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cells. Promoters can be maximally reactivated within 10 min of phosphate addition
(
Botella
et al.
, 2011
). These studies illustrate the additional insights into promoter
behaviour that can be obtained by high-resolution temporal analysis using GFP
fusion technology.
High temporal resolution of promoter activity formed part of the analysis of the
global network reorganization that occurs during adaptation of
B. subtilis
to different
carbon sources (
Buescher
et al.
, 2012
). Malate was added to
B. subtilis
cultures
growing on glucose as sole carbon source (glycolytic metabolism), while glucose
was added to cultures growing on malate as sole carbon source (gluconeogenic
metabolism). The reorganization of the regulatory network that ensued was
established using transcriptome, promoter activity (
300 transcriptional fusions),
proteome and metabolome data, combined with statistical- and model-based ana-
lyses. The analysis showed that adaptation of glucose-grown cells to the addition
of malate occurred rapidly and mainly at a post-transcriptional level. In contrast,
the adaptation of malate-grown cells to the addition of glucose was slow and
occurred mainly at a transcriptional level, involving almost half the genes in the
regulatory network (
Buescher
et al.
, 2012
).
While luciferase reporters have not been used for high-throughput analysis of
gene expression in
B. subtilis
, several studies have demonstrated their applicability
to, and utility in, this bacterium. The Dubnau group has adapted the firefly luciferase
(
luc
from plasmid pGL3; Promega Corp. Wi, USA) for use in
B. subtilis
, generating
transcriptional fusions to examine the expression of the
rapH
and
phrH
genes. RapH
is a response regulator aspartate phosphatase whose activity is modulated by the
PhrH protein (
Mirouze
et al.
, 2011a,b, 2012
). They also generated
spo0A
and
comK
promoter fusions with the firefly luciferase
luc
gene to establish the expression of
these promoters during growth and the onset of competence development and spor-
ulation (
Mirouze
et al.
, 2011a,b, 2012
). Promoter activity was established at very
high temporal resolution (1.5-min intervals) providing insight into the kinetics of
promoter activity in unprecedented detail, and with unexpected results. For example,
spo0A
transcription fluctuates during the developmental transition period in a manner
that correlates with pauses in growth rate that may be caused by the release of RNA
polymerase from ribosomal RNA transcripts during this period. It is proposed that
these bursts of
spo0A
transcription may provide the temporal gate for entry of cells
into the competent state (
Mirouze
et al.
, 2011a,b, 2012
) and explain why competence
development has a bimodal distribution in cell populations. This temporal gating
mechanism differs fundamentally from the involvement of Spo0A in sporulation
development where the cellular level of phosphorylated Spo0A is the determining
factor. The cellular ATP level is an important consideration when using firefly lucif-
erase, as outlined in the supporting material of the
Mirouze
et al.
(2011a)
study. The
K
M
value of ATP binding to the light generating binding site of firefly luciferase is
100
M(
DeLuca and McElroy, 1984
). Thus, when comparing promoter activities
under different experimental conditions, it is important that cellular ATP levels do
not deviate to the extent that they influence light production. The Losick group have
adapted and optimized the
luxABCDE
operon from
P. luminescens
for use in
m
