Taur.vil Week 11

Week 11 Individual Journal

Group Presentation

Team Journal Club Presentation

Journal Club Preparation

Terminology

1. Coccus: A bacterium of round, spheroidal, or ovoid form, less than 1 μm in diameter. [1]
2. Cariogenic: Tending to produce caries (decay of a bone or tooth, especially dental). [2] [3]
3. Otitis media: Infection of the middle ear space, behind the eardrum (tympanic membrane), characterized by pain, dizziness, and partial loss of hearing. [4]
4. Commensal: Living on or within another organism, and deriving benefit without harming or benefiting the host. [5]
5. Bacteriocins: Proteins produced by certain bacteria that exert a lethal effect on closely related bacteria; in general, bacteriocins have a narrower range of activity than antibiotics and are more potent. [6]
6. Efflux Pump: An active transport system for the removal of some antibiotics (such as tetracyclines, macrolides, and quinolones) from bacterial cells. [7]
7. Lipoprotein: Any of a class of conjugated proteins consisting of a protein combined with a lipid. [8]
8. Transpeptidase: An enzyme that catalyzes the transfer of an amino group from one peptide chain to another. [9]
9. Fastidious: In bacteriology, having precise nutritional and environmental requirements. [10]
10. ABC Transporter: Transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out certain biological processes including translocation of various substrates across membranes and non-transport-related processes such as translation of RNA and DNA repair. [11]

Note: For many of these definitions I had a general notion of the concept, but did not have a concrete definition that I could call to mind. There were very few words in the article I had never come across before.

Outline

Importance of S. pneumoniae

S. pneumoniae is one of the leading causes of death, both world-wide and in the United States. However, efforts to treat pneumonia have stalled due to increasing antibiotic resistance. When this article was published (2001), one third of S. pneumoniae isolates within the US were resistant to penicillin and many were resistant to multiple antibiotics. One of the goals for this study was to combat this problem and identify new targets for vaccine and antibiotic development.

S. pneumoniae has many sub-strains and in this project, the R6 strain was sequenced. R6 is an unencapsulated, avirulent strain which is safe for researchers to work with. Additionally, it's lack of an outer capsule and complete complement of competence genes grants it extreme genetic malleability, making it one of the preferred models for gram-positive coccus.

Methods Used

In this study, S. pneumoniae R6 was isolated from pure colonies grown on media. The DNA was purified through phenol extractions, ethanol precipiation, and spooling, sheared, and fragments were shot-gun sequenced. Fragments were aligned in PHRED, PHRAP, and CONSED and sequence-spanning PCR and direct sequencing using custom primers were used to form a complete sequence. This sequence was confirmed by Southern Blotting, restriction digests, PCR, and electrophoresis.

Open reading frames were determined using gene prediction programs (particularly GLIMMER) to search for sequence regions matching predictive models. ORFs were labeled spr#### starting at 0001. BLAST searches were performed on all ORFs to identify functional domains, orthologous proteins, and provide possible identifications. Non-translated RNA genes were identified using a set of programs to scan for sequence similarity to known functional RNA products.

Tables and Figures

• Table 1 lists the proteins found in R6 that have been studied as part of S. pneumoniae virulence or antibiotic antigens. In addition to the gene number in the R6 genome, the table also provides the gene name and a description of its function.

• Table 2 lists possible drug efflux pumps found in the R6 genome. However, it should be noted that S. penumoniae antibiotic resistance appears to be a result of transformation and lateral transfer, not innate resistance. That would indicate these genes are potentially important in drug resistance, but also likely ineffective or the result of prior transformations. Again, this table includes gene number, gene name, and a description of gene function.

• Table 3 is a long list of truncated or fragmented genes in the R6 strain. Partial genes account for 2% of ORFs in the genome, likely the result of prior transformations or now non-functional parental genes.

• Table 4 examines the transcriptional orientation of genes adjacent to BOX and RUP (2 transposons). It looks at the number of elements which go the same direction, both go into the BOX/RUP, and both move away from the BOX/RUP. The majority of sequencing appear to go in the same direction on each side of the transposons which may have implications for gene regulation.

