Dwilliams Week 11 assignment

Article 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 Article

• Spirochetes -- A microscopic bacterial organism, a spirochete apperars worm-like, spiral-shaped, and wiggles vigorously when viewed under a microscope. Treponema pallidum, the cause of syphilis, is a particularly well-known member of the spirochaeta family. The term spirochete is an odd hybrid of greek and latin roots, the latin spira for coil and the greek chaite for long flowing hair, formed because the spirochete looked like a coil of hair.

<http://www.biology-online.org/dictionary/Spirochetes> Accessed 10 November, 2013

• Porphyrin -- porphyrins are pigments found in both animal and plant life. They are all chelates with metals (fe, mg, co, zn, cu, ni) and constituents of haemoglobin, chlorophyll, cytochromes. <http://www.biology-online.org/dictionary/Porphyrin> Accessed 10 November, 2013
• pseudouridylate synthase activity -- Catalysis of the reaction: D-ribose 5-phosphate + uracil = H(2)O + pseudouridine 5'-phosphate.

<http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0004730> Accessed 10 November, 2013

• Elementary body -- "Miyagawa bodies" A term previously used to refer to chlamydia trachomatis (miyagawanella lymphogranulomatosis), the elementary body's that develop in the intracytoplasmic microcolonies of lymphogranuloma venereum.

<http://www.biology-online.org/dictionary/Miyagawa_bodies> Accessed 10 November, 2013

• Reticulate body -- The chlamydia have two forms, transforming into the reproductive reticulate body after being endocytosed by a host cell.

<http://www.atsu.edu/faculty/chamberlain/Website/Lects/Rickett.htm> Accessed 10 November, 2013

• Domain protein -- (1) An autonomously folding functional unit of a protein.

(2) A part of protein that can fold, function and exist independently of the rest of the protein chain or structure. <http://www.biology-online.org/dictionary/Protein_domain>

• Mycobacteria -- bacteria with unusual cell walls that are resistant to digestion, being waxy, very hydrophobic and rich in lipid, especially esterified mycolic acids. Staining properties differ from those of gram-negative and gram-positive organisms, being acid-fast. Many are intracellular parasites, causing serious diseases such as leprosy and tuberculosis. Cell wall has strong immunostimulating (adjuvant) properties due to muramyl dipeptide (MDP). Mycobacterium bovis causes tuberculosis in cattle, attenuated strain is bacille Calmette-Guerin (bCG), used for immunisation. Mycobacterium leprae is the causative agent of leprosy. Mycobacterium microti is a mycobacterium that causes tuberculosis like disease in small rodents (Microtus microtus is the vole), will infect mice but not humans and is therefore much used as a laboratory model. Releases large amounts of cAMP which may inhibit lysosome phagosome fusion. Mycobacterium tuberculosis Is an obligate anaerobic nonmotile bacterium, causative agent of tuberculosis in humans. Lives intracellularly in macrophages.

<http://www.biology-online.org/dictionary/Mycobacteria>

• glycyl-tRNA synthetase --

A multimeric enzyme complex which, in bacteria, is usually a tetramer of two alpha and two beta chains and in eukaryotes, is usually a homodimer. Functions in the ligation of glycine and tRNA(Gly) to form glycyl-tRNA(Gly ). <http://www.ebi.ac.uk/QuickGO/GSearch?q=glycyl-tRNA%20synthetase>

• peptidoglycan -- (1) A glycan (a polysaccharide) attached to short cross-linked oligopeptides in the cell wall of eubacteria

(2) A crystal lattice structure in the bacterial cell wall that is made up of linear chains of alternating amino sugars, namely N-acetylglucosamine and N-acetylmuramic acid, and short peptide chains of three to five amino acids (attached to the N-acetylmuramic acid). The peptide chains form cross-links between them resulting in a 3D mesh-like structure.

(3) A polymer that serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm. It is also involved in binary fission during bacterial cell reproduction. <http://www.biology-online.org/dictionary/Peptidoglycan>

• operon -- A group of genes or a segment of DNA that functions as a single transcription unit. It is comprised of an operator, a promoter, and one or more structural genes that are transcribed into one polycistronic mRNA.

<http://www.biology-online.org/dictionary/Operon>

• horizontal gene transfer -- "Horizontal transmission" transmission occurring generally within a population, but not including vertical transmission.

