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Volume 5, Issue 2 (Suppl)
Transcriptomics, an open access journal
ISSN: 2329-8936
Molecular Biology 2017
August 31-September 01, 2017
2
nd
International Conference on
August 31-September 01, 2017 Philadelphia, USA
Molecular Biology, Nucleic Acids &
Molecular Medicine
Transposon-mediated directed mutation in
E. coli
Milton H Saier
and
Zhongge Zhang
University of California, San Diego, USA
E
scherichia coli
cells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because
cAMP-Crp is a required activator of the glycerol utilization operon, glpFK. We have previously shown that a transposon,
Insertion Sequence 5 (IS5), can insert into the upstream regulatory region of the operon to activate the glpFK promoter and
enable glycerol utilization. GlpR, which represses glpFK transcription, binds to the glpFK upstream region near the site of IS5
insertion and inhibits insertion. By adding cAMP to the culture medium in ΔcyaA cells, the cAMP-Crp complex, which also
binds to the glpFK upstream regulatory region, inhibits IS5 hopping into the activating site. Control experiments show that the
frequencies of mutations in response to cAMP were independent of parental cell growth rate and the selection procedure. These
findings led to the prediction that glpFK-activating IS5 insertions can also occur in wild-type (Crp+) cells under conditions
that limit cAMP production. Accordingly, IS5 insertion into the activating site in wild-type cells is elevated in the presence of
glycerol and a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. The resultant IS5 insertion
mutants arising in this minimal medium become dominant constituents of the population after prolonged periods of growth.
Thus, DNA binding transcription factors can reversibly mask a favored transposon target site, rendering a hot spot for insertion
less favored. Such mechanisms could have evolved by natural selection to overcome environmental adversity. We have further
shown that IS elements can insert upstream of the flagellar master regulator operon, flhDC, to activate transcription in a
process that depends on viscosity (agar concentration). Documentation of these processes shows that IS elements can direct
mutations (IS insertions) to specific sites in response to environmental stress.
msaier@ucsd.eduTranscriptomics 2017, 5:2 (Suppl)
DOI: 10.4172/2329-8936-C1-013