A genome-wide strategy for the identification of essential genes in Staphylococcus aureus.
To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.
Forsyth
R Allyn
RA
Elitra Pharmaceuticals, San Diego, CA 92121, USA.
Haselbeck
Robert J
RJ
Ohlsen
Kari L
KL
Yamamoto
Robert T
RT
Xu
Howard
H
Trawick
John D
JD
Wall
Daniel
D
Wang
Liangsu
L
Brown-Driver
Vickie
V
Froelich
Jamie M
JM
C
Kedar G
KG
King
Paula
P
McCarthy
Melissa
M
Malone
Cheryl
C
Misiner
Brian
B
Robbins
David
D
Tan
Zehui
Z
Zhu Zy
Zhan-yang
ZY
Carr
Grant
G
Mosca
Deborah A
DA
Zamudio
Carlos
C
Foulkes
J Gordon
JG
Zyskind
Judith W
JW
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D018390:Gene Targeting; D020043:Genes, Essential; D016680:Genome, Bacterial; D009174:Mycoplasma / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D014169:Transformation, Bacterial; D014994:Xylose / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D014994:Xylose