b.
a.
A B C D E
10kb
5kb
4kb
3kb
2kb
1.5kb
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 .
F
F z r
G
-C
-1 w s p
R
-1
R w s p
R
-6 w s p
R
-5

QuickTime™ and a
TIFF (U ncompressed) decompressor are needed to see this picture.
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 o C;

-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) (S TOCK and S URETTE 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 (D JORDJEVIC et al.
1998; F ALKE 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 .

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