|
1108 K. Hollands, D. J. Lee, G. S. Lloyd and S. J. W. Busby ■ Table 2. Strains and plasmids and promoter fragments. | ||
|
Name |
Description |
Reference |
|
E. coli K-12 strains |
F- l- ilvG rfb-50 rph-1 |
Blattner etal. (1997) |
|
JW1907-1 |
fliA::kan rrnB3 DlacZ4787 hsdR514 D(araBAD)567 D(rhaBAD)568 rph-1 |
Baba etal. (2006) |
|
M182 |
lacX74 galK galU strA |
Casadaban and Cohen (1980) |
|
M182 Dcrp |
Dcrp lacX74 galK galU strA |
Busby etal. (1983) |
|
M182 D fliA |
DfliA lacX74 galK galU strA |
This study |
|
M182 Dcrp D fliA |
D fliA Dcrp lacX74 galK galU strA |
This study |
|
BL21(DE3) |
F-ompT hsdSB (гв-n-) gal dcm l(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) |
Studier and Moffat (1986) |
|
Plasmids |
Broad-host-range lacZexpression vector used for cloning EcoRI-HindIII |
Lodge etal. (1992) |
|
pSR |
promoter fragments; contains the RK2 origin of replication and encodes TcR |
Kolb et al. (1995) |
|
pET21a |
upstream of the loop terminator |
Novagen |
|
pKXH100 |
pET21a carrying fliA gene cloned on an NdeI-XhoI fragment |
This study |
|
Promoter fragments’ |
168 bp EcoRI-HindIII fragment carrying the aer regulatory region |
Hollands etal. (2007) |
|
aer206 |
Derivative of aer200 with CGA to GCT changes from positions -11 to -9 |
This study |
|
aer213 |
in the promoter -10 element Derivative of aer200 with a T to C substitution at position -32 in the promoter |
This study |
|
aer214 |
-35 element Derivative of aer200 with an A to T substitution at position -30 in the promoter |
This study |
|
aer224 |
-35 element Derivative of aer200 with a G to A substitution at position -28 in the promoter |
This study |
|
aer212 |
-35 element Derivative of aer200, in which the DNA site for CRP is moved to position -41.5 |
This study |
|
aer211 |
Derivative of aer200, in which the DNA site for CRP is moved to position -61.5 |
This study |
|
aer226 |
Derivative of aer200, in which the DNA site for CRP is moved to position -44.5 |
This study |
|
aer227 |
Derivative of aer200, in which the DNA site for CRP is moved to position -54.5 |
This study |
|
fliC100 |
EcoRI-HindIII fragment carrying the regulatory region of the fliC operon |
This study |
|
flgM100 |
EcoRI-HindIII fragment carrying the regulatory region of the flgMN operon |
This study |
|
flgK100 |
EcoRI-HindIII fragment carrying the regulatory region of the flgKL operon |
This study |
|
motA100 |
EcoRI-HindIII fragment carrying the regulatory region of the motABcheAWoperon |
This study |
|
tar100 |
EcoRI-HindIII fragment carrying the regulatory region of the tar tap cheRB |
This study |
|
tsr100 |
CheYZflgMN operon EcoRI-HindIII fragment carrying the regulatory region of the tsr operon |
This study |
|
trg100 |
EcoRI-HindIII fragment carrying the regulatory region of the trg operon |
This study |
a. The base sequence of each of the promoter fragments is shown in Fig. S2.
The DNA sequence of each promoter fragment is shown in
Fig. S2. Promoter fragments were amplified by PCR from
genomic DNA of E. coli K-12 strain MG1655, using primers
that introduce flanking EcoRI and HindIII sites (listed in
Table S1). For promoter activity assays, EcoRI-HindIII frag-
ments were cloned into the lac expression vector, pRW50. To
construct templates for in vitro transcription assays, and to
generate DNA fragments for electromobility shift assays and
footprinting, promoter fragments were cloned into plasmid
pSR. Derivatives of the aer200 fragment carrying point muta-
tions in the -10 or -35 elements (aer206, aer213, aer214 and
aer224) were constructed by megaprimer PCR. In a first-
round PCR reaction, a megaprimer was synthesized from
pSR/aer200 as a template, using a mutagenic primer carrying
the desired mutation, and a flanking primer (either D51598 or
D53041: see Table S1). The megaprimer was then used in a
second-round PCR with the opposing flanking primer and
pSR/aer200 as a template to generate a full-length promoter
fragment containing the required mutation, which was then
cloned into pRW50. The aer212, aer211, aer226 and aer227
fragments were constructed by inserting or deleting DNA
between the DNA site for CRP and the -35 element of the aer
promoter. First, two PCR products were synthesized using
pSR/aer200 as a template: one generated using upstream
primer D53041 and a downstream primer carrying the inser-
tion or deletion, and a second generated using the down-
stream primer D51598 and an upstream primer carrying the
insertion or deletion (see Table S1). The two PCR products
were then annealed via their 26-32 bp overhangs, and the
two strands were extended using DNA polymerase to gener-
ate a full-length promoter fragment carrying the insertion or
deletion. This product was then amplified by PCR using
primers D53041 and D51598 and cloned into pRW50.
b-Galactosidase assays
b-Galactosidase levels in cells carrying promoter::lacZ
fusions, cloned in pRW50, were measured using the method
of Miller (1972). Cells were grown aerobically at 37°C in LB
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 75, 1098-1111