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Projects
External Guide Sequences (EGSs) as a means of
gene down regulation in bacteria. We use a novel technique to
dynamically down-regulate bacterial gene expression and thereby
alter bacterial phenotype. This inducible gene product disruption
employs RNA molecules called external guide sequences (EGS), which
are expressed from plasmid vectors introduced into bacteria of
interest. The EGS oligonucleotides target complementary mRNA for
single-site cleavage by endogenous RNase P in cells and disrupt
downstream protein expression. Top: schematic of molecular events.
Bottom: Cartoon of cellular effects of EGSs on gene expression.
(Graphics by C. McKinney)
EGSs and Bacterial Viability. EGSs
complementary to mRNAs of two essential E. coli genes can inhibit
viability in the E. coli. This inhibition exhibits reproducible
time course dynamics, dose-response, and synergy and is EGS
oligonucleotide sequence specific. Top: Cartoon of experimental
system used for bacterial growth studies. In E. coli, IPTG is used
as the induction agent to turn on EGS expression (graphic by C.
McKinney). Bottom: Quantitative plating of E. coli transformants
containing plasmids encoding various EGS constructs designed to
cleave target mRNA at the nucleotides listed (e.g., EGyrA 241/211
EGS is designed to cleave gyrase A mRNA at nucleotides 241 and
211). E. coli viability is inhibited following induction of EGSs
against the essential genes encoding Gyrase (gyr A) or the protein
component of RNase P (C5). Negative controls included E. coli
with EGSs designed to cleave synthetic (non-naturally occurring)
mRNA for C5 at nucleotides 21 and 45; and an E. coli with an EGS
expression vector lacking its T7 promoter and EGS encoding
segments.
Controlled EGS Expression in Salmonella. A
fully virulent Salmonella strain has been modified to permit
tightly controlled EGS intracellular expression, turned on and off
in sequence by arabinose and glucose, respectively. Top: Cartoon
of Arabinose-inducible expression of a T7 RNA polymerase gene that
has been integrated with an adjacent araC-PBAD control element
into the bacterial chromosome. This construct allows dynamic
control of T7 promoter-driven RNA transcription (graphic by C.
McKinney). Bottom: Basal and induced gene expression in E. coli
BL21(DE3) and the Salmonella serovar Typhimurium construct.
Northern blots of constitutive and induced RNA transcription are
shown for two systems: E. coli lac-based induction with IPTG
(lanes 1 and 2) and the new Salmonella serovar Typhimurium system,
ara-based induction with arabinose (lanes 3 to 6). The Salmonella
serovar Typhimurium in lanes 3 and 4 has a PCR-detectable T7 RNA
polymerase gene in genomic DNA, whereas the Salmonella in lanes 5
and 6 lacks an integrated T7 RNA polymerase gene.
Disruption of Salmonella Pathogenesis Steps.
Relevant EGSs interfere with Salmonella’s invasion of host cells.
Here, EGSs have been designed to disrupt the expression of
Salmonella pathogenicity island 1 (SPI-1) genes invB or invC,
believed important in early stages of mucosal invasion. The
effects of these EGSs have been demonstrated at the levels of
specific Salmonella mRNA and protein expression, type-III
secretion activity and host cell invasion in tissue culture.
Table: Decreased rate of invasion into cultured host cells, to
roughly 3% of inoculated Salmonella (compared to 25-30% rates for
invasion competent controls, or for negative control EGSs)
following concurrent expression of pairs of relevant invB or invC
EGSs.
Top Western Blot: Disappearance of immunoreactive InvC from whole
cell bacterial preps, following concurrent expression of pairs of
relevant invB or invC EGSs. Bottom Western Blot: Decrease in
protein immunoblot signal for assay of InvC powered, type-III
secretion of SipB into culture supernatants for matched Salmonella
cultures, following concurrent expression of pairs of relevant
invB or invC EGSs. Salmonella strain SB300A#1 without an EGS
expression plasmid (no EGS) and a SB300 invC deletion mutant which
does not express InvC (delta invC) are shown to the left of
protein molecular weight kD markers.
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SCHOOL OF MEDICINE
