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Journal of Bacteriology, December 1998, p. 6090-6100, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Expression of lacZ from the Promoter of the
Escherichia coli spc Operon Cloned into Vectors Carrying the
W205 trp-lac Fusion
Sung-Tzu
Liang,1
Patrick P.
Dennis,2,* and
Hans
Bremer1
Molecular and Cell Biology Programs,
University of Texas at Dallas, Richardson, Texas
75083-0688,1 and
Department of
Biochemistry and Molecular Biology, University of British Columbia,
Vancouver, British Columbia, Canada V6T 1Z32
Received 20 July 1998/Accepted 18 September 1998
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ABSTRACT |
The expression of lacZ has been analyzed and compared
in a series of promoter cloning vectors by measuring the amount of
lacZ mRNA by hybridization and the amount of
-galactosidase by standard enzymatic assay. Expression was driven by
the promoter, Pspc, of the spc
ribosomal protein operon. The vectors contained either the standard
W205 trp-lac fusion with the trp operon
transcription terminator, trpt, located in the
lacZ leader sequence, or a deletion derivative that
functionally inactivates trpt. In the presence of
trpt, lacZ expression was temperature dependent
so that increasing the growth temperature reduced the accumulation of
lacZ mRNA and -galactosidase activity. The frequency of
transcript termination at trpt was estimated to be near
zero at 20°C and at about 45% at 37°C. The amount of
Pspc-derived lacZ mRNA and the amount of -galactosidase produced per lacZ mRNA varied,
depending on the mRNA 5' leader sequence between
Pspc and lacZ. These results
demonstrate that the quantitative assessment of promoter activities
with promoter cloning vectors requires careful analysis and
interpretation. One particular construct without trpt did
not seem to contain fortuitous transcription or translation signals
generated at the fusion junction. In this strain, lacZ expression from Pspc was compared at the enzyme
activity and mRNA levels with a previously constructed strain in which lacZ was linked to the tandem P1 and P2 promoters of the
rrnB operon. At any given growth rate, the different
activities of -galactosidase in these two strains were found to
reflect the same differences in their amounts of lacZ mRNA.
Assuming that the promoter-lacZ fusions in these strains
reflect the properties of the promoters in their normal chromosomal
setting, it was possible to estimate the absolute transcription
activity of Pspc and the relative translation
efficiency of Pspc-lacZ mRNA at
different growth rates. Transcription from the spc promoter was found to increase from about 10 transcripts per min at a growth rate of 1.0 doublings/h to a maximum plateau of about 23 transcripts per min at growth rates above 1.5 doublings/h. The translation frequency of lacZ mRNA expressed from
Pspc was unaffected by growth rates.
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INTRODUCTION |
To study gene regulation in bacteria
and to define the basic properties of a promoter isolated from its
normal control sites, the promoter of interest is often linked on a
plasmid or phage vector to a reporter gene such as lacZ. The
promoter activity in lacZ-based vectors is then assessed
from measurements of -galactosidase, an enzyme that is very stable
and easy to assay. The amount of enzyme produced depends in part on the
strength of the inserted promoter and in part on other factors,
including the termination or antitermination properties of the
transcribing RNA polymerase, the stability of lacZ mRNA, and
the ability of the lacZ mRNA to compete with bulk mRNA for
the initiation of translation. The latter factors may be affected by
particular, unnatural sequence combinations at the junction of the
operon fusion. In the work described below, we have attempted to
identify and quantify some of these effects by measuring with various
lacZ vectors the amounts of lacZ mRNA and of
-galactosidase produced from the promoter of the spc
ribosomal protein operon.
Two widely used lac-based promoter cloning vectors, the
phage 
RS205 (3) and the plasmid pRS415 (33),
were derived from the trp-lac fusion W205, isolated by
Mitchell et al. (25). In these vectors, the W205 fusion is
located downstream of the promoter cloning site and contains the end of
trpA, followed by one of the two trp operon
transcription terminators, trpt (29, 35, 36).
Termination at trpt is independent of rho and occurs with an
efficiency of about 37% in vivo (27) and of about 25% in vitro (36). The trpt element also serves as a
pause or stop signal for 3'-to-5' exonucleolytic degradation of mRNA
(27). The second, rho-dependent transcription terminator,
trpt', is located 250 bp downstream of trpt in
the normal trp operon (27). The trpt'
element is not present in the W205 fusion.
Expression from plasmid-cloned genes is difficult to quantify because
of plasmid copy number effects; therefore, expression of the
lacZ reporter gene is often studied after integration of the
promoter-lac fusion into the chromosome, e.g., by using the RS205 system. Lysogens constructed with these phage vectors carry a
temperature-sensitive repressor to aid in preparing phage lysates for
moving the operon fusion from one strain to another. Because of lysis
induction at higher temperature, such strains are grown and analyzed at
30°C. In our laboratory, an alternative system to study gene
expression was developed in which the plasmid-borne promoter-lacZ fusion is flanked by sequences of the
Escherichia coli maltose (mal) genes. This
allows for insertion of the plasmid-constructed promoter-lacZ fusions into the mal locus of the
chromosome by a double recombination event (16, 38). These
strains can be grown at any temperature.
We have observed that promoter-lacZ operon fusions
derived from the original W205 trp-lac fusion exhibit
temperature-sensitive expression of -galactosidase activity. The
temperature sensitivity in expression was seen with a number of
promoters, including the P1 or P2 promoters from rrnB, the
replication primer promoter of plasmid pBR322, and the promoter of the
spc ribosomal protein operon. We show here that the
temperature sensitivity in lacZ expression is caused by the
presence of the trpt transcription terminator within the
W205 trp-lac fusion. In addition, we show that new sequence
combinations generated at the fusion junction can have other dramatic
effects on lacZ expression and therefore may adversely
affect the quantitation of promoter activity. The analysis allowed us
to identify and characterize a particular Pspc-lacZ construct in which these anomalous
effects were minimal or absent and in which the expression of
lacZ appears to reflect the properties of
Pspc in its natural chromosomal setting.
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MATERIALS AND METHODS |
Plasmids and strains.
The plasmids and bacterial strains
used and details of their construction are presented in Tables
1 and 2. To
delete trpt from plasmid pXZ09-B, a 2.3-kb fragment from
this plasmid, spanning the region from the center of the
trpt palindrome to a site beyond a unique SacI
site within lacZ, was amplified by PCR. One of the primers
added a BamHI site at the end bordering the trp
terminator. After cleavage of the PCR product with BamHI and
SacI, the resulting fragment was inserted between the
BamHI and SacI sites of plasmid pXZ09-B, thereby
deleting 50 bp of the trp region, including the end of
trpA and half of the trpt terminator repeat (Fig.
1). The resulting new plasmid without a
functional trpt transcription terminator is pSL03. The
correct deletion was verified by DNA sequencing.
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FIG. 1.
