Jennifer Okonta

Shared Assignment

Defined Words

1. transposases-Transposase is an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. en.wikipedia.org/wiki/Transposases
2. chemontaxis-movement by a cell or organism in reaction to a chemical stimulus. wordnetweb.princeton.edu/perl/webwn
3. prophage-A prophage is a phage genome inserted as part of the linear structure of the DNA chromosome of a bacterium. en.wikipedia.org/wiki/Prophage
4. integron-A genetic unit that, among others, encodes proteins that splice gene cassettes into chromosomes, where the cassettes can become functional. www.nature.com/nrg/journal/v5/n3/glossary/nrg1292_glossary.html
5. aetiological- the cause of a disease. wordnetweb.princeton.edu/perl/webwn
6. heterotrophic- obtaining nourishment from organic substances, not from food produced within the organism.wordnetweb.princeton.edu/perl/webwn
7. Transposon- a segment of DNA that can become integrated at many different sites along a chromosome (especially a segment of bacterial DNA that can be translocated as a whole).wordnetweb.princeton.edu/perl/webwn
8. pathogenesis- the origination and development of a disease. wordnetweb.princeton.edu/perl/webwn
9. Permease - The permeases are membrane transport proteins, a class of multipass transmembrane proteins that facilitate the diffusion of a specific molecule in or out of the cell.en.wikipedia.org/wiki/Permease
10. Rho-Independent terminator- The rho-independent signal is found on the DNA template strand and consists of a region that contains a section that is then repeated a few base pairs away in the inverted sequence. http://www.sparknotes.com/biology/molecular/dnatranscription/section2.rhtml

Outline

What is Vibrio Cholerae

• the aetiological agent of cholera
• includes pathognenic and non pathognenic strains that vary in gene content
• contains a wide variety of strains and biotypes
• receives and transfers genes for toxins
• has colonization factors
• is resistant to antibiotics
• has capsular polysaccharides that provide resistance to chlorine and new surface antigens
• it represents a significant portion of the culturable heterotrophic bacteria of oceans, coastal waters and estuaries
• these bacteria strongly influence nutrient cycling in the marine environment

Purpose of the Work

• To determine and analyze the genome sequence of Vibrio Cholerae.
• It is an important step toward the complete molecular description of how this free-living environmental organism became a human pathogen by horizontal gene transfer.

Genome Analysis & Comparative Genome Analysis

• Sequenced by the whole genome random sequencing method.
• Two circular chromosomes make up this genome
• 3,885 predicted open reading frames (ORFs) and 792 predicted Rho-independent terminators
• Most genes required for growth and viability are located on chromosome 1
• two-chromosome structure of V. cholerae allows for comparisons
• There is pronounced asymmetry in the distribution of genes known to be essential for growth and virulence between the two chromosomes
• Chromosome 2 contains a larger fraction (59%) of hypothetical genes and genes of unknown function, compared with chromosome
• Most genes known to be essential in bacterial pathogenicityare located on chromosome 1

Figure Analysis

Figure 1

Linear representation of the V. cholerae chromosomes

• Shows the location of the predicted coding regions, colour-coded by biological role, RNA genes, tRNAs, other RNAs, Rho-independent terminators and Vibrio cholerae repeats.
• Arrows represent the direction of transcription for each predicted coding region

Figure 2

Circular representation of the V. cholerae genome

• 2 chromosomes
• the first and second circles show predicted protein-coding regions on the plus and minus strand
• The third circle shows recently duplicated genes on the same chromosome and on different chromosomes
• The fourth circle shows transposon-related (black), phage-related (blue), VCRs (pink) and pathogenesis genes (red
• The fifth circle shows regions with significant values for trinucleotide composition in a 2,000-bp window
• The sixth circle shows percentage G+C in relation to mean G+C for the chromosome.
• The seventh and eighth circles are tRNAs and rRNAs, respectively.

Figure 3

• Pathways for energy production and the metabolism of organic compounds, acids and aldehydes are shown.
• Transporters are grouped by substrate specificity.
• Question marks associated with transporters indicate a putative gene, uncertainty in substrate specificity, or direction of transport
• Export or import of solutes is designated by the direction of the arrow through the transporter
• Gene location on the two chromosomes, for both transporters and metabolic steps, is indicated by arrow color.

Table 1

General features of the Vibrio cholerae genome.

• self explanatory
• the size, number of ORF, the number of tRNA, the number rRNA, the number of significant and unsignificant proteins and other things are listed.

Figure 4

Percentage of total Vibrio cholerae open reading frames (ORFs) in biological roles compared with other -Proteobacteria

• V. cholerae, chromosome 1 (blue)
• V. cholerae, chromosome 2 (red)
• Escherichia coli (yellow)
• Haemophilus influenzae (pale blue)

Figure 5

Comparison of the V. cholerae ORFs with those of other completely sequenced genomes

• All V. cholerae ORFs (large chromosome, blue; small chromosome, red) were searched against all other genomes with FASTA

Figure 6

Phylogenetic tree of methyl-accepting chemotactic proteins (MCP) homologues in completed genomes

• Amino-acid sequences of the proteins were aligned using CLUSTALW
• Neighbor-joining phylogenetic tree was generated from the alignment using the PAUP program

Methods

• Grown from a single isolated colony
• Cloning, sequencing and assembly were as described for genomes sequenced by TIGR
• Sequences from both ends of served as a genome make up to show the orientation, order and integrity of the contigs
• Sequence gaps were closed by editing the end sequences and/or primer
• The initial set of ORFs was identified with GLIMMER and those shorter than 30 codons were eliminated
• ORFs were searched against a non-redundant protein database
• Frameshifts and point mutations were detected and corrected when needed
• Paralogous gene families were made by searching the ORFs against themselves using the program BLASTX
• Probability values for this analysis are based on the assumption that the DNA composition is relatively uniform throughout the genome
• Homologues of the genes of interest were identified using the BLASTP and FASTA3 search programs
• All homologues were then aligned to each other using the CLUSTALW program with default settings
• Phylogenetic trees were generated from the alignments using the neighbour-joining algorithm

Conclusions

• New starting point for the study of V. Cholerae environmental and pathobiological characteristics
• The genomic sequence of V. cholerae should facilitate the study of this model multi-chromosomal prokaryotic organism
• Might provide important clues to understanding the metabolic and regulatory networks that link genes on the two chromosomes
• Represents a promising genetic system for studying how several horizontally acquired loci located on separate chromosomes can still efficiently interact at the regulatory, cell biology and biochemical levels

Jennifer Okonta 13:32, 17 October 2010 (EDT)