Aherman week 7
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Definitions of terms
- Reductase- an enzyme that catalyses the biochemical reduction of some specified substance. [http://www.mondofacto.com/facts/dictionary?reductase}
- Megaplasmid- extrachromosomal genetic elements in the size range of 100 kb and larger. [1]
- Rho-independat terminators- A DNA sequence signaling the termination of transcription [2]
- Aetiological- deals with the causes or origin of disease, the factors which produce or predispose toward a certain disease or disorder. [3]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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
- The completed genome sequencing of Vibrio cholerae
- 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
- Vibrio harveyi and Escherichia coli:
- co-localization of genes found near origin of prokaryotic types, dnaA/N, recF, and gyrA
- 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
- products that could provide drug resistance:
- 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
- Suggests high importance in V. cholerae biology:
- Chemotaxis and solute transport
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)