Structure of the promoter cloning vector containing the
W205 trp-lac reporter system and the mal
insertion sequences. (a) Arrangement of genes in pSL03. Filled areas
are the functional genes lacZ, kan, and
bla, and the positions of the tandem rrnB
transcription terminators, rrnB T1T2 are indicated. The
malE' and 'malK sequences are indicated by open
boxes. The EcoRI-SmaI-BamHI sites are
used for promoter insertion to drive expression of lacZ. The
SacI site was used for construction of the trp
deletion (see Materials and Methods). (b) Structure of the
spc promoter-trp-lac fusion region on plasmid
pSL02: the 110-bp fragment containing the spc ribosomal
protein promoter from  51 to +59 relative to the transcription start
site (+1) was inserted between the EcoRI and
BamHI sites of plasmid pXZ09-B to give plasmid pSL02 (Table
1). This plasmid contains 75 nt of trp operon
sequence (open box) that includes the rho-independent
transcription terminator trpt. The coding region of
lacZ begins at nt +160. The 10 and 35 elements of
Pspc are indicated. (c) Structure of the
spc promoter-trp-lac fusion region in plasmid
pSL04. The same 110-bp fragment containing the spc promoter
was cloned between the EcoRI and BamHI sites of
plasmid pSL03 that lacks 50 bp of trp sequences to give
plasmid pSL04. The deletion removes half of the trpt
palindromic sequence. The coding region of lacZ begins at nt
+110. (d) Structure of the spc promoter-trp-lac
fusion region on plasmid pSL05: a 504-bp fragment from nt 51 to +453
(relative to the transcription start) containing the
Pspc promoter and the rplN gene (from
nt +73 to +441) was cloned between the EcoRI and the
BamHI sites of plasmid pXZ09B to give plasmid pSL05. The
coding region of lacZ begins at nt +554. (e) Structure of
the spc promoter-trp-lac fusion region on plasmid
pSL06: a 504-bp fragment from nt 51 to +453 (relative to the
transcription start) containing the Pspc
promoter and the rplN gene (from nt +73 to +441) was cloned
between the EcoRI and BamHI sites of plasmid
pSL03 to give plasmid pSL06. The coding region of lacZ
begins at nt +504.
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A 110-bp
spc promoter fragment (from
51 to +59 relative to
the transcription start; Fig.
1) and a 504-bp fragment, carrying
the
spc promoter and the coding sequence of r-protein L14
(
rplN;
from
51 to +453), were obtained by PCR with
appropriate primers
by using
d
spc-1 DNA (
12)
as a template and adding
EcoRI and
BamHI
restriction sites at the ends. After cleavage with
EcoRI
and
BamHI, these fragments were inserted between the
EcoRI and
BamHI sites of pXZ09-B and pSL03 to
yield pSL02, pSL04, pSL05,
and pSL06 (Table
1).
All plasmids were constructed in duplicate with independently
synthesized PCR products and checked for correct length of restriction
fragments and enzyme activity. The promoter-
lacZ fusions
from
these duplicate plasmids were first recombined into the
mal genes
of the
recBC sbc strain JC9387 (Table
2) as described previously
(
16). The
P
spc-lacZ-kan fusions were then transduced
with
phage P1 into the
lac deletion strain HB181, selecting for
kanamycin resistance (Km
r). The correct insertion was
verified by streaking onto MacConkey
maltose and MacConkey lactose
plates (
24). Before transduction,
the HB181 recipient was
positive for
mal but lacked
lac; after
transduction the strains lacked
mal but were positive for
lac.
The absolute enzyme activities in bacteria transformed with plasmids
pSL02 and pSL04 varied considerably from transformant
to transformant,
which probably resulted from variations in plasmid
copy number. When
the P
spc-lacZ fusions from different
transformants (including plasmids with independently prepared
PCR
products) were recombined into the
mal locus of the
chromosome
so that they were present as a single copy, there was no
significant
variation (less than 10%) in the
-galactosidase
activity expressed
from P
spc in replicate
cultures and strains (Table
3).
As a background control, the host strain was separately transformed
with vector plasmids without promoter inserts. In pXZ09-B
and pSL03
transformants, a low level of
-galactosidase activity
is expressed
from an upstream
tet promoter. After recombination
of
lacZ from these plasmids into the chromosomal
mal
locus, the
tet promoter is excluded and the resulting
strains showed a
lacZ activity near
zero.
Growth conditions.
Cultures were grown in Medium C
(15) supplemented with either 0.2% (vol/vol) glycerol or
0.2% (wt/vol) glucose (with or without 0.8% Difco Casamino Acids plus
50 µg of tryptophan per ml), or they were grown in LB medium
(24) with 0.2% glucose. Minimal media were supplemented
with phenylalanine and threonine at 50 µg/ml. Experimental cultures
were inoculated from overnight cultures in glycerol minimal medium by
diluting at least 250-fold into minimal medium or 2,000-fold into amino
acid-supplemented medium.
Growth was followed as the increase in turbidity at 600 nm with a 1-cm
light path (i.e., the optical density at 600 nm [OD
600]).
Since the turbidity is not exactly proportional to the culture
density,
the observed values, after subtraction of the medium
blank, were
corrected for nonlinearity (
4). The corrected OD
values
deviated by less than 1% from the average exponential curve,
so that
the accuracy of the average OD used for determination
of the specific
enzyme activity was about 1%. For measurements
of mRNA decay (see Fig.
5), rifampin was used at a concentration
of 300 µg/ml and was added
when the culture had reached an OD
600 of about 0.32.
Determination of -galactosidase specific activities.
For
determination of -galactosidase content, several 10- or 20-µl
samples were taken from an exponential culture over a period of two or
three generations, and -galactosidase was assayed as described
earlier (38). The volume of the final reaction mixture before the addition of an equal volume of stopping solution was 1.0 ml.
The assays were incubated at 30°C, generally for 50 to 90 min.
Similar assays were performed with media blanks. The -galactosidase activity was determined as the increase in A420
per hour of assay time per sample volume. In the exponential cultures,
the -galactosidase activity always increased in parallel with the
culture mass, and the observed points scattered by about 2% around the
average exponential curve. The specific activity was calculated as
-galactosidase activity per OD600 unit of culture mass
in the assay. One unit defined in this manner corresponds to 16.7 (1,000/60) Miller units (24). For a given culture, the
specific activity was determined with an accuracy of about ±2%, but
variations of about 10% were observed for cultures grown on different days.
RNA hybridization methods.
