Biology Reference
In-Depth Information
duplicated sequences during chromosomal replication or by integration of a multi-
meric plasmid (
Janniere
et al.
,1985
;
Young, 1984
). This results in individual transfor-
mants having similar expression profiles but different levels of promoter activity
(usually multiples of 2-4
, E Botella and KM Devine, unpublished). The most desir-
able placement of a promoter fusion is at a neutral heterologous locus by a double-
crossover event. A locus such as
amyE
of
B. subtilis
(that encodes
a
-amylase) is useful
in that successful integration can be monitored by loss of
-amylase production with a
simple iodine-starch plate assay. Promoter fusions placed on the chromosome by this
mechanism are stable and present in single copy. Chromosomal integration of pro-
moter fusions by a double-crossover event is best achieved with linear DNA fragments,
generated either by linearizing plasmids or by overlapping PCR. This approach is less
amenable to high-throughput analysis because of the additional DNA manipulations
required and because double-crossover integration events occur at significantly lower
frequencies than do single-crossover events.
a
5
CHOICE OF SYSTEM
Undertaking a global study of promoter activity in an organism requires careful
consideration of the system most appropriate to fulfill the objectives of the study.
Accurate determination of promoter activity requires that the reporter protein folds
rapidly and be either highly stable or unstable in the organism under study. While
GFPmut3 (stable) and firefly luciferase (unstable) have these properties in
B. subtilis
,
they may behave differently in other bacterial species. When choosing between GFP
and luciferases, it is important to evaluate the likely strengths of the promoters under
study: very high-level production of GFP may be toxic, while very low-level produc-
tion may not be detectable over background autofluorescence. Luciferases are more
suited to studying low-activity promoters because of the higher signal-to-noise
ratios. However, firefly luciferase requires the addition of substrate, while bacterial
luciferases require additional genes to generate its substrate intracellularly, which
makes plasmid constructs more complex. The decision on where to place the
promoter fusion is a critical one with an inverse relationship between the ease of
construction and avoidance of potential artefacts. Integration of promoter fusions
into a neutral chromosomal locus is optimal but more difficult to achieve than inte-
gration by a single crossover or establishing the promoter fusions on a replicating
plasmid. However, the latter approach can give artefactual results in some instances.
6
USE OF PROMOTER FUSIONS TO STUDY BIOLOGICAL
PROCESSES AT HIGH TEMPORAL RESOLUTION IN BACTERIA
6.1
Analysis of promoter activity in E. coli and Salmonella species
Promoter activity analyses in
E. coli
and
Salmonella typhimurium
have tended to use
gfpmut2
,
gfpmut3
and
lux
reporter genes located on low copy number (pSC101 deriv-
atives) replicating plasmids. Two approaches can be used to generate promoter
