Kmeilak Week 11

Group Presentation

isolate - to obtain (a substance or microorganism) in an uncombined or pure state. [1]
transpeptidase - an enzyme that catalyzes the transfer of an amino acid residue or a peptide residue from one amino compound to another [2]
beta-hemolytic - capable of causing a sharply defined clear colorless zone of hemolysis surrounding colonies of certain streptococci on blood agar plates [3] [4]
otitis media - acute or chronic inflammation of the middle ear; especially : an acute inflammation especially in infants or young children that is caused by a virus or bacterium, usually occurs as a complication of an upper respiratory infection, and is marked by earache, fever, hearing loss, and sometimes rupture of the tympanic membrane [5]
intergenic - occurring between genes : involving more than one gene [6]
fosmid - An f-factor cosmid, which is like a plasmid (circular DNA), but it is capable of containing much larger pieces of DNA, up to 50 kb compared to about 10 kb in a plasmid. [7]
contigs - Group of clones representing overlapping regions of a genome. [8]
bacteriocins - any of a group of substances, e.g., colicin, released by certain bacteria that kill other strains of bacteria by inducing metabolic block. [9]
efflux pumps - An active transport system for the removal of some antibiotics (such as tetracyclines, macrolides, and quinolones) from bacterial cells. [10]
sortase - Any of a group of prokaryotic enzymes that form pili through cleavage and isopeptide bonding of precursors. [11]

Streptococcus pneumoniae causes over 1 million deaths worldwide annually, and despite a wide array of antibiotics and a vaccine, it remains among the top 10 leading causes of death in the United States. Nearly one-third of patient isolates in the United States are resistant to penicillin and there are a growing number of multiple antibiotic resistant strains. The sequencing of its genome was done to find new antibiotic targets to aid in drug therapy and treatment for patients, and to prevent this organism from continuing to cause such high numbers of deaths even in a developed country with an excellent healthcare system. The bacteria is a Gram positive coccus as well as being a lactic acid bacteria.

Methods: S. pneumoniae strain R6 was used for sequencing. Its lack of a capsule renders it avirulent and therefore safe to work with in a laboratory setting. It was also selected for its high genetic malleability, which provides greater utility. Genomic DNA was isolated, purified using multiple phenol extractions and ethanol precipitations, sheared, sized fractioned, and used to create plasmid and fosmid libraries. DNA sequences were analyzed by PHRED, PHRAP, and CONSED. The sequence was analyzed and annotated using several programs for gene prediction, similarity searching, and functional assignment, and the results were imported into a relational database based on the Microsoft SQL server. Glimmer was used to determine potential protein-encoding sequences and to create organism-specific open reading frames. BLAST searches were used on all predicted ORFs, parsed using BioPerl tool kit, and imported into the SQL server for analysis. The Prosite motif library and Blocks database of protein families were used to identify functional domains. tRNAscan-SE was used to identify genomic sequences that coded for tRNAs. rRNAs were identified based on their similarity to genes in the Ribosome Database progject sequence database. The genomic sequence was added to GenBank (accession number AE007317) and the annotated genome and supplementary data can be found at http://www.lilly.com/s.pneumoniae.

Table 1: a list of genes encoding putative virulence factors present in the R6 genome despite its avirulence.

Table 2: a list of genes encoding probable efflux pump proteins that do not provide antibiotic resistance.

Table 3: a list of truncated/fragmented genes in R6, which may be nonfunctional remnants of parental genes or a result of transformation.

Table 4: transcriptional orientations of genes close to BOX and RUP repetitive elements that may affect transcriptional termination.

Figure 1: a diagram of substrate transport, carbohydrate and glutamine metabolism, and cell surface proteins that function in signalling, attachment sites, proteins recognized by choline-binding domains, many of which represent potential antibiotic targets.

Figure 2: A bar graph comparing predicted R6 ORFs with those of other completely sequenced genomes. R6 is most similar to S. pyogenes, a beta-hemolytic human pathogen.

A 7,504-bp deletion noted in the region that encodes for capsule biosynthesis confirmed the work of Iannelli et. al. (1999) as R6 has no capsule. A single gene, spr1098, was determined to be likely to code for a sortase although no previous work had identified any sortases present in S. pneumoniae. 7 novel genes coding for proteins containing the LPXTG motif and other sortase substrate features were discovered. While no D-alanine is present in S. pneumoniae, a complete dltABCD operon homologous to those adding D-alanine to LTA in Bacillus subtilis was found, though it may be silent or defective. R6 has all of the genes induced during competence as noted by Lee and Morrison. Due to this competence, the R6 genome is littered with genes derived from other bacteria as anticipated for a competent species. As expected, R6 contains all of the genes necessary to oxidize carbohydrates to pyruvate via glycolysis. R6 has the highest percentage of repetitive elements of any bacterial genomes sequenced to date.

The database contains nucleotide sequences (DNA/RNA), specific gene information, strain information, the medical relevance of the strain, and information about its metabolism. It is a meta database that is electronically curated and then has manual quality control in house by the UAB center for AIDS research.
The University of Alabama Birmingham Center for AIDS research.
Funded by the NIH 1991-2008 (NIH P30 AI027767) and the UAB Health Services Foundation General Endowment Fund (2000-2004) (last update was 17 Feb. 2005)
The database is public and contains no access restrictions or licensing information.
The last update to the database was February 17, 2005.
There are links to GenBank and to the CDC database website.
The information can be downloaded in the FastA format (text).
The website is very well organized, clean and easy to navigate. It lacks any help, tutorial, or explanation section. The results of a sample query looking for genes relating to amino acid biosynthesis returned a list of related genes and information about the genes which was very easy to interpret.
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Kmeilak (talk) 21:20, 12 September 2013 (PDT)