Biology Reference
In-Depth Information
gene, encoded on the integrating plasmid pBaSysBioII, by LIC cloning (
Botella
et al.
, 2010
). This allowed transcriptional fusions to be established on the chromo-
some at the homologous locus by a non-mutagenic Campbell-type insertion (
Botella
et al.
, 2010
). The GFPmut3 protein has a half-life of
10 h in
B. subtilis
, making it
an ideal reporter for measuring promoter activities at high temporal resolution. The
Botella
et al.
(2011)
study focused on cell wall metabolism in wild-type and
phoPR
mutant cells growing under three experimental conditions: a defined high phosphate
medium (HPDM), under phosphate-limiting conditions (LPDM) and during phos-
phate replenishment of phosphate-limited cells in LPDM. GFP levels were measured
at 10-min intervals and all experiments were performed with three independent
clones of each promoter fusion, with the expression profile of each clone determined
at least in duplicate using two different Biotek2 plate readers. This data set comprised
a minimum of 150,000 data points. Expression profiles were visualized in heat map
format, with promoter activity values normalized to the maximum observed under
those conditions and expressed on a scale of 0-1. By documenting the numerical
maximum activity attained by each promoter, the strengths of different promoters
can be compared under these experimental conditions. A second important feature
of this study is that promoter activities were compared with the RNA abundance
of each operon determined using high-density microarrays. Discrepancies between
promoter activities and RNA abundances (e.g. a high promoter activity with a low
RNA abundance) can indicate post-transcriptional regulation. The very high degree
of reproducibility with which promoter activity profiles and RNA levels were estab-
lished is a notable feature of this study. Significant promoter activity (
D
25 units
above the background threshold) was detected in 109 of the 135 cell wall-associated
promoters examined. Several novel features of cell wall metabolism emerged from
these studies that could only have been revealed by determining promoter activity at
high temporal resolution. Of the 109 promoter activity profiles established, those of
WalRK (YycFG)-controlled genes are unique in terms of their both kinetics and
activity levels: they are the first genes to be turned on when stationary phase cells
are diluted into fresh medium and are expressed at a significantly higher level than
all other genes involved in cell wall metabolism. This study also revealed the tem-
poral heterogeneity with which PhoPR regulon promoters are activated upon phos-
phate limitation (
Botella
et al.
, 2011
). Of particular note is the early activation of the
tuaABCDEFGH
operon (encodes the enzymes for teichuronic acid biosynthesis)
promoter that is turned on before the culture ceases exponential growth. Its promoter
reaches maximum activity up to 30 min before the majority of the other PhoPR-
controlled promoters (that control expression of genes encoding phosphate scaveng-
ing activities) are activated. This promoter profile suggests that teichuronic acid syn-
thesis plays a distinctive role in the cellular response to phosphate limitation. This
result also resonates with the diauxic shift study of Alon and colleagues who
observed increased
lac
and
gal
promoter activities before growth slows as glucose
level falls beneath the utilizable threshold (
Zaslaver
et al.
, 2006
). A further notable
feature of the
B. subtilis
response to phosphate limitation is the speed with which
promoters are reactivated when phosphate is added to cultures of phosphate-limited