Total bacterial RNA was prepared
with the glass fiber filter method (5, 7) by using
commercial columns (RNAqueous; Ambion, Inc., Austin, Tex.). Samples (5 or 12.5 ml) of exponential culture were taken at an OD600
between 0.30 and 0.35 and added to a one-fifth volume of ethanol-phenol
stopping solution (5% phenol in ethanol) at 22°C. The bacteria were
pelleted by low-speed centrifugation (5 min at 5,000 rpm) and
homogeneously resuspended in 0.3 ml of guanidinium thiocyanate lysis
medium provided with the glass fiber column kit. After dilution with
0.3 ml of 64% (RNase-free) ethanol, the lysate was filtered through
the RNAqueous column by centrifugation (15 s at 10,000 rpm at room
temperature). The bound nucleic acids were washed with
high-salt-concentration and ethanol-containing buffers provided with
the columns. Essentially protein-free RNA was then eluted twice with 60 µl of 0.1 mM EDTA in diethyl pyrocarbonate (DEPC)-treated water.
After the UV absorption spectra were measured at pH 12 in 10 mM NaOH,
the preparations were diluted to an A260 of 10.0 with 0.1 mM EDTA in DEPC-treated water. For cultures grown in LB and
glycerol minimal medium, the preparations yielded about 1.2 or 0.6, respectively, A260 U of RNA per
OD600 U of culture mass, corresponding to about 150 or 75 µg, respectively, of RNA per 12.5-ml sample. The
A280/A260 ratio was
between 0.48 and 0.49, and the
A235/A260 ratio was
between 0.55 and 0.57. A sample of each preparation was subjected to
agarose gel electrophoresis. The presence of the 23S and 16S rRNAs in
ratios of greater than 1.5:1 suggested minimal RNA degradation.
To remove some residual DNA, 0.45
A260 U of RNA
(45 µl) were treated for 1 h at 37°C with 10 U of RNase-free
DNase I (Boehringer
Mannheim) and 80 U of an RNase inhibitor (RNaseout;
BRL Laboratories)
in a total volume of 50 µl of reaction buffer with
final concentrations
of 10 mM MgCl
2, 50 mM Tris-HCl (pH
7.9), 50 mM NaCl, and 1 mM
dithiothreitol (DTT). After this treatment,
the RNA was diluted
with 1 mM DTT (in DEPC-treated water) to a total
volume of 270
µl to give an
A260 of 1.67, which corresponded to 50 µg of RNA/ml.
It was found to be unnecessary
to remove or inactivate the DNase
or RNaseout enzymes. Again, a sample
of the preparations was subjected
to agarose gel electrophoresis to
verify the removal of chromosomal
DNA. Northern blot analysis with a
lacZ probe showed several bands
with molecular weights of
greater than 3,000 nucleotides (23S
rRNA marker) in addition to smaller
RNAs. Aliquots (50 µl containing
2.5 µg of RNA) of the DNA-free RNA
preparations were stored at
70°C. For the dot blot analysis (see
below), 5 µl of these dilutions
was added to 995 µl of 5× SSC (1×
SSC is 0.15 M NaCl plus 0.015
M sodium citrate), so that 100 µl of
this dilution used in a single
well of the dot blot apparatus contained
25 ng of total
RNA.
For quantitation of
lac-mRNA, a digoxigenin (DIG)-labeled
probe complementary to the 5' end of
lacZ (308 nucleotides
from
44 to +264, setting the beginning of the
lacZ coding
region to
+1) was used. To prepare the probe, DIG-labeled dUTP
(Boehringer
Mannheim) at a 1/20 dilution in TTP was included in a PCR
reaction
with appropriate primers and linearized vector DNA as a
template.
Known amounts of bacterial RNA (between 12.5 and 50 ng), each
in duplicate, were bound to a nylon membrane by slow vacuum filtration
by using a dot blot manifold. The RNA was fixed to the membrane
by heat
treatment (40 min at 80°C under vacuum), followed by UV
irradiation
(900 J/m
2). Hybridization and detection protocols used were
those provided
by Boehringer Mannheim. For the final detection by
chemiluminescence,
the membranes were exposed to X-ray film for 4, 8, and 16 min
(in some cases also for 2 or 32 min). The chemiluminescent
product
formed in the solution surrounding the membrane binds tightly
to the membrane; however, before this binding, the product may
be moved
from the site of its formation by convection, which causes
a smearing
and broadening of the signal on the membrane. To minimize
this effect,
the sealed plastic envelope was kept pressed flat,
with essentially no
liquid above and below the membrane during
overnight incubation, i.e.,
until the amount of chemiluminescent
product had reached its maximum
steady-state
concentration.
The photographic images were digitized by using either the ScanJet 4c
(Hewlett-Packard Corp.) or Gel Print 2000i apparatus
(BioPhotonics) and
then analyzed with the ImageQuant program (Molecular
Dynamics). The
method yields relative values of darkness of the
photographic images
within defined areas (circles around hybridization
"dots"). The
values were plotted as both functions of exposure
time and amount of
RNA spotted. The values given in Fig.
2
and
3 and Table
4 represent the slopes from these plots
in relative
units and were obtained in the linear range of exposure
time and
RNA amounts spotted. As controls, RNA preparations from
lac+ cultures grown in the presence and absence
of IPTG (isopropyl-
-
D-thiogalactopyranoside)
inducer
were analyzed with the Northern blot and dot blot methods.
No
detectable signals were obtained with RNA from uninduced cultures,
corresponding to less than 1% of the signal from the induced culture.
For the experiments presented in Fig.
2 to
5, eight
RNA preparations
were compared at a time, each represented by four
samples (e.g.,
either 25 and 50 ng or 12.5 and 25 ng, each in
duplicate) to give
a total of 32 hybridization areas ("dots") on
one 5-by-10-cm nylon
membrane. For background values, 45 additional
circular areas
between the dots (four areas surrounding each dot) were
measured,
averaged, and subtracted from the values obtained from the
RNA-containing
dot areas.
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FIG. 2.
Temperature-dependent expression of lacZ from
Pspc in the presence or absence of
trpt and rplN. Four strains in which the
Pspc promoter fusions were recombined into the
mal locus of the chromosome were used: SL102
(Pspc-trpt-lacZ; left panels,  ); SL104
(Pspc-lacZ; left panels,  ); SL105
(Pspc-rplN-trpt-lacZ; right panels, ); and
SL106 (Pspc-rplN-lacZ; right panels, ).
Cultures were grown at different temperatures in LB medium supplemented
with glucose, and -galactosidase specific activities (panels a and
b) and lacZ mRNA per total RNA (panels c and d) were
measured. The ratio of -galactosidase specific activity and
lacZ mRNA per total RNA (panels e and f) is a measure for
the translation efficiency of lacZ mRNA (from the data in
panels a and c or in panels b and d, respectively). The ratios of the
amounts of lacZ mRNA observed in the presence or absence of
a functional trpt (panels g and h,  ) at temperatures
between 20 and 42°C are illustrated.
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FIG. 3.
