Biology Reference
In-Depth Information
These studies were extended by construction of a comprehensive library of tran-
scriptional promoter fusions (
2000) for
E. coli
(
Zaslaver
et al.
,2006
). Intergenic
regions were amplified by PCR and cloned into plasmid pUA66 (low copy number
pSC101-based derivative) that encodes a promoterless
gfpmut2
. GFPmut2 is a fast-
folding variant of GFP that becomes fluorescent within 5 min of expression and is
highly stable in
E. coli
. The authors established the kinetics of promoter activity for
this library of transcriptional fusions during a diauxic shift, monitoringGFP production
at 7-min intervals over a period of 20 h. Cells were grown in medium containing lim-
iting glucose (0.03%) and non-limiting lactose, conditions under which
E. coli
utilizes
glucose first and then lactose. More than 60% of the promoter fusions showed activity
under these conditions with many genes being strongly expressed during the early and
mid-logarithmic growth phases. Expression of the
lac
and
gal
operons was particularly
interesting, as the activity of their promoters increased before growth of the culture
slows down and peaked at the point of growth rate reduction.
This library of transcriptional fusions has been utilized by the Alon group to
investigate several additional features of promoter activity in
E. coli
. They have
investigated the distribution of promoter activities in cells growing at different
growth rates and under different growth conditions (
Zaslaver
et al.
, 2009
). A partic-
ularly interesting study examined the variance of promoter activity in cell popula-
tions by flow cytometry for the
2000 fusion-containing strains in their library
(
Silander
et al.
, 2012
). It has been widely observed that some genes show significant
fluctuations in expression in individual cells within a population. Such differences in
expression level (called phenotypic noise) can result in different phenotypes being
manifest within a cell population. Important observations are that the promoter alone
can contribute to the generation of phenotypic noise while essential and highly
conserved genes have lower levels of phenotypic noise than do non-essential and
lowly conserved genes (
Silander
et al.
,2012
).
The library of transcriptional promoter fusions generated by the Alon group was
also used to establish the toxicity of naphthenic acids (cyclopentyl and cyclohexyl
carboxylic acids) that are components of effluents from oil production facilities
(
Zhang
et al.
, 2011
). The library of strains with promoter
gfpmut2
fusions was
exposed to the range of naphthenic acid concentrations typically observed in the
environment, and changes in promoter activity profiles were established. Results
showed that the main cellular response of
E. coli
to naphthenic acids was to increase
expression of genes involved in the pentose phosphate pathway and to down-regulate
expression of an ABC-type transporter complex. Exposure to naphthenic acids also
affected adenylate cyclase metabolism and the SOS response.
6.2
Analysis of promoter activity in B. subtilis
Global analyses of promoter activities at high temporal resolution have also been
performed in
B. subtilis
(
Botella
et al.
, 2011; Buescher
et al.
, 2012
). Transcriptional
reporter fusions were generated in a high-throughput manner: promoter-containing
DNA fragments were directionally inserted upstream of a promoterless
gfpmut3
