Table S1. E. Coli Strains and Plasmids Used in This Work

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli; Structural and Functional Studies

Donata Figaj, Artur Gieldon, Agnieszka Polit, Anna Sobiecka-Szkatula, Tomasz Koper, Milena Denkiewicz, Bogdan Banecki, Adam Lesner, Jerzy Ciarkowski, Barbara Lipinska and Joanna Skorko-Glonek

Table S1. E. coli strains and plasmids used in this work.

Strain or plasmid Relevant characteristic Reference or source
BL20 W3110 htrA63 galE sup+ (1)
BL21DE3 F- ompT hsdSB(rB –mB-) gal dcm Novagen
DH5α supE44 lacU169 (80 lacZΔM1 ) (2)
hsdR17 endA1 gyrA96 thi-1 relA1
K38(pGP1-2 ) HfrC(λ),T7 polymerase, KanR (3)(4)
Plasmids
High copy number
pTA3 pQE60 htrA C57S C69S kindly provided by M. Ehrmann
pJS13 pT7-5 htrA (5)
pJS14 pT7-5 htrA S210A (5)
pJS19 pT7-5 htrAC57S C69S S210A this work
pJSFW pT7-5 htrA F50W S210A this work
pDF1 pT7-5 htrA P43G this work
pDF2 pT7-5 htrA R44D this work
pDF3 pT7-5 htrA R44L this work
pDF3A pT7-5 htrA R44L S210A this work
pDF4 pT7-5 htrAR44P this work
pDF5 pT7-5 htrAF46Y this work
pDF5A pT7-5 htrAF46Y S210A this work
pDF6 pT7-5 htrAQ47L this work
pDF6A pT7-5 htrAQ47L S210A this work
pDF7 pT7-5 htrAF63Y this work
pDF7A pT7-5 htrAF63Y S210A this work
pDF8 pT7-5 htrAQ64A this work
pDF9 pT7-5 htrAQ64I this work
pDF10 pT7-5 htrAF68Y this work
pDF10A pT7-5 htrAF68Y S210A this work
pDF11 pT7-5 htrAQ70A this work
pDF11A pT7-5 htrAQ70A S210A this work
pDF12 pT7-5 htrAD52A this work
pDF13 pT7-5 htrAD53A this work
pDF14 pT7-5 htrAS54A this work
pDF15 pT7-5 htrAF56S this work
pDF16 pT7-5 htrAE59V this work
pDF17 pT7-5 htrAS65Y this work
pDF18 pT7-5 htrAS66Y this work
pDF19A pT7-5 htrAR44A F50W S210A this work
pDF20A pT7-5 htrA C57S Q64C C69S S210A this work
pDF21A pT7-5 htrA S210A L229C this work
pDF22A pT7-5 htrA this work
S54C C57S C69S S210A R325C
pA1wt pT7-5 htrA F49Y F50Y this work
pA1 pT7-5 htrA F49Y F50Y S210A this work
pA14wt pT7-5 htrA S65A S66A this work
pMD2 pT7-5 htrA R44A S210A this work
pMD2wt pT7-5 htrA R44A this work
Low copy number
pGB2 general cloning vector (6)
pJS5 pGB2 the wild-type htrA (5)
pJS6 pGB2 htrA S210A (5)
pJS7 pGB2 the wild-type htrA (7)
pDF23 pGB2 htrA R44A this work
pDF24 pGB2 htrA F68Y this work
pDF25 pGB2 htrA F49Y F50Y this work

Table S2. Primers used to construct the htrA gene mutants.