• Figure 1 illustrates the transport proteins, metabolic pathways, and two categories of cell surface proteins. Many of these external feature may represent vaccine or drug targets and may also be used to deduce the mechanism of virulence is S. pneumoniae.

• Figure 2 shows the amount of ORF similarity between S. pneumoniae and other bacteria genomes. Generally, R6 ORFs show a large degree of homology with other bacteria. This is particularly true for B. subtilis with whom it shares a large degree of homology for all metabolic processes.

Comparison of Results with Other Studies

The major difference between R6 and other S. pneumoniae already sequenced is a 7.5kbp deletion in the capsule biosynthesis genes. This confirms the work of Iannelli et. al. (1999) which found that R6, unlike other S. pneumoniae, has no capsule. Also consistent with prior studies was that R6 had copies of all genes needed for competency as deduced by Lee and Morrison. As predicted by high competency, an abnormally high percentage of repetitive elements were found. Predictions about R6 metabolism where also met as genes were found for the metabolic pathway of carbohydrate to pyruvate to lactic acid.

An unexpected difference was the presence of a sortases (a subgroup of transpeptidases) that no previous studies had identified.

Model Organism Database SpR6

1. Both nucleotide and protein sequences are available in this database along with protein structures and notes for particular genes. It is possible to search through the database using gene name, keywords, or other gene-specific information. The database can also be searched using nucleotide or protein sequence blasts.
   • This is a meta database that combines some of the authors original results with information available on GenBank and other studies.
   • The database underwent an initial electronic curation where all data was collected but was then manually processed in-house to form the final pages and perform quality assurance.
2. This database is maintained by John I. Glass from the University of Alabama-Birmingham out of the lab run by Elliot Lefkowitz. However, it has not been updated in 7+ years.
3. This database was funded by the NIH and through the University of Alabama Health Sciences Fund.
4. There is no license agreement and no restrictions on access to the database.
5. This database has not been updated since 2007.
6. There are links to GenBank and the Center for Disease Control.
7. Information for nucleotide sequences, protein sequences, sequences for specific ORFs, RNA sequences, and repetitive elements can be downloaded in FastA text files.
8. This database is very use-friendly. There are multiple, intuitive ways to query the database and the layout is simple yet effective. The one thing that could make the database easier to work with is to provide more information about the cration and maintenance, but I doubt that was the designer's primary focus.
9. The web site is well organized although the changing link locations are a little disconcerting at first.
10. There is no help section or tutorial, but one does not appear to be necessary.
11. Yes, the results of a sample query make sense and are easily understandable.
12. The main type of Gene ID is spr####, the same as in our paper.

Lab Journal:

On Lab computer, downloaded updated GenMapp Builder v. 2.0b71

Opened pgAdmin III, created new data base titled tATK_20131107

Opened gmbuilder.sql in SQL window and executed

Produced the predicted 159 tables

Opened GenMappBuilder.bat v. 2.0b71

-Linked to our database tATK_20131107

Imported 20131107_UniProtXML_tATK_TIGR4_AJV.xml

• Took 1.62 minutes

Imported CUserskeckuserDesktop20131107_GO-OBO_tATK_KM.obo-xml

• Took 5.46 minutes
• Proceeded with additional processing: took 4.09

Imported 20131107_UniProtXML_tATK_TIG4_AJV.goa

• Took 0.03 minutes

Exported to desktop under the name of 20131107_GenMappExport_tATK_TIGR4_TPV.gdb

Duties

As team leader: timeline and milestones -deadlines: final project presentation -deadlines: final project deliverables, files and reports -weekly deadlines for each wk's wiki

Determine with team what are some of the milestones we want to hit to finish on time -Exports, QA, deliverable files --accelerate and leave down time --usually at least 2 or 3 iterations of the analysis we did for vibrio

-everybody will be writing electronic lab notebooks -want easy links to everybody narratives (in template?)

-Group action items --what and who's doing them

-Presentations next week

-When do we need drafts of the report by

Raw and edited files --need a naming convention

Main Job: need Gene IDs that will match gene database QA needs to become ID expert Coders will all work as a group, editing GenMAPP and xmlPipeDB

Personal Template

By Tauras Vilgalys

As part of Biological Databases

Please Remember the Harassing of Deities is Strictly Prohibited

Never Forget Samson