SME3358 ECL116 ârhaS λΦ(Phts-lacZ) recA::cat. This study. SME3359 SME3358 + pHG165lacI. This study. SME3632 SME3000 malP::lacIq zhc-511::Tn10 ...
SUPPLEMENTARY MATERIAL TABLE Table S1. Bacterial strains and plasmids Strain a
Genotype
Acella™
E. coli B F ompT hsdSB(rB mB ) gal dcm λ(DE3)
Source or Reference -
-
-
MoBiTec
ΔendA ΔrecA ArcticExpress E. coli B F– ompT hsdS(rB– m B–) dcm+ Tetr gal
Agilent Technologies
λ(DE3) endA The [cpn10 cpn60 Gentr] ECL116
F- ∆lacU169 endA hsdR thi
1
MC4100
F- araD139 Δ(argF-lac)U169 rpsL150 relA1
2
flhD5301 deoC1 ptsF25 rbsR SG22166
MC4100 malP::lacIq ftsH1(ts) zgj::Tn10
SME1048
ECL116 recA::cat
Laboratory collection
SME1085
ECL116 ∆rhaS recA::cat
Laboratory collection
SME1088
ECL116 λΦ(rhaB-lacZ)Δ84b ∆rhaS recA::cat
Laboratory collection
SME1403
ECL116 ∆crp-3 zhc-511::Tn10
Laboratory collection
SME3000
ECL116 λΦ(rhaB-lacZ)Δ84b Δ(rhaSR)::kan
Laboratory collection
SME3006
SME1088 + pHG165rhaS
Laboratory collection
SME3358
ECL116 ∆rhaS λΦ(Phts-lacZ) recA::cat
This study
SME3359
SME3358 + pHG165lacI
This study
SME3632
SME3000 malP::lacIq zhc-511::Tn10 recA::cat
This study
SME3634
SME3632 + pHG165rhaS
This study
SME3635
SME3632 + pHG165rhaS(163-278)
This study
1
3
Plasmid
Relevant characteristics
Source or Reference
pHG165
lacα+ rop+ Ampr, essentially pUC8 with pBR322
4
copy number pHG165rhaS expresses wild-type RhaS from vector lac promoter
5
pHG165rhaS(163-278)
This study
expresses RhaS residues 163-278
from vector lac promoter pHG165lacI
expresses wild-type LacI from vector lac promoter L. Swint-Krusec
pRS414
T14 ‘lacZ (lacYA)+ Ampr
6
a
All bacteria were strains of Escherichia coli K-12, except for two E. coli B strains, as noted. The promoter of Φ(rhaB-lacZ)Δ84 contains the full RhaS binding site, but not the binding site for CRP, identical to the fusion described in 7. c Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS. b
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Backman, K., Chen, Y. M., and Magasanik, B. Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981, 78, 3743-3747. Casadaban, M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976, 104, 541-555. Jubete, Y., Maurizi, M. R., and Gottesman, S. Role of the heat shock protein DnaJ in the Lon-dependent degradation of naturally unstable proteins. J Biol Chem. 1996, 271, 30798-30803. Stewart, G. S., Lubinsky-Mink, S., Jackson, C. G., Cassel, A., and Kuhn, J. pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression. Plasmid. 1986, 15, 172-181. Kolin, A., Balasubramaniam, V., Skredenske, J. M., Wickstrum, J. R., and Egan, S. M. Differences in the mechanism of the allosteric L-rhamnose responses of the AraC/XylS family transcription activators RhaS and RhaR. Mol Microbiol. 2008, 68, 448-461. Simons, R. W., Houman, F., and Kleckner, N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987, 53, 85-96. Egan, S. M., and Schleif, R. F. A regulatory cascade in the induction of rhaBAD. J Mol Biol. 1993, 234, 87-98.
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FIGURES Figure S1.
Figure S1. Growth curves in the presence (+) and absence (-) of OSSL_051168. Strains SME3634 (rhaB-lacZ, pHG165rhaS) and SME3359 (hts-lacZ, pHG165lacI) were grown in the absence or presence of 44 uM OSSL_051168. Results were averaged from two independent experiments with three replicates each.
3
Figure S2.
Figure S2. Model for OSSL_051168 inhibition. Our results indicate that OSSL_051168 inhibits by binding to the RhaS (and RhaR) proteins, and blocking its ability to bind to DNA. As a result, RhaS would not activate transcription in the presence of rhamnose. Labeling same as in Fig. 1 except small black rectangles: OSSL_051168.
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