<http://www.biology-online.org/dictionary/Horizontal_transmission>

Outline of Article

Importance of Chlamydia trachomatis genome

• C. trachomatis causes several diseases in human beings, including trachoma, which is a leading cause of preventable blindness.
• C. trachomatis infections in humans are estimated to increase the risk of HIV infection.
• At the time of the release of the article, little was known about the two developmental stages of C. trachomatis. These two developmental stages will come to be known as the elementary body and the reticulate body. Relatively little was also known about bacterial pathogen 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.
• Following 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 the sequencing of 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 than previously sequenced chlamydial plasmids.
• Two methods were used to validate the assembled sequence:
• 1) The predicted restriction digest map of the sequence as compared to the physical genome map of NotI and SgrAI.
• 2) 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 Chlamydia trachomatis Genome

• The programs PEPDATA and FRAMES were used to translate the chlamydial genome.
• BLASTP was used to find open reading frames in the C. trachomatis genome.
• RNAse P RNA, tRNAs, and rRNAs were identified using BLASTN.
• BLASTP-(-mp4-option) and CLUSTALW were used to estimate the location of start codons by looking for conserved sequences.
• GeneMark and Glimmer were used to evaluate the predicted start codons.
• When more than one start codon was identified for a given protein sequence, the first of the codons to appear in the genome was set as the start codon.
• PSI-BLAST was used to detect homologs to chlamydial protein sequences.
• EMOTIF was used to analyze sequence motifs.
• The COGNITOR program was used to compare chlamydial protein sequences to Clusters of Orthologous Groups.
• The above analysis resulted in the identification of 894 protein-coding genes and functional assignment for 604 of these genes.
• 35 chlamydial protein-coding genes were found to be similar to known genes in other bacteria.
• There are 256 chlamydial proteins that are paralogs belong to 58 families of similar genes.

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 and Macromolecule Synthesis

• Genes were missing for both the Entner-Doudoroff pathway and the TCA cycle, so the cycle was said to be incomplete.
• Due to the presence of a complete glycogen synthesis and degradation system, glycogen is hypothesized to be the primary carbon source, and may even 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 paper hypothesized that the bacterium's genome codes for the entire peptidoglycan biosynthesis pathway. This would suggest that the bacterium utilizes peptidoglycan differently than other bacteria.
• The C. trachomatis genome lacks genes that are involved in purine and pyrimidine nucleotide synthesis, but does contain genes to synthesize and convert deoxyribonucleotides.

Membrane, Intracellular Vacuole, and Pathogenesis

• 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.

• The C. trachomatis genome contains genes orthologous to type III secretion systems that determine that virulence of Gram-negative bacterial pathogens. It is proposed that these genes are involved in modifying host cell processes to facilitate invasion and remodeling the inclusion membrane.
• Two genes homologous to those found in Chlamydia psittaci were found in C. trachomatis that, along with a third protein, form an operon that may be involved in the inclusion (i.e. intracellular vacuole) membrane remodeling and transport.
• Six paralogous chlamydial proteins were found in C. trachomatis that belong to the HKD superfamily and may be involved in the modification of host cell phospholipids. However, Figure 2 shows that these proteins show little sequence similarity to other proteins previously identified in the HKD superfamily.

Phylogeny and Horizontal Gene Transfer

• Within the inclusion, C. trachomatis present today likely do not exchange genetic information with the host often because there are no genes in the chlamydial genome that are homologous to genes that are transposons or are involved in transformation or the acquirement of foreign DNA.
• Despite the aforementioned, Table 1 in the paper provides evidence that a majority of the genes in C. trachomatis are a result of horizontal gene transfer with bacterial ancestors or eukaryotic hosts.
• Figure 3 traces the origins of the chlamydial enoyl-acyl carrier protein reductase through various organisms supporting the theory that some chlamydial genes originated from horizontal gene transfer with eukaryotes.
• The mechanisms of chromatin condensation-decondensation may be related to that in eukaryotes as evidenced by the discovery of SET and SWIB domains in the chlamydial genome previously only found in eukaryotes.

EnsemblBacteria Database for Chlamydia trachomatis A/HAR-13

• NOTE: The Model Organism Database was found for the strain A/HAR-13 rather than the strain in the genome paper because the former strain is the one for which we will be analyzing the microarray data.

1. This database contains the full genome sequence of the bacterium including a map of the different genes with the option to search for particular genes. In addition, the database contains information on cDNA, non-coding RNA, and protein sequences. This is electronically curated, meta database.
2. The European Molecular Biology Laboratory maintains the database.
3. The database is funded by the European Molecular Biology Laboratory, United Kingdom Biotechnology and Biosciences Research Council, The Bill and Melinda Gates Foundation, and The Wellcome Trust.
4. There is no license on the data that would restrict downloading it.

Individual Powerpoint Slides for Group Presentation

PowerPoint Slides