Aherman week 7

Powerpoint Presentation

Definitions of terms

1. Reductase- an enzyme that catalyses the biochemical reduction of some specified substance. [http://www.mondofacto.com/facts/dictionary?reductase}
2. Megaplasmid- extrachromosomal genetic elements in the size range of 100 kb and larger. [1]
3. Rho-independat terminators- A DNA sequence signaling the termination of transcription [2]
4. Aetiological- deals with the causes or origin of disease, the factors which produce or predispose toward a certain disease or disorder. [3]
5. Transposition- The ability of genes to change position on chromosomes, a process in which a transposable element is removed from one site and inserted into a second site in the DNA. [4]
6. Integron island- DNA elements that acquire open reading frames embedded in exogenous gene cassettes and convert them to functional genes by ensuring their correct expression. [5]
7. Proteobacteria- A bacterial phylum containing 1534 species or 32.3% of all known bacteria. Proteobacteria are all gram negative, but otherwise represent a diverse range of organisms. [6]
8. Entertoxigenic states- Refers to an organism that produces toxins in the gastrointestinal tract that cause such things as vomiting, diarrhea, and other symptoms of food poisoning. [7]
9. Lipases- Any of a group of lipolytic enzymes that cleave a fatty acid residue from the glycerol residue in a neutral fat or a phospholipid. [8]
10. Haemolysins- An agent or a substance, such as an antibody or a bacterial toxin, that causes the destruction of red blood cells, thereby liberating hemoglobin. [9]

Outline of Article 3

1. The completed genome sequencing of Vibrio cholerae
2. 2 circular chromosomes that encode 3,885 open reading frame's

The Large chromosome

• 2,961,146 base pairs that controls most important cellular functions

The Small chromosome

• 1,072,314 base pairs that contains a high percentage of hypothetical genes, a gene capture system, and some host addiction genes
• These characteristics suggest that the small chromosome was a megaplasmid captured far in the past by an ancestral Vibrio cholerae strain
• The genetic sequencing of this strain is critical for its understanding

What Vibirio cholera is

• Vibrio cholera is the cause of the human disease known as cholera
• There are many different strains including, pathogenic and non-pathogenic
• Bacterium thrives in oceans, coastal waters and estuaries
• Known to transfer genes horizontally

Genomic comparative analysis

• Sequencing performed using the the whole genome random sequencing model on the two circular chromosomes with 3,885 open reading frame's
• 792 predicted Rho-independent terminators
• Large chromosome contains- 2,961,146 base pairs, Av G+C (46.9%), 2,770 open reading frame’s, 599 Rho-independent terms
• Smaller chromosome contains- 1,072,314 base pairs, Av G+C 47.7%, encoded intermediary metabolic pathways

1,115 open reading frame’s, 193 Rho-independent terms

Methods

• Whole-genome random sequencing procedure- V. colerae grown in single isolated colony; with cloning, sequencing and assembly described by TIGR
• ORF predition determined
• Paralogous gene families determined
• Distribution of all 64 trinucleotides determination
• Homologues determined

Results

• Linear representation of the V. cholerae chromosomes
• Location of predicted coding regions coded by- biological role, RNA genes, tRNA, Rho-independent terminators, VCR

Circular representation of the V. cholerae genome

• Two chromosomes:
  • First and second circles- predicted protein-coding regions on the plus and minus strands by role
  • Third circle- recently duplicated genes on same chromosome and on different chromosomes
  • Fourth circle- transposition-related (black), phage-related (blue), VCR's (pink) and pathogenesis genes (red)
  • Fifth circle- regions with significant values for trinucleotide composition of 2,000 base pairs
  • Sixth circle- % C+G in relation to the mean G+C for the chromosome
  • Seventh circle- tRNA
  • Eighth circle- rRNA

Overview of metabolism nd transport in V. cholerae

• Pathways for energy production and the metabolism of organic compounds:
  • Transporters are grouped by substrate
  • Green = cations
  • Red = anions
  • Yellow = carbohydrates
  • Purple = nucleosides, purines and pyrimidines
  • Blue = amino acids, peptides, and amines
  • Question marks indicate:
    • putative genes
    • uncertainties in substrate specificity
    • directions of transport

Gene location of both transporters and metabolic steps indicated by colored arrow

• Black = genes located on large chromosome
• Blue = genes located on small chromosome
• Purple = all genes needed for complete pathway on one chromosome but a duplicate copy on one or more genes on other chromosome
• Red = required genes on both chromosomes
• Green = complete pathway on both chromosomes