Growth rate dependency of lacZ expression
from Pspc. Four strains containing
Pspc promoter fusions recombined into the
mal locus were used: SL102
(Pspc-trpt-lacZ; left panels, ); SL104
(Pspc-lacZ; left panels, ); SL105
(Pspc-rplN-trpt-lacZ; right panels, ); and
SL106 (Pspc-rplN-lacZ; right panels, ). The
media used to give increasing growth rates were glycerol minimal,
glucose minimal, glucose-amino acids, and LB medium supplemented with
glucose. All cultures were grown at 37°C, and culture growth rates,
-galactosidase specific activities, and lacZ mRNA per
total RNA were measured.
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TABLE 4.
Expression of lacZ from the spc
ribosomal protein promoter in different strains grown at
37°C in LB medium or glycerol minimal medium
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FIG. 4.
Comparison of lacZ expression from
Pspc and from the P1 and P2 promoters of
rrnB. Two strains, SL106
(Pspc-rplN-lacZ, ) and XZ231 (rrnB
P1-P2-lacZ, ) were grown in four different media
supporting growth rates of between 1.0 and 3.0 doublings/h (see
legend to Fig. 3). Two samples were removed from each culture for
measurement of -galactosidase specific activity (panel a), and one
sample was removed for preparation of total RNA. Each RNA preparation
was used in two independent hybridization assays for determination of
lacZ mRNA per total RNA (panel b). The -galactosidase
activity per mRNA (panel c, solid symbols) was obtained in relative
units by first forming the quotient of the data in panels a and b and
then dividing this quotient by the amount of RNA/OD600 (at
growth rates of 0.97, 1.23, 2.20, and 2.90 doublings/h the
RNA/OD600 values were 5.8 × 1016,
6.6 × 1016, 9.4 × 1016, and
10.7 × 1016 RNA nucleotides per OD U, respectively
(4, 6). The division by these values corrects for
the different reference units used for enzyme specific activity
(OD600) and hybridization (total RNA). The rate of
translation per lacZ mRNA (panel c, open symbols) was
obtained by multiplying the data represented by the solid symbols by
the growth rate (ln2/ ).
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FIG. 5.
Residual accumulation of -galactosidase and decay of
lacZ mRNA in cultures treated with rifampin. Rifampin (300 µg/ml) was added to cultures of strains SL106 (), XZ231 (), and
HB123 () that were growing exponentially (OD600 = 0.32)
in either glycerol minimal medium (panels a and c) or LB medium (panels
b and d). The residual accumulation of -galactosidase (panels c and
d) and the levels of lacZ mRNA (panels c and d) were
monitored. In addition, the accumulation of total RNA was determined
(only in LB medium; panel d, open symbols). Due to the linear ordinate
scale in panels c and d (no exponential growth in the presence of
rifampin), the enzyme activity before zero time increases nonlinearly
(i.e., exponentially) with time. The zero time slopes were calculated
from the culture doubling times (63 min for glycerol minimal medium and
20.5 min for LB medium).
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RESULTS |
Features of promoter cloning vectors pRS415, pXZ09,
pXZ09-B, and pSL03.
Plasmids pRS415, pXZ09, pXZ09-B, and
pSL03 (Table 1 and Fig. 1) are promoter cloning vectors,
designed specifically to detect and measure promoter activities.
The plasmid pRS415 (33) was derived from plasmid
pBR322 and carries the W205 trp-lac fusion (25);
its cloning site is located between four upstream rrnB transcription terminators and a downstream promoterless
lacZ gene. The DNA section between the cloning
site and the start codon of lacZ consists of 75 bp of
trp sequence (including the distal 28 bp of trpA
and the 47-bp trailer sequence containing the trp
operon transcription terminator, trpt; refs.
28, 37) and the 17-bp lacZ leader sequence (including the wild-type ribosome
binding site, but not the promoter and operator sequences). Without an insert, this plasmid expresses very low background -galactosidase activity; with an insert containing a promoter, -galactosidase activity is elevated, but the extent of elevation is temperature sensitive. That is, -galactosidase activity is substantially reduced
at higher temperatures; this effect has been observed with several
different promoters and has been attributed to the presence of
trpt in the cloning vector (see below).
Both pXZ09 and pXZ09-B carry the cloning site and the W205
trp-lacZ fusion of pRS415. In addition, these plasmids carry
a
kan gene and segments of the
E. coli mal genes,
which permits
the recombinatorial insertion of operon fusions
constructed on
the plasmids into the
mal locus of the
chromosome (
16). Plasmid
pXZ09-B was obtained from pXZ09
(
38) by removing two
BamHI sites
flanking the
kan gene; the remaining
BamHI site on pXZ09-B
forms
part of the multiple cloning site. The plasmid pSL03 (Fig.
1)
was
obtained from pXZ09-B by deleting 50 bp of
trp sequences,
beginning at
BamHI of the cloning site and ending between
the
palindromic sequences that form the
trp operon
transcription terminator,
trpt (
37). By numerous
criteria, this deletion appears to inactivate
trpt and
results in elevated expression of the downstream
lacZ gene.
Moreover, the temperature sensitivity of
-galactosidase
expression
was greatly reduced or eliminated by the deletional
inactivation of
trpt (see
below).
Promoter constructs with and without trpt and with and
without rplN.
To illustrate the effects of trpt
and other features of operon fusions that might
complicate the quantitation of promoter activity, two sets of promoter
fusions were constructed by inserting DNA fragments containing
the promoter for the spc ribosomal protein operon
into pXZ09-B (with trpt) and into pSL03 (without
trpt). In the first set, pSL02 and pSL04, the insert
contained only the spc promoter (from nucleotides [nt]
51 to +59 relative to the transcription start site), whereas in the
second set, pSL05 and pSL06, the insert contained the spc
promoter and the first gene (rplN) of the spc
operon (from nt 51 to +453; see Fig. 1). To eliminate plasmid
copy number effects, the Pspc-lacZ fusions in
pSL02 and pSL04 and the Pspc-rplN-lacZ fusions in pSL05 and pSL06 were recombined into the mal locus of the
chromosome of the lacZ deletion strain HB181. With
these chromosomal constructs (strains SL102, SL104, SL105, and
SL106, respectively; Table 2), there was little variation in the amount
of -galactosidase specific activity in replicate constructs or
replicate cultures (see standard deviations of enzyme activity in Table
3).
lacZ expression from Pspc
at different temperatures.
Cultures of the four different
strains, SL102 (Pspc-lacZ with trpt),
SL104 (Pspc-lacZ without trpt), SL105 (Pspc-rplN-lacZ with trpt), and
SL106 (Pspc-rplN-lacZ without trpt)
were grown at four different temperatures between 20 and 42°C. The
-galactosidase specific activities and the relative amounts of
lacZ mRNA for the cultures were determined. In the two
strains carrying the trpt element upstream of
lacZ, expression of both lacZ mRNA and
-galactosidase enzyme was temperature sensitive (Fig. 2a
through d, circles). However, enzyme activity and mRNA were not
strictly proportional, as seen from the nonparallel curves in the
semilog plots used. This suggests that the amount of -galactosidase produced per amount of lacZ mRNA varies with the
temperature. This is best illustrated by visualizing the ratio of
-galactosidase specific activity to the amount of lacZ
mRNA as a function of temperature (Fig. 2e and f, circles). The values
in these curves decrease with increasing temperature. This suggests
that, in the presence of sequences associated with
trpt, lacZ mRNA translation is severalfold more
efficient at 20°C than at 42°C.
