Download Identification of Critical Staphylococcal Genes Using Conditional

Identification of Critical
Staphylococcal Genes Using
Conditional Phenotypes
Generated by Antisense RNA
Ji, et al
Shannon Davis & Tim Miller
Question
I've fractionated the H. pylori genome (rather
than S. aureus) and isolated a gene that is
homologous to rplN, a 50S ribosomal protein.
If I grow my colony on TSA-ERM with ATc,
what phenotype will I get?
Defect or Growth Defective
Staphylococcus aureus
Genome is approximately 2.8 Mb
Gram positive
Spherical bacterium
Appear in pairs, short chains, or bunched, grape-like clusters
Scanning electron micrograph of Staphylococcus aureus
Staph infections
Staph is in air, water, milk, food, humans, and
animals
Humans and animals are the primary reservoirs

present in the nasal passages, throats, hair and skin of
~50% of healthy individuals
Most common cause of hospital infections
Sickens over 2 million people per year
Some strains produce a heat-stable protein toxin that
causes illness in humans
Enterotoxins produce Staph food poisoning


caused by less than 1.0 microgram of toxin or 100,000 S.
aureus per gram
usually from meat, poultry, egg, milk and dairy
Antisense regulation
Developed ~10 years ago by Dr. Meng-Chao Yao
Used to analyze physiological consequences associated with
selective elimination of a particular protein
A complimentary (antisense [AS]) RNA sequence binds to a
(sense [S]) mRNA, thus preventing transcription of the mRNA
 specifically blocks the normal process of gene expression
without affecting the expression of other genes
 selectively turns off production of certain proteins because
ribosomes
cannot access dsRNA
Not siRNA
or RNAi!!
The experiment
Created a randomly cloned library of 200 – 800 bp fragments
tetR = tet Repressor
Colony selection
Library was replica plated and screened with ATc –
androtetracycline

ATc is a weak tet analog
Selected colonies that grew without ATc but were
absent or growth defective in ATc
L – lethal colony
D – growth
defective colony
Phenotype confirmation
Selected 600 colonies (3%) and rescreened them
Single colonies were restreaked, isolated and
transformed into wt S. aureus and tested with ATc
YJ335 (wt)
JY12 (L)
JY57 (L)
ATc
(mg/ml)
0.0
1.0
Identify cell viability fragments
600 plasmids were PCR’d (from 20,000)
Database comparisons (?) showed 1/3 of the colonies
contained AS fragments from different ORF regions

Remaining 2/3 contained S ORFs, non-ORFs, AS non-ORFs,
or a mix of S/AS chimeric fragments
This method allows for isolation and maintenance of
conditional strain
Identified 150 critical staph genes



Table 1
40% homologs to known essential bacteria genes
30% homologs to functional bacteria genes
 includes transcription, translation, & biochem activities like methyl/acyl
transferases

30% critical genes with unknown function
More gene identification
Several L colonies are known virulence factors



YJ69-1 fibronectin binding protein
YJ15-9 virulence extracellular factor
JSB162 ABC transporter
Selecting D colonies allows detection of important
nonessential genes


suboptimal AS efficiency would not result in L colonies
some essential genes may not be detected
Inducible & titratible vector
system in vitro
AS colonies were induced by dose-dependent ATc

Shows gene product relevance to cell viability
 wt
Inducible & titratible vector
system in vivo
Used pyelonephritis model – localized kidney infection
that bacteria are easily recovered from
Mice were injected with 10,000,000 CFU  ATc and
kidneys recovered 72 H later
All wt and ATc– mice recovered 500,000 CFU
Mice treated with increasing ATc showed a dosedependent effect on recoverable bacteria
ATc treated mice
Mice treated with increasing ATc showed a dosedependent effect on recoverable bacteria
Criticisms
Antisense technology is still somewhat controversial.

unreliable and results unpredictable
Where are the statistics??
Why didn’t they show if the mice lived or died after
infections with  ATc?
Criticisms cont’d
They are equating gene expression with cell viability
yet they never show any gene expression

How about a Northern?
A paper they cited showed that as ATc is increased,
the expression of their reporter decreased
And more criticisms
Why not use transposons (until they hop into the L
genes) to prove these genes really cause cell death?

Or any other test to show these genes are essential
Aren’t papers designed so that results can be
repeated??

How can “Bioinformatics was used” be repeated?
References
http://vm.cfsan.fda.gov/~mow/chap3.html
http://www.bact.wisc.edu/Bact330/lecturestaph
http://www.fhcrc.org/pubs/center_news/1996/Jul3/Antisense.htm