selective pressure
to suppress integration events
– Environmental conditioning causes a difference in copy number
– Chromosome 2 may have accumulated genes that are better
expressed at higher or lower copy number
– In response to environmental cues aberrant segregation of
genes on each chromosome
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Outline
• What is Vibrio cholerae?
• A genomic comparative analysis of VC
• Methods and results of genomic
comparisons.
• The biological significance of the data.
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FIGURE 1. Linear representation of
the V. cholerae chromosomes
• Shows the location of the predicted
coding regions
• Color-coded by biological role
• Arrows represent the direction of
transcription
High-Res Image and Legend
• Numbers represent the # of tRNA
• # of GES represent the number of
membrane-spanning domains
Methods
a)Vibrio cholerae N16961 was grown from a single isolated colony.
b)Cloning, sequencing and assembly were as described for genomes sequenced by TIGR
c) One small-insert plasmid library (2–3 kb) was generated by random mechanical shearing of genomic DNA.
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FIGURE 2. Circular representation of
the V. cholerae genome
• 2 chromosomes
• 1st and 2nd circle shows the predicted protein-coding
regions on the plus and minus strand
• 3rd circle shows recently duplicated genes on the
same chromosome (black) and on different
chromosomes (green)
High-Res Image and Legend
• 4th circle shows transposon-related (black), phagerelated (blue), VCRs (pink) and pathogenesis genes
(red)
• 5th circle shows regions with significant 2 values for
trinucleotide composition in a 2,000-bp window
• 6th circle shows percentage G+C in relation to mean
G+C for the chromosome
• 7th and 8th circles are tRNAs and rRNAs
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FIGURE 3. Overview of metabolism
and transport in V. cholerae
• Shows the pathways for energy production and
the metabolism
• Transporters are grouped by substrate
• Permeases are represented as ovals
High-Res Image and Legend
• Gene location on the two chromosomes, for
both transporters and metabolic steps, is
indicated by arrow color
• Gene numbers on the two chromosomes are in
parentheses and follow color-scheme for gene
location
Methods
a)In the initial sequence phase, approx. sevenfold sequence coverage was achieved with 49,633 sequences from plasmid clones.
b) Sequences from both ends of 383 clones served as genome scaffold, verifying the orientation, order and integrity of the contigs.
c) Physical gaps were closed by direct sequencing of genomic DNA, or combinatorial polymerase chain reaction (PCR) followed by
sequencing the PCR product.
d) The final genome sequence is based on 51,164 sequences.
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Table 1. General features of the
Vibrio cholerae genome
• This table shows the size of both chromosomes.
• The total number of sequences.
• G + C percentage
• Total number of open reading frames & size
• Number of ribosomal (rRNA) & transfer (tRNA).
Full Table
• Number similar to known & unknown proteins
• Number of Rho-Independent terminators
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FIGURE 4. - V. cholerae ORFs %
comparison other Proteobacteria
• Majority of V. cholerae genes very
similar to E. coli genes (1,454 ORFs)
• 105 duplications with at least one ORF
on each chromosome
High-Res Image and Legend
• Significant duplication of scavenging
behavior genes
Method:
a) Initial set of ORFs from GLIMMER. ORFs searched against non-redundant protein database.
b) ORFs also analyzed with two set of Markov Models (HMMs) constructed for a number of conserved protein families.
c) TopPred used to identify membrane-spanning domains in proteins.
d) Paralogous gene families by searching ORFs against themselves in BLASTX, clustering matches into multi-gene families.
e) Multiple alignments generated with CLUSTALW
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FIGURE 5. - V. cholerae ORFs vs.
other sequenced genomes
• V. cholerae ORFs (chromosome 1 in
blue and chromosome 2 in red)
compared against all other genomes
with FASTA3
High-Res Image and Legend
• Number of V.cholerae ORFs with
greatest similarity displayed
proportionately to the total ORFs of that
genome
Method:
a) Initial set of ORFs from GLIMMER. ORFs searched against non-redundant protein database.
b) ORFs also analyzed with two set of Markov Models (HMMs) constructed for a number of conserved protein families.
c) TopPred used to identify membrane-spanning domains in proteins.
d) Paralogous gene families by searching ORFs against themselves in BLASTX, clustering matches into multi-gene families.
e) Multiple alignments generated with CLUSTALW
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FIGURE 6. Phylogenetic tree of
(MCP) homologues
• ORFS with seemingly identical
functions exist on both chromosomes
which suggest acquisition by lateral
gene transfer
High-Res Image and Legend
• Example: glyA found on both
chromosomes
Method:
a) Homologues of genes identified using BLASTP and FASTA3 db searches.
b) Homologues aligned using CLUSTALW program.
c) Phylogenic trees generated from alignments using neighbor-joining algorithm implemented by the PAUP application
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Outline
• What is Vibrio cholerae?
• A genomic comparitive analysis of VB
• Methods and results of genomic
comparisions.
• The biological significance of the data.
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The Biological Significance of Vibrio
cholerae
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Provides a new starting point for the study of this organism's environmental
and pathobiological characteristics
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The study of gene expression patterns involved in survival and replication
during
– human infection
– environmental factors
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The genomic sequence facilitates the study of this model multi-chromosomal
prokaryotic organism
•
Comparative genomics provides a better understanding of the origin of the
new small chromosome
– What role does it plays in Vibrio biology
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The Biological Significance of Vibrio
cholerae (cont.)
•
May also provide understanding to metabolic and regulatory networks that
link genes on the two chromosomes
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Horizontally acquired loci located on separate chromosomes still interacting
at the:
– regulatory
– cell biology
– biochemical levels.
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