Difference between revisions of "Ksherbina Week 11"

Revision as of 00:25, 12 November 2013

Paper for Journal Club:

Stephens, R.S., Kalman, S., Lammel, C., Fan, J., Marathe, R., Aravind, L., Mitchell, W., Olinger, L., Tatusov, R., Zhao, Q., Koonin, E. V., Davis, R.W. (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282: 754-759. doi: 10.1126/science.282.5389.754

10 Biological Terms from Paper

• Biovar - A strain differentiated from other strains by biochemical or other non-serological means.

Abedon, S. T. (1998) Supplemental Lecture (98/04/14 update). <http://www.mansfield.ohio-state.edu/~sabedon/biol3010.htm>. Accessed 10 November 2013.

• Contig - Copies of pieces of DNA that represent the overlapping regions of a chromosome.

U.S. Department of Energy Human Genome Project (2013) The Human Genome Project Information Archive 1990-2003. <http://web.ornl.gov/sci/techresources/Human_Genome/glossary.shtml>. Accessed 10 November 2013.

• Entner-Doudoroff pathway - A pathway that converts glucose to pyruvate and glyceraldehyde-3 phosphate by producing and then dehydrating 6-phosphogluconate.

EMBL-EBI (2013) GO:0009255 Entner-Doudoroff pathway. <http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0009255>. Accessed 10 November 2013.

• Hydrodynamic shearing - A technique that fragments DNA molecules by forcing them through a small orifice or the bore of a small-diameter tube at high velocity.

Joneja, A. & Huang, X. (2009) Supplementary Material For: A device for automated hydrodynamic shearing of genomic DNA. BioTechniques 46: 553–556.

• Serology - A type of laboratory medicine that analyzes blood serum for signs of infection by looking at antigen-antibody interactions in vivo.

Serology. (n.d.). Medical-Dictionary. <http://medical-dictionary.thefreedictionary.com/serology>. Accessed 10 November 2013.

Outline of the Article

Importance of sequencing the Chlamydia trachomatis genome

• C. trachomatis causes several diseases in human beings, including trachoma, which leads to blindness.
• C. trachomatis infections in humans may increase the risk of HIV infection.
• At the time the article was released, little was known about the two developmental stages of C. trachomatis (i.e. the elementary body and the reticulate body) as well as the bacterial pathogen's physiology and genetics.

Method used to sequence the genome of Chlamydia trachomatis strain D/UW-3/CX

• Chlamydial elementary bodies (EBs) were isolated from infected host cells using sonication on ice and then centrifuged to create pellets that were suspended in 5 mL of Hank's balanced salt solution.
• After two washes through centrifugation, extracellular DNA was removed by incubating the cells in 5µg/mL of DNase and RNase at 37°C for 30 min.
• The cells were purified using 30%, 40%, 45%, and 50% Renografin and then suspended in HBSS or SPG and stored at -70°C.
• After hydrodynamic shearing, the fragmented DNA was cloned into M13 phage.
• 28,458 sequencing reactions were performed using dye-labeled primers with the ABI Catalyst 800 Turbo robot followed by 4,688 dye-terminator reactions.
• 23 contigs from 4 to 164 kbp were observed using the Phrap and Phred software.
• Physical gaps in the genome were closed by either sequencing a PCR product that spans the gap or by using custom oligonucleotide primers.
• The plasmid that was sequenced from strain D/UW-3/CX had two less codons that previously sequenced chlamydial plasmids.
• Two methods were used to validate the assembled sequence: the predicted restriction digest map of the sequence as compared to the physical genome map of NotI and SgrAI and the restriction map and fragment sizes were analyzed after digesting oligonucleotides spaced 15kbp apart with BamHI .
• The results showed that the strain contains a 1,042,519 base pair chromosome and a 7493 bp plasmid.

Methods used to annotate the genome

Analysis of gene expression pathways

• Enzymes were discovered in C. trachomatis orthologous to enzymes in other bacteria that are involved in DNA replication, repair, transcription, and translation.
• The bacterium seems to have DNA repair and recombination systems, as is evident by the presence of two DNA helicases of the Swi2/Snf2 family.
• Genes were identified in the bacterial genome that code for aminoacyl-transfer RNA synthetases, two identical ribosomal RNA operons, RNA modification enzymes, translation factors, and a complete set of ribosomal proteins.
• The chlymadial genome codes for two alternative sigma factors, σ²⁸ and σ⁵⁴, which are likely involved in initiating differentiation from one developmental stage to another.
• The presence of an σ-factor regulatory system in C. trachomatis suggests that his system regulates different stages within the developmental cycle, monitors ATP status, or participates in the heat-shock response similar to that in Bacillus subtilis.

Analysis of metabolic pathways

• Genes were missing for both the Entner-Doudoroff pathway and the TCA cycle.
• As a result of the presence of a complete glycogen synthesis and degradation system, the authors suggest that glycogen is a the primary carbon source and may be involved in developmental stage differentiation.
• Genes were found that are necessary for aerobic respiration and that protect the bacterium from the toxic oxygen intermediates created through respiration.
• In contrast to the prevailing belief that the bacterium only obtains ATP from its host, genes were found in the chlamydial genome that are involved in ATP synthesis.
• While few genes were found that are involved in amino acid biosynthesis, many genes were discovered that encode enzymes involved in fatty acid and phospholipid biosynthesis.
• Contrary to past belief that C. trachomatis lacks peptidoglycan, the authors of the paper found that the bacterium's genome codes for the entire peptidoglycan biosynthesis pathway. This finding suggests that the bacterium utilizes peptidoglycan differently than other bacteria.

Membrane Composition, Proteins Part of the Intracelluar Vacuole, and Nutrient Uptake Across the Vacuole

• The presence of numerous amino acid and peptide transporters as well as porins and membrane transport components suggest that C. trachomatis obtains the nutrients it needs from its host through membrane transport systems.
• Figure 1 discusses the identification of nine paralogous genes encoding Pmp outer membrane proteins and proteases located in two clusters (one of the genes was not found in either cluster).

• Part A of the figure shows the orientation of the genes within each cluster indicating the direction of the coding strand.
• Part B of the figure displays the predicted molecular mass and pI for each the nine Pmp proteins. While the proteins differed in their sequence overall, two types of tetrameric amino acid repeat motifs were found in each of the proteins toward the N-terminus: FXXN and GGAI.
• Part C of the figure shows that certain motifs are conserved between the the proteases encoded in the C. trachomatis genome and adenovirus proteases.

Model Organism Database

http://efaecalis.mlst.net/ http://liferay.csb.univie.ac.at/portal/web/chlamydiaedb

• http://mbgd.genome.ad.jp/htbin/MBGD_whole_html.pl?spec=ctr for strain in genome paper not in microarray paper
• http://pedant.gsf.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r58333_Chl_trach cannot search for genes but gives a complete list of gene
• General database for many genomes but you can search for genes within a particular strain: http://www.biocyc.org/organism-summary?object=CTRA315277