Table 1 - General features of the Vibrio cholerae genome

• Replicative origin in chromosome 1

1. Vibrio harveyi and Escherichia coli:

• co-localization of genes found near origin of prokaryotic types, dnaA/N, recF, and gyrA

1. GC nucleotide skewed distribution:

• GC = (G-C/G+C) analysis conclusions:
  • 1 - designated base pair 1 in an intergenic region located in origin of replication
  • 2 - skew was useful to identify a putative origin on 2nd chromosome
• Genomic sequence displayed presence of large integron island on chromosome 2
• Integron island contains all copies of the VCR sequences and 216 open reading frame’s
• Among recognizable genes are those that encode:
  • products that could provide drug resistance:
    • chloramphenicol acetyltransferase
    • fosfomycin resistance protein
    • glutathione transferase
• DNA metabolism enzymes:
  • MutT
  • transposase
  • an integrase
• Virulence genes:
  • haemagglutinin
  • lipoproteins
• 'host addition' proteins which plasmids use to select their maintenance from host cells:
  • higA
  • higB
  • doc

Comparative genomics

• Comparison types used between the two V. cholerae chromosomes
  • Assymetrical distribution of genes known for growth and virulence between the chromosomes
• Chromosome 1 encode’s DNA replication and repair
  • transcription and translation
  • cell-wall synthesis
  • several central catabolic and biosynthetic pathways
  • bacterial pahogenicity
• Chromosome 2 encode’s
  • Greater number (59%) of hypothetical genes and those of unknown function
  • The partitioning of hypothetical genes proteins is highly localized in the integron island on chromosome 2
  • Carries 3-hydroxyl-3-methylglutaryl CoA reductase - most likely acquired from an archaea

V. cholerae chromosomes and chromosomes of other microbial species

• Percentage of total Vibrio cholerae open reading frames in biological roles compared with general Proteobacertia (figure 4)
  • Majority of V. cholerae genes very similar to E. coli genes
  • 499 ORFs showed highest similarity to other V. cholera genes suggesting recent dupes
  • functions related to:
    • regulatory functions, chemotaxis, transport and binding, transposition, pathogenicity
  • Significant duplication of scavenging behavior genes involved in:
    • Chemotaxis and solute transport
      • Suggests high importance in V. cholerae biology:
        • Ability to exist in in many diverse environments, environments may have selected genes for duplication and divergence of genes to support function
        • Various strains have different numbers and location of these genes
        • Virulence gene numbers are subject to pressures which affect copy numbers and location

Comparison of the V. cholerae ORF's with those of other completely sequenced genomes

• Protein sequences from NCBI, TIGR and Caenorhabditis elegans database
• V. cholerae open reading frames compared against all other genomes
• Number of V.cholerae open reading frames similarity displayed proportionatly to the total open reading frames of that genome

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

• Homologues of MCP, identified through FASTA3
• Sequences aligned using CLUSTALW
• The neighbor-joining phylogenetic tree generated using a PAM-based distance calculator
• Hypervariable regions of alignment and position with gaps excluded
• ORFS with seemingly identical functions exist on both chromosomes which suggest acquisition by lateral gene transfer
  • glyA found on both chromosomes

Conclusions of biological significance

• Origin small V.cholerae chromosome is likely a megaplasmid absorbed by a strain.
• Transport and energy metabolism- Resides in water, zooplankton, and the human gastrointestinal tract. Contains many transport proteins that cover a large general substrate/pathway specificity.
• Interchromosonal regulation- Both chromosomes interact with one another in response to signals of environmental stress, such as starvation and entertoxigenic states.
• DNA repair- Many homologues are shared between the two chromosomes, that can both serve to maintain functioning DNA such as nucleotide excision, mismatch excision, and alkylation transfer. (not found in E. coli)
• Pathogenicity- Strain contains a single copy of cholera toxin gene CTX (on chromosome 1). Other potential toxin genes are present as well, such as haemolysins proteases and lipases.

Conclusion

• V.colerae genome sequence serves as starting point to study environmental and pathobiological characteristics
• Attention should be focused on the gene expression patterns that govern its survial and replication during human infection as well as the various earthly environments in which it is found
• DNA sequencing will assist greatly the continued study of this model organism
• Origins of the new smaller chromosome and its specific functions
• Understanding metabolic and regulatory link between two chromosomes
• Basis to study how several horizontally acquired loci on each chromosome can still interact at regulatory, cell and biochemical levels.

Andrew Herman 20:24, 17 October 2010 (EDT)