Removal of the transcription terminator
trpt from the
respective promoter
lacZ fusions altered both the
-galactosidase specific
activity and the amount of
lacZ
mRNA produced (Fig.
2a through
d, triangles). In this case, the
response of the enzyme activity
was nearly proportional to that of the
mRNA, so that the ratio
curves (Fig.
2e and f, triangles) are
essentially flat. These
results indicate that the presence of
trpt at the
spc-lac junction
directly or
indirectly influences translation initiation at the
downstream
lacZ ribosome binding site in a temperature-dependent
manner. Removal of
trpt abrogates this effect. It is also
apparent
that the efficiency of translation of
lacZ is
influenced by 5'
leader sequences in a temperature-independent manner.
For example,
the presence of the
rplN sequence in the leader
resulted in a
twofold reduction in
lacZ translation when the
two fusions lacking
a functional
trpt are compared (Fig.
2e
and f,
triangles).
Effect of different temperatures on transcript termination at
trpt.
To visualize the effect of trpt on
transcript termination at different temperatures, the ratio of observed
transcripts +trpt/trpt has been plotted as a
function of growth temperature (Fig. 2g and h; obtained from the data
in Fig. 2c and d). With or without rplN sequences, this
ratio decreased with increasing temperature. This suggests that the
efficiency of termination at trpt increases with increasing
temperature. The value of this ratio was expected to be maximal 1.0 when trpt is totally inactive, and this ratio should be less
than unity when any portion of the transcripts terminate at
trpt. When rplN was present upstream of
lacZ, the results were consistent with this expectation
(Fig. 2h): termination at trpt was about 10% efficient at
20°C but more than 60% efficient at 42°C. Surprisingly,
however, when rplN was absent, the ratio was greater
than unity at low temperatures (2.2 during growth at 20°C; Fig. 2g).
In this case the amount of lacZ transcript was increased,
rather than decreased, by the presence of trpt. This
suggests that the particular spc-trp-lac fusion without both trpt and rplN generates a fortuitous signal at
the fusion junction that reduces the accumulation of transcripts,
particularly at lower temperatures (Fig. 2e). This signal, although
undefined, could either cause transcript termination or reduce
transcript stability. When rplN sequences are present
between Pspc and the trp-lacZ
fusion, indications of this additional signal were not present (Fig.
2h).
Effect of different growth rates on lacZ expression and
termination at trpt.
Using the same four strains, the
effects of both (i) the presence or absence of trpt and (ii)
the presence or absence of rplN on lacZ
expression (enzyme and mRNA) were determined during growth at 37°C in
different nutritional media (Table 4; Fig. 3). At all growth rates of
between 1.0 and 3.0 doublings/h, the deletion of trpt
resulted in the expected increase in lacZ mRNA/total RNA and
in -galactosidase specific activity (e.g., compare strains SL104 and
SL102 or strains SL106 and SL105 in Table 4 or Fig. 3). The simplest
interpretation of this observation is that at all growth rates a
fraction of transcripts initiated at Pspc is
terminated at trpt when it is present. Assuming that only
the transcript termination function of trpt is responsible
for the reduction in the amount of lacZ mRNA, the fraction
of transcripts terminated was estimated by comparing the amounts of
lacZ mRNA (per total RNA) in the two isogenic
(+trpt/trpt) strain pairs (i.e., strains SL102 and SL104
and strains SL105 and SL106). With one exception, the efficiency of
transcript termination at trpt was estimated to be
between 43 and 46% in both fast- and slow-growing cultures
(Table 4). The exception occurred in the SL102-SL104 pair in
glycerol medium, where a higher value of 74% was found. This higher
value apparently results from an exceptionally high accumulation of
lacZ mRNA in the reference strain SL104 without trpt (compare hybridization data in Fig. 3c and d), which
may be related to the abnormality described above for this strain and
illustrated in Fig. 2g. Therefore, in the absence of other complicating
factors, the transcript termination function of trpt is
probably not growth rate dependent.
With increasing growth rate, the
-galactosidase specific
activity was not strictly proportional to the amount of
lacZ mRNA
(Fig.
3). The main reason for this is that
the specific activity
represents enzyme activity per culture mass,
whereas the hybridization
data represent transcripts per amount of
total RNA. The two reference
units, culture mass and total RNA, change
differently with respect
to the exponential growth rate (see
below).
Comparison of lacZ expression from different
promoters.
Because of different sequences at the junction of the
spc-trp-lac operon fusion, the
-galactosidase activities per amount of lacZ mRNA
expressed from Pspc were different in the four strains examined (Table 4). The results in Fig. 2g and h suggest that lacZ expression in strain SL106 (deletion of
trpt; inclusion of rplN upstream of
lacZ) was less influenced by artificial or fortuitous
transcription and translation signals at or near the fusion junction
than was expression in the other three strains. To test this
supposition, lacZ expression from
Pspc in strain SL106
(Pspc-rplN-lacZ) was compared with
lacZ expression from the P1-P2 tandem promoters of the
rrnB operon by using strain XZ231 (rrnB
P1-P2-lacZ [38]). The rRNA promoters were
chosen because their absolute activity is known and their importance
for the control of ribosome synthesis has been established.
Expression from the rRNA promoters increased with
increasing growth rate at both enzyme and mRNA levels (Fig.
4a
and b, circles),
whereas expression from P
spc
decreased at the enzyme
level and was nearly constant at the mRNA level
(Fig.
4a and b,
triangles). Despite these differences, the amounts of
-galactosidase
made per
lacZ mRNA were essentially
the same for both promoters
(Fig.
4c, filled circles and
triangles; the data are normalized
for the different reference units as
indicated in the figure legend).
Thus, in these two strains,
differences in
-galactosidase activity
at a given growth rate were
correctly reflected by differences
in
lacZ mRNA
accumulation.
These values reflect the amount of
-galactosidase per
lacZ mRNA; they may be multiplied with the rate of culture
growth (ln2/
,
where
is the culture doubling time) to obtain the
relative rate
of
-galactosidase accumulation per
lacZ
mRNA. This rate was approximately
constant (Fig.
4c, open symbols). As
anticipated, the translation
per
lacZ mRNA was independent
of the promoter from which
lacZ was expressed, i.e.,
rrnB P1-P2 or P
spc (Fig.
4c, circles
and triangles,
respectively).
Lifetime of lacZ mRNA.
