Aherman week 7

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.
 * 3) Rho-independat terminators- A DNA sequence signaling the termination of transcription
 * 4) Aetiological-  deals with the causes or origin of disease, the factors which produce or predispose toward a certain disease or disorder.
 * 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.
 * 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.
 * 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.
 * 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.
 * 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.
 * 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.

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

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