Difference between revisions of "Kmeilak Week 11"

Revision as of 00:43, 11 November 2013

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.

Kmeilak (talk) 21:20, 12 September 2013 (PDT)

@@ Line 22: / Line 22: @@
 *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 used to remove antibiotics and therefore allow antibiotic resistance.
+*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.
@@ Line 32: / Line 32: @@
 *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 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.
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