To find the relative
rate of lacZ mRNA synthesis expressed from
Pspc and rrnB P1-P2, the decay of
lacZ mRNA was determined by following the disappearance of
the lacZ hybridization signal during growth in the presence
of the antibiotic rifampin (Fig. 5a and b, circles and triangles,
respectively). The hybridization probe, a 308-bp section that includes
the 5' end of the lacZ coding region, was the same as in the
preceding experiments. For comparison, the decay of lacZ
mRNA expressed from its natural promoter, Plac, was observed with the isogenic strain HB123 (Table 2) carrying a
wild-type lac operon (Fig. 5, diamonds). To obtain
information about the functional life of lacZ mRNA under
these conditions, the residual accumulation of -galactosidase during
rifampin treatment was also measured (Fig. 5c and d). The cultures were
grown in either glycerol minimal or LB medium (Fig. 5, left and right
panels). In all three strains (i.e., SL106, XZ231, and HB123) the
accumulation of total RNA (mainly rRNA) stopped immediately after the
addition of rifampin (Fig. 5d, open symbols), indicating that the
strains used were rifampin sensitive.
After the addition of rifampin to cultures grown in glycerol minimal
medium,
lacZ mRNA expressed from P
spc
and
rrnB P1-P2 decayed initially at about the same rate,
corresponding
to an average life of 1.8 min (Fig.
5a). However, for
P
spc-derived
mRNA, the decay slowed down after 1 min, leading to a plateau
of apparently stable mRNA (i.e., of the
5'-terminal region of
lacZ mRNA) at about 40% of the zero
time (exponential growth)
level (Fig.
5a). In LB medium, the initial
decay rates appear
to be slightly lower than in the minimal medium
(about a 2.4-min
average life), and the plateau of
P
spc-derived mRNA was
at about 64% of the zero
time level (Fig.
5b). The decay of
rrnB P1-P2-derived mRNA
also slowed. In this rich medium, the stability
of
P
spc- and
rrnB P1-P2-expressed
lacZ mRNA was reflected
in a continuing synthesis of
-galactosidase in the presence of
rifampin at a rate corresponding
to 15% of the enzyme synthesis
rate observed immediately before the
addition of rifampin (Fig.
5d). We cannot explain why
lac
mRNA from heterologous promoter
constructs fails to decay completely in
the presence of rifampin
(see
Discussion).
In control experiments, mRNA expressed from the
lac
operon promoter, P
lac, decayed
exponentially and apparently completely
(Fig.
5a and b, diamonds). The
decay rates for P
lac-derived
mRNA were identical
to the initial decay rates observed for
P
spc-
and
rrn P1-P2-derived mRNAs in
the two media. We assume that these
rates (ca. 1.8 min in glycerol
medium and 2.4 min in LB medium)
reflect the decay rate of
lac mRNA during exponential
growth.
In further control experiments (unpublished data), the decay of
rplN and
rplX mRNA (first and second genes in the
spc operon)
was measured in the strains HB123 and
SL106 with appropriate probes.
These mRNAs, when derived from the
spc operon, decayed exponentially
and identically in
the two strains used (the same RNA preparations
were used as for
Fig.
5). This indicates that the
spc promoter
is not
resistant to rifampin inhibition and that the construction
of SL106 did
not cause a special mutation that affects mRNA decay.
Therefore, the
stabilization of mRNA from the
spc-lac fusion appears
to be
specific for the fusion mRNA. It might reflect some special
properties
of r-protein mRNAs with respect to the control of their
decay rates and
the absence of those control sites in the fusion
mRNA (see
Discussion).
Absolute activity of the spc promoter.
The
activity of Pspc relative to the combined
activity of the two rRNA promoters rrnB P1-P2 was obtained
as the ratio of either the amounts of lacZ mRNA expressed
from Pspc and rrnB P1-P2 (ratio of
the two curves in Fig. 4b) or the corresponding -galactosidase
specific activities (ratio of the two curves in Fig. 4a). Either ratio
decreased with increasing growth rate from about 1.5 at a growth rate
of 1.0 doubling/h to about 0.3 at a growth rate of 3.0 doublings/h
(Fig. 6a, circles and triangles, respectively). The results shown in Fig. 5 suggest that
Pspc- and rrn P1-P2-derived
lac mRNA decayed at essentially equal rates during
exponential growth so that, for a given medium, the different amounts
of Pspc- and rrnB P1-P2-derived
lacZ mRNAs reflect the differences in their relative
synthesis rates. The absolute activity of the rRNA promoters in rRNA
transcripts per minute was determined previously (4,
6) and is illustrated in Fig. 6b (open symbols). The rRNA gene
activity increases from about 3 to over 60 initiations/min in the range
between 0.6 and 3.0 doublings/h. By multiplying the rRNA gene activity
with the relative Pspc activity (relative to the
rrnB P1-P2 activity), absolute Pspc
activities were obtained (Fig. 6b, circles). According to these
estimates, the Pspc activity increased from a
value of about 10 transcripts/min at a growth rate of 1.0 doubling/h to
a plateau of about 23 transcripts/min at growth rates of above 1.5 doublings/h.
View larger version (25K):
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FIG. 6.
Absolute activities of Pspc and
rrnB P1-P2 promoters as a function of growth rate. The
activity of Pspc relative to the activity of
P1-P2 promoters of rrnB (panel a) was obtained from the
ratios of -galactosidase specific activities () or
lacZ mRNA values () by using the data presented in Fig.
4a and b, respectively. The absolute activities of the rrnB
P1-P2 promoters (panel b, diamonds) were obtained from literature data
that were based on measurements of stable RNA synthesis and of rRNA
gene dosages (4, 6); the units are the transcripts per
minute per rrn operon. The absolute activity of the
spc promoter (panel b, solid symbols) was obtained by
multiplying the absolute activity of the rRNA promoters with the
activity of Pspc relative to the activity of the
rrnB P1-P2 promoters shown in panel a.
|
|
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DISCUSSION |
Transcription termination and other effects of trpt.
The
expression of -galactosidase activity from promoter cloning vectors
based on the classical W205 trp-lac fusion has been used in
the past to study promoter activities under different physiological
conditions. Generally, the promoter-lacZ fusions are
integrated into the bacterial chromosome by using phage vectors
(3), and the resulting lysogens are grown at 30°C because of the presence of a temperature-sensitive repressor.
rrnB promoters have been studied by using the mal
chromosomal integration system (16) at 37°C (38,
39), which is the standard temperature for physiological
experiments with E. coli. However, when we tried to use this
system to study the pBR322 replication primer promoter, we noticed
that the -galactosidase activity expressed from the primer promoter
was temperature sensitive (unpublished observations). Subsequently this
temperature sensitivity was confirmed with other promoters, including
the rrnB P1 and P2 promoters and the ribosomal protein
Pspc promoter (unpublished observations). Here we have traced the temperature sensitivity to the presence of the
rho-independent trpt transcriptional terminator that is
located immediately upstream of lacZ in the W205
trp-lac fusion (Fig. 1).
