TABLE 1.
Strain or plasmida | Genotype or characteristics | Source or reference |
---|---|---|
E. coli strains | ||
BL21(DE3) | E. coli B, F−ompT hsdSB(rB− mB−) gal dcm (chromosomal T7 RNA polymerase on λDE3) | Novagen |
BTH101 | F−cya-99 araD139 galE15 galK16 rpsL1 (Strr) hsdR2 mcrA1 mcrB1 | 19 |
SPC105 | MC4100 (ΔlacU169 araD rpsL relA thi flbB) containing a chromosomal PmarII::lacZ fusion at the λ attachment site and a wild-type mar locus | 11 |
Plasmids | ||
Two-hybrid system | ||
pKT25 | IPTG-inducible expression vector encoding the N-terminal T25 fragment (aa 1 to 224) of CyaA in frame with a multiple cloning site; Kmr; ori p15A | 19 |
pUT18 | IPTG-inducible expression vector encoding the C-terminal T18 fragment (aa 225 to 339) of CyaA in frame with multiple cloning site; Ampr; ori colE1 | 18 |
Two-hybrid analysis of MarR interactions | ||
pUT18-marR | pUT18 derivative bearing the full-length marR as T18-ORF fusion | 12 |
pKT25-gyrA | pKT25 derivative bearing the full-length gyrA as T18-ORF fusion | 12 |
pKT25-marR | pKT25 derivative bearing the full-length marR as T25-ORF fusion | 12 |
pKT25-marR R77H | pKT25-marR containing the Arg77His mutation of MarR | 12 |
pTK25-marR G116S | pKT25-marR containing the Gly116Ser mutation of MarR | 12 |
pTK25-marR SP34FS | pKT25-marR containing the Ser35Phe/Phe35Ser mutations of MarR | 12 |
pTK25-marR D26N | pKT25-marR containing the Asp26Asn mutation of MarR | 12 |
pTK25-marR G95S | pKT25-marR containing the Gly95Ser mutation of MarR | 12 |
pTK25-marR L135F | pKT25-marR containing the Leu135Phe mutation of MarR | 12 |
Analysis of tktA mutants | ||
pSPOK | Kanamycin resistance derivative of pSPORT1; T7 promoter, lacO lacI | 21 |
pSPOK-gyrA | pSPOK expressing gyrA | From Xiaowen R. Bina |
pDrive | PCR cloning vector | Qiagen |
pEGyr241/223 | pUC18 with inducible external guide sequence 241/223 for GyrA | Gift from Sidney Altman (17) |
pDrive-EGyr241/223 | pDrive containing the inducible external guide sequences 241/223 for GyrA from pEGyr241/223 | This study |
His pulldown | ||
pET13a | Expression cloning vector; Kanr; T7 promoter, lacO lacI | Novagen |
pETmarR | marR cloned in pET13a | 2 |
pET21b | Cloning vector for making C-terminal six-His fusion proteins; Ampr; T7 promoter, lacO lacI | Novagen |
pETgyrA | pET21b containing gyrA | This study |
Gyrase activity assay | ||
pUC19 relaxed | Expression cloning vector; Ampr; relaxed by topoisomerase I of E. coli | New England Biolabs |
The strains used were E. coli K-12, unless noted otherwise in the text.