Biology Reference
In-Depth Information
B. subtilis
to establish expression of
sinI
at high temporal resolution (
Schmalisch
et al.
, 2010; McLoon
et al.
, 2011
). A particular novelty of this study is that the light
emitted by the bacterial luciferase was measured in real time in growing colonies and
in biofilm pellicles during their formation and maturation (
McLoon
et al.
, 2011
).
In summary, these studies illustrate the versatility of both the GFP and luciferase
reporters showing how the establishment of promoter activity at high resolution can
reveal the dynamics of genetic control mechanisms in unprecedented detail and
establish novel features of regulation and regulatory networks.
7
METHODOLOGY FOR HIGH-THROUGHPUT ANALYSIS OF
PROMOTER ACTIVITY WITH FINE TEMPORAL RESOLUTION
IN B. subtilis
The following sections discuss important practical aspects associated with the
determination of global promoter activity at high temporal resolution in
B. subtilis
.
The available plasmids, procedures and methodologies are outlined, with emphasis
on their advantages and disadvantages.
7.1
Promoter fragment selection and generation
With more than 12,000 prokaryotic genomes now partially or completely sequenced,
most studies would use a directed approach to identify and clone promoter-containing
DNA fragments. In the absence of detailed transcriptome knowledge, the majority of
promoters can be assumed to be located within intergenic regions. However, it should
be noted that the number of promoters was higher than predicted in a recent transcrip-
tomic study in
B. subtilis
using high-density microarrays, with a significant number
located 3
0
to the operon of interest generating antisense RNA (
Nicolas
et al.
, 2012
).
Delimiting the promoter-containing fragment to be cloned depends on the type
of fusion to be made and its placement within the cell: that is, whether it is to be cloned
into a replicating plasmid or integrated into the chromosome by a single-crossover
event at the homologous locus or a double-crossover event at a heterologous neutral
locus.
The ease with which constructs can be integrated into the chromosome has made
this the method of choice in
B. subtilis
. In a recent study, promoter fusions were inte-
grated into the chromosome at homologous loci by a single-crossover event (
Botella
et al.,
2010, 2011
). Promoter-containing fragments spanning each intergenic region
were amplified by PCR (
Botella
et al.
, 2010, 2011; Buescher
et al.
, 2012
). The 3
0
-end
of each PCR-generated fragment was located 18 bp upstream of the start codon of the
first gene of the operon to avoid the ribosome binding site and to prevent formation of
translational fusions. For intergenic regions of less than 400 bp, a fragment of 400 bp
was chosen despite the 5
0
-end containing the 3
0
-end of the previous operon. There are
several reasons for these choices. Integration directed by such a DNA fragment
is non-mutagenic, generating a transcriptional promoter fusion while leaving the