The frequency of transcription termination at
trpt was
estimated by comparing the amount of
lacZ mRNA present in
isogenic
P
spc-rplN-lacZ fusion constructs with
or without a functional
trpt upstream of
lacZ. At
37°C, transcript termination was estimated
to be 43 to 46% and
independent of the growth rate (Table
4).
This value is similar to the
previously reported value of 37%
(
27) based on
galK expression from P
lac with or
without
trpt. In that study, neither the growth temperature
nor the growth
medium were indicated. In another study also based on
enzyme activity
data, a higher termination efficiency of 83% at
trpt was estimated
at 30°C (17% readthrough
[
1]). Our comparison of strains SL106
(P
spc-rplN-lacZ) and SL105
(P
spc-rplN-trpt-lacZ)
indicates that very
little, if any, termination at
trpt occurred
at 20°C but
that with increasing temperature the termination efficiency
increased to about 60% at 42°C (Fig.
2h). An alternative
interpretation
of these results cannot be ruled out, namely, that
trpt or the
associated sequences that have been deleted in
pSL03 (Fig.
1b)
produces a temperature-dependent mRNA stability
signal that causes
the observed temperature
effects.
A comparison of
lacZ expression at the enzyme and mRNA
levels in isogenic strain constructs with or without
trpt
and with
or without an
rplN sequence in the
lacZ
leader revealed a number
of anomalous effects that remain
uncharacterized. We suggest that
these anomalies arise from
artificial sequence combinations at
the
spc-trp-lac fusion junction and that they
influence features
such as termination and antitermination
properties of transcribing
RNA polymerase, mRNA stability,
or translation of the
lacZ cistron.
These indirect
effects defeat the purpose of promoter-
lacZ fusions,
i.e.,
to obtain information about the promoter activity. We therefore
focused
our attention on the P
spc fusion containing
rplN and lacking
trpt. In this strain (SL106),
such anomalous effects
appeared to be minimal if not completely
absent.
Expression of -galactosidase from Pspc
and P1-P2rrnB.
In the strain SL106
(Pspc-rplN-lacZ without trpt), the
-galactosidase activities per amount of lacZ mRNA
observed at different growth rates were the same as in a previously
constructed strain XZ231 (Fig. 4c) in which the P1-P2 tandem promoters
of the rRNA operon rrnB are linked to
lacZ (38). The rrnB
P1-P2-lacZ fusion includes trpt but, in addition,
contains the antitermination elements of rRNA genes which are assumed
to prevent or at least greatly reduce termination at trpt:
the readthrough at trpt at 30°C has been reported to
be fourfold increased, from 17 to 73%, by the presence of the
rrnE antitermination sites (1). The rrnB P1-P2-lacZ fusion also includes 1,120 bp of
phage DNA between the rrnB P1-P2 promoters and the
trp-lacZ section. Insertion of this DNA "spacer" was
necessary for the initial cloning of strong rRNA promoters on
pBR322-derived plasmids (38). The DNA sequences inserted
do not contain known promoters or ribosome binding sites. The
observation that the differences in -galactosidase synthesis in the
strains SL106 and XZ231 correctly reflect the differences in the
accumulation of 5'-terminal lacZ mRNA (Fig. 4c) suggests that fortuitous translation signals at the trp-lac fusion
junction are either the same or absent for these two constructs. The
anti-transcription termination sites of the rRNA promoters apparently
do not affect the expression of -galactosidase activity by
suppressing polarity within lacZ. Furthermore, the initial
decay rates of lacZ mRNA after the addition of rifampin were
about the same in SL106 and XZ231 (Fig. 5a and b), suggesting that
the different lacZ leaders in these two strains do not
differently affect lacZ mRNA decay during exponential
growth; at later times after rifampin addition lacZ mRNA
decay was clearly different. For these reasons, the use of the
rrn P1-P2-lacZ construct in XZ231 as a reference
for comparison with the Pspc-rplN-lacZ fusion in
SL106 appears to be justified, despite the differences in the leader regions.
The
-galactosidase specific activity expressed from
rrnB
P1-P2 in strain XZ231 increased with increasing growth rate (Fig.
4a, circles), as was expected in view of the increased ribosome
synthesis at high growth rates and in agreement with previous
reports
(
38). On the other hand, the
-galactosidase specific
activity expressed from P
spc in strain SL106
decreased
with increasing growth rate (Fig.
3b and Fig.
4a,
triangles).
A decreasing
-galactosidase expression from r-protein
promoters
with increasing growth rate is in contrast to the increasing
amounts
of r protein made per total protein (
r
[
8,
10,
32]). A
similar decrease
-galactosidase specific activity with increasing
growth rate
has been observed previously with the promoter of
another major
r-protein operon, S10, linked to
lacZ
(
19). As
had been suggested in that study, it is possible
that this discrepancy
results from the omission on the fusion
constructs of certain
control sites located distally in these
operons. These sites are
thought to regulate the decay of
r-protein mRNA via translational
repression, translational coupling,
and endonucleolytic cleavage
followed by 3'-to-5' exonucleolytic mRNA
degradation ("retroregulation";
for a review, see reference
18). In
the
spc operon, such sites
have been located
downstream of
rplN (
22) and are expected to
stabilize the mRNA when the production of 16S rRNA exceeds or
equals
the production of the S8 regulatory protein. Based on the
arguments
above, we suggest that, in the absence of fortuitous
transcription and
translation signals at the junction of the operon
fusion, the
-galactosidase expression from r-protein promoters
such as
P
spc decreases with increasing growth rate, as
seen in Fig.
4a (triangles). This is not in contradiction to an
increasing transcriptional activity of P
spc (see
below).
Other investigators have reported that the
-galactosidase specific
activity expressed from P
spc is growth rate
independent
(
2,
14,
26). In those studies the
RS205
system carrying
trpt was used. The same result was obtained
here with a similar
construct carrying
trpt and
P
spc directly linked to
lacZ (strain SL102). With this strain, we also observed that
-galactosidase
specific activity was independent of the growth rate
(Fig.
3a,
circles). We suggest that those earlier results
(
2,
14,
26;
Fig.
3a, circles) were influenced either by the presence of
trpt or by the artificial sequence combinations generated at
the fusion
junction.
Transcriptional activity of Pspc.
The rate
of spc mRNA synthesis has previously been measured per rate
of total transcription (rspc/rt) by
RNA pulse-labeling and with a hybridization probe that included
spc mRNA (9). In that study,
rspc/rt was found to decrease by
about 20% (from 2.15 to 1.74%) when the growth rate increased
threefold from 0.67 to 2.1 doublings/h. Here we observed the amount of
Pspc-derived lacZ mRNA per amount of total RNA,
which also decreased by about 20% in the range of growth rates studied
(Fig. 4b, triangles). After the amounts of lacZ mRNA shown
in Fig. 6b were converted into relative synthesis rates (by using the
mRNA decay data of Fig. 5) and the different reference units were taken
into account the decrease becomes somewhat greater than 20%.
