1 SUPPLEMENTARY MATERIAL TABLE Table S1 ...

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


SME1088

ECL116 λΦ(rhaB-lacZ)Δ84b ∆rhaS recA::cat


SME1403

ECL116 ∆crp-3 zhc-511::Tn10


SME3000

ECL116 λΦ(rhaB-lacZ)Δ84b Δ(rhaSR)::kan


SME3006

SME1088 + pHG165rhaS


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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