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R- 1
1
R- 6
w sp
R- 5
R
w sp
cDNA
DNA
F
1kb Marker
E
frzG-C-1 + wspR-6
D
cDNA
DNA
b.
1kb Marker
C
RNA
B
wspR-1 + wspR-5
A
w sp
G- C
F rz
wsp operon
RNA
a.
10kb
5kb
4kb
3kb
0.5kb
2kb
1.5kb
0.7kb
1kb
0.5kb
Supplementary Figure 1. RT-PCR analysis of the wsp operon.
a. To test whether wspF and wspR are transcribed as a single unit, RT-PCR was performed using
primers FrzG-C-1 and wspR-6, which flank the non-coding region between wspF and wspR. Primers
wspR-1 and wspR-5, which lie within wspR, were used as controls to confirm transcription of wspR
and accuracy of the RT-PCR. The location and orientation of primers are marked as arrows. The
expected PCR products are shown as double-headed arrows underneath the relevant primer sets.
b. A reverse transcription step was performed on total RNA using the reverse primers wspR-6 and
wspR-5 individually in order to obtain the desired cDNA fragments from wsp mRNA. PCR was
performed on the obtained cDNA with the appropriate primers. PCR was also performed on total RNA
(as a negative control) in order to test for DNA contamination that would result in false-positive
signals, and on DNA as a positive control. The templates and primers used are shown above the
relevant lanes. The 1 kb Marker (New England Biolabs) was used as a DNA size marker; the sizes of
the individual fragments (kb) are marked next to the relevant DNA bands. The expected ~0.7 kb
fragment was obtained from DNA and cDNA samples with primers wspR-1 and wspR-5, confirming
transcription of wspR. The ~0.5kb fragment obtained from the DNA and cDNA samples with primers
FrzG-C-1 and wspR-6 confirmed the presence of mRNA transcript spanning the non-coding 50bp
region between wspF and wspR.
-galactosidase activity (Miller units)
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Supplementary Figure 2. Transcriptional activity of the wsp operon in SM and LSWS genotypes.
Overnight cultures of the ancestral (SM) and derived (LSWS) genotypes both containing identical
chromosal fusions between wspR and promoterless lacZ (EB01 is wspR-lacZ fusion in SM and EB02
is wspR-lacZ fusion in LSWS) were used to inoculate 50 ml of fresh King’s Medium B containing
tetracycline (25 μg ml-1). Cultures were grown shaking at 28 oC; -galactosidase activity was
quantified after 4, 12, 24 and 48 hours of growth. Data are means and standard errors of three
replicates.
Supplementary Figure 3. Alignment of the predicted amino acid sequence of WspF with E. coli
CheB. The N-terminal response regulator receiver domain of WspF spans residues 1 to 122 and
contains the major active site aspartate residues, which in CheB are Asp-10, Asp-11 and Asp-56
(Asp56 is thought to be the phosphorylation site) (STOCK and SURETTE 1996). The C-terminal
methylesterase domain of WspF spans 184 residues (residues 153-332) and contains the major
methylesterase active sites, which in CheB are Ser-164, His-190 and Asp-286 (DJORDJEVIC et al.
1998; FALKE et al. 1997). All described sites of functional significance are highlighted in red.
Identical and similar residues residues are highlighted with black and grey, respectively
Supplementary Figure 4. The location of the non synonymous wspF mutations mapped onto the
crystal structure of CheB from Salmonella typhimurium.
REFERENCES
DJORDJEVIC, S., P. N. GOUDREAU, Q. XU, A. M. STOCK and A. H. WEST, 1998 Structural basis for
methylesterase CheB regulation by a phosphorylation-activated domain. Proc. Natl. Acad. Sci.
USA 95: 1381-1386.
FALKE, J. J., R. B. BASS, S. L. BUTLER, S. A. CHERVITZ and M. A. DANIELSON, 1997 The twocomponent signaling pathway of bacterial chemotaxis: A molecular view of signal
transduction by receptors, kinases, and adaptation enzymes. Annu. Rev. Cell Dev. Biol. 13:
457-512.
STOCK, J. B., and M. G. SURETTE, 1996 Chemotaxis, pp. 123-145 in Escherichia coli and Salmonella
typhimurium: Cellular and Molecular Biology, edited by F. NEIDHARDT. American Society
for Microbiology, Washington, DC.