Mutation / Oligonucleotide sequence (5’-3’)
P43G / GCGTATGGGCCGTAATTTCCAGCAGTTCTTCGG
CCGAAGAACTGCTGGAAATTACGGCCCATACGC
R44A / CCGTTAATACGCCGCGTATGCCGGCGAATTTCCAGCAG
CTGCTGGAAATTCGCCGGCATACGCGGCGTATTAAGG
R44D / GCGTATGCCGGATAATTTCCAGCAGTTCTTCGG
CCGAAGAACTGCTGGAAATTATCCGGCATACGC
R44L / CCGTTAATACGCCGCGTATGCCGCTGAATTTCCAGCAG
CTGCTGGAAATTCAGCGGCATACGCGGCGTATTAACGG
R44P / CGTATGCCGCCGAATTTCCAGCAGTTCTTCGG
CCGAAGAACTGCTGGAAATTCGGCGGCATACGC
F46Y / CGTATGCCGCGTAATTACCAGCAGTTCTTCGG
CCGAAGAACTGCTGGTAATTACGCGGCATACG
Q47L / CGTATGCCGCGTAATTTCCTGCAGTTCTTCGG
CCGAAGAACTGCAGGAAATTACGCGGCATACG
F49YF50Y / GCGTAATTTCCAGCAGTACTACGGTGATGATTCTCC
GGAGAATCATCACCGTAGTACTGCTGGAAATTACGC
F50W / CGTAATTTCCAGCAGTTCTGGGGTGATGATTCTCCG
CGGAGAATCATCACCCCAGAACTGCTGGAAATTACG
D52A / CCAGCAGTTCTTCGGTGCGGATTCTCCGTTCTGC
GCAGAACGGAGAATCCGCACCGAAGAACTGCTGG
D53A / GCAGTTCTTCGGTGATGCGTCTCCGTTCTGC
GCAGAACGGAGACGCATCACCGAAGAACTGC
S54A / GCAGTTCTTCGGTGATGATGCGCCGTTCTGCCAGG
CCTGGCAGAACGGCGCATCATCACCGAAGAACTGC
S54CR325C / GCAGTTCTTCGGTGATGATTGCCCGTTCTCCCAGG
CCTGGGAGAACGGGCAATCATCACCGAAGAACTGC
GCTTTGCCGCACTGTGCGCTCAGGTGGGTACTATGC
GCATAGTACCCACCTGAGCGCACAGTGCGGCAAAGC
F56S / GGTGATGATTCTCCGAGCTGCCAGGAAGGTTCTCC
GGAGAACCTTCCTGGCAGCTCGGAGAATCATCACC
E59V / CCGTTCTGCCAGGTGGGTTCTCCGTTCCAGAGC
GCTCTGGAACGGAGAACCCACCTGGCAGAACGG
F63Y / GGAAGGTTCTCCGTACCAGAGCTCTCCGTTCTGC
GCAGAACGGAGAGCTCTGGTACGGAGAACCTTCC
Q64A / GGAAGGTTCTCCGTTCGCGAGCTCTCCGTTCTGC
GCAGAACGGAGAGCTCGCGAACGGAGAACCTTCC
Q64C / GGTTCTCCGTTCTGCAGCTCTCCGTTCTCCCAGG
CCTGGGAGAACGGAGAGCTGCAGAACGGAGAACC
Q64I / GGAAGGTTCTCCGTTCATCAGCTCTCCGTTCTGCCAGG
CCTGGCAGAACGGAGAGCTGATGAACGGAGAACCTTCC
S65Y / GGTTCTCCGTTCCAGTACTCTCCGTTCTGCCAGG
CCTGGCAGAACGGAGAGTACTGGAACGGAGAACC
S65AS66A / GGTTCTCCGTTCCAGGCCGCTCCGTTC TGCCAGGGTGG
CCACCCTGGCAGAACGGAGCGGCCTG GAACGGAGAACC
S66Y / GGTTCTCCGTTCCAGAGCTACCCGTTCTGCCAGG
CCTGGCAGAACGGGTAGCTCTGGAACGGAGAACC
F68Y / CCAGAGCTCTCCGTACTGCCAGGGTGGCCAGG
CCTGGCCACCCTGGCAGTACGGAGAGCTCTGG
Q70A / GCTCTCCGTTCTGCGCGGGTGGCCAGGGCGGTAATGG
CCATTACCGCCCTGGCCACCCGCGCAGAACGGAGAGC
L229C / GGTATCAACACCGCGATCTGCGCACCGGACG
CGTCCGGTGCGCAGATCGC GGTGTTGATACC

SUPPLEMENTAL REFERENCES

1. Lipinska, B., Fayet, O., Baird, L. and Georgopoulos, C. (1989) Identification, characterization and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures. J. Bacteriol. 171, 574-584

2. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580

3. Tabor, S. and Richardson, C. C. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078

4. Russel, M. and Model, P. (1984) Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J. Bacteriol.159, 1034-1039

5. Skorko-Glonek, J., Wawrzynow, A., Krzewski K., Kurpierz, K. and Lipinska, B. (1995) Site-directed mutagenesis of the HtrA (DegP) serine protease, whose proteolytic activity is indispensable for Escherichia coli survival at elevated temperatures. Gene163, 47-52

6. Churchward, G. Belin, D. and Nagamine, Y. (1984) A pSC01-derived plasmid which shows no sequence homology to other commonly used vectors, Gene, 31, 165-171

7. Skorko-Glonek, J., Zurawa, D., Tanfani, F., Scire, A., Wawrzynow, A., Narkiewicz, J., Bertoli, E. and Lipinska, B. (2003)The N-terminal region of HtrA heat shock protease from Escherichia coli is essential for stabilization of HtrA primary structure and maintaining of its oligomeric structure.Biochim. Biophys. Acta1649, 171-182