However, because of the differences in methods, growth media, and
hybridization probes, these data sets are not strictly comparable and
it is not clear whether the modest discrepancy between them is significant.
The rate of
spc mRNA synthesis relative to the rate of total
mRNA synthesis (
rspc/rm) has been
reported to increase with increasing
growth rate similar to
r (
13,
21). This has suggested that
r-protein
synthesis is primarily regulated at the transcriptional
level, so that
the translational regulation only provides a "fine-tuning"
to
accurately adjust r-protein synthesis to rRNA synthesis (
6,
13,
21). This interpretation was based on the plausible but
unproven
assumption that the rates of translation and degradation
of bulk mRNA
change with growth rate in a way similar to that
of translation and
degradation of
spc mRNA. However, this assumption
may not be
warranted, especially since the rate of
spc mRNA degradation
appears to be subject to a special regulation dependent on the
synthesis of rRNA (
22).
The absolute activity of P
spc was estimated
above in transcripts initiated per minute per promoter by comparison
with the known absolute activity of rRNA promoters. At low growth
rates, the P
spc activity increased approximately
in proportion
to the rRNA promoter activity and then became constant
above 1.5
doublings/h at about 23 transcripts/min (Fig.
6b). In view of
the 1.1 kb of
DNA spacer between the promoters and
lacZ
in the
operon fusion on strain XZ231, it seems possible that a
fraction
of the transcripts originating at
rrnB P1-P2
terminates before
reaching
lacZ. In that case transcription
from
rrnB P1-P2 in strain
XZ231 would be underestimated, so
that the P
spc activities
in Fig.
6b would be
overestimates. However, because of the transcription
antitermination elements associated with the rRNA promoters, this
may
not be significant, so that the
spc promoter activities in
Fig.
6b should be essentially
correct.
Decay of lacZ mRNA in the presence of rifampin.
An
attempt was made to determine the average lifetime of
Pspc- and rrnB P1-P2-derived
lacZ mRNA sequences by using rifampin to inhibit
transcription initiation. Surprisingly, in the presence of rifampin
lacZ mRNA expressed from Pspc did not
completely disappear (Fig. 5b), and in LB medium some residual
-galactosidase synthesis from Pspc and
rrnB P1-P2 continued (Fig. 5d). A number of control
experiments demonstrated that the rifampin used was fully active and
that the bacterial strains were fully sensitive. First, the
accumulation of stable RNA (rRNA and tRNA) ceased immediately after the
addition of rifampin in all strains used (Fig. 5d). Second,
lacZ mRNA derived from the lactose operon
promoter in the isogenic strain HB123 decayed exponentially and
completely in the presence of rifampin (Fig. 5a and b). Third,
spc mRNA sequences derived from transcription of the
spc operon in the spc-lac fusion strain
decayed exponentially and completely in the presence of rifampin (data
not shown). Finally, in the presence of rifampin, all bacterial
cultures stopped growth immediately and none accumulated
rifampin-resistant bacteria (data not shown). We therefore conclude
that initiation of all RNA chains ceased in the presence of rifampin
and that the incomplete or nonexponential decay of
Pspc- and rrnB P1-P2-derived lacZ mRNA observed at later times was due to mRNA
stabilization. The mechanism responsible for this stabilization of
fusion mRNA is not known. In part, it might be caused by a crowding of
mRNA with ribosomes when bulk mRNA gradually vanishes during rifampin treatment. Similar decreased rates of mRNA decay as a result of ribosome crowding have been reported (23, 28). Conversely, when translation of the mRNA was reduced, the rate of lacZ
mRNA decay has been found to increase (17). Again, this
indicates that increased translation can result in a decreased rate of
mRNA decay.
Based on the preceding arguments, we assume that the initial decay
rates in the presence of rifampin reflect the decay rates
during
balanced exponential growth. The initial decay kinetics
of
lacZ mRNA derived from P
lac,
P
spc, and
rrnB P1-P2 for a given
medium were virtually identical. For glycerol
medium, the initial rate
corresponded to an average lifetime of
about 1.8 min and for LB medium
it was about 2.4 min. Since in
a given medium the decay rates for
P
spc- and
rrnB P1-P2-derived
mRNAs
were the same, we were able to estimate the absolute activity
of
P
spc from the observed accumulations of
lacZ mRNA
(see
above).
Features of new cloning vector pSL03.
When a reporter
system is used it does not seem prudent to include a transcription
termination signal upstream of the reporter gene, particularly if the
terminator activity is variable and affected by conditions such as
temperature and growth media. For these reasons, we and other
investigators (20) have removed trpt from
W205-derived vectors. The presence of trpt in the W205 fusion might not have been apparent to all previous investigators; for
example, when RS205 was used as a cloning vector for
Pspc by Miura et al. (26), they
stated that the fusion W205 removes the transcription termination
signal of the trp operon. Clearly, trpt'
was removed but trpt was not.
In addition to the higher expression values due to the absence of the
transcription terminator, pSL03 has several other desirable
features.
(i) In contrast to phage
-based vectors with a temperature-sensitive
repressor, the
mal-inserted constructs can be grown at
any temperature.
Although
-based vectors with a
temperature-independent repressor
are available, the presence of
the prophage may not always be
desirable. (ii) In the absence of a
cloned promoter, there is
very little background
-galactosidase
activity when the construct
is integrated into the chromosome.
(iii) The location of
mal close
to
oriC on the
E. coli chromosome produces a relatively constant
gene
dosage (
11), in contrast to
att near the
middle of the
E. coli replicon, which shows
considerable changes in gene dosage
at different growth rates
(
4). (iv) The orientation of the
lacZ insertion
into the chromosome at the
mal locus is such that
the
directions of transcription and replication are aligned. This
may be an
advantage for active promoters since most operons with
strong
promoters are oriented in this
manner.
A number of investigators (see, for example, reference
30) have also observed that in fusion constructs the
translation
of the reporter gene may be affected by fortuitous signals
that
arise at the junction of the fused operons. Linn and St.
Pierre
attempted to alleviate this problem by including in their vector
an RNase III cleavage site upstream of
lacZ so that all
reporter
gene mRNAs had the same 5' terminus (
20). However,
it is not
certain even with their vector that RNase III cleavage
and RNA
polymerase transcription
termination-antitermination properties
are completely independent
of growth conditions and sequences
at the fusion junction. Therefore,
for accurate quantitation a
careful analysis of fusion gene expression
is necessary with any
vector.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from NIH and MRC.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, University of British Columbia,
2146 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Phone: (604) 822-5975. Fax: (604) 822-5227. E-mail:
pdp1{at}unixg.ubc.ca.
| |
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Journal of Bacteriology, December 1998, p. 6090-6100, Vol. 180, No. 23
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
