Article Figures & Data
Figures
- Fig. 1.
Expression of thegntT::lacZ operon fusion in the wild type (NP100) and in a gntR mutant strain (PR201). The strains were growing in LB plus 0.4% of the indicated carbon sources. β-Galactosidase activities were measured in late log growth phase.
- Fig. 2.
Expression of thegntT::lacZ fusion in wild-type andgntR mutant strains in the presence and absence of cAMP. The cells were grown in LB medium with carbon sources as indicated. Symbols: circles, wild type (NP100); squares, gntR mutant (PR201); open and filled symbols, in the absence and presence, respectively, of 5 mM cAMP. β-Galactosidase was measured in the cultures at the indicated OD600 points.
- Fig. 3.
Schematic representation of the gntT promoter region showing the site-directed mutations within the two operator sites and the cAMP-CRP binding site. The binding sites are shown in boldface; the specific mutations are indicated below the wild-type binding sequences. The deletion subclones (PN400, PN401, and PN404) of the gntT promoter region are shown under the line map.
- Fig. 4.
Overexpression and purification of GntR and CRP, demonstrated by SDS-PAGE of the purified His-tagged proteins. Lanes A and F, molecular weight standards; lanes B and E, cell extracts of strains M15(pNP-41) and M15(pNP-52) after IPTG induction; lanes C and D, purified GntR and CRP, respectively.
- Fig. 5.
Electrophoretic mobility of the GntR-operator complexes. Increasing amounts of purified GntR were added to a 456-bpgntT fragment containing the putative regulatory region of the gntT gene in the absence or presence of different concentrations of gluconate. In the presence of GntR, two GntR-operator complexes (R1 and R2) were formed. F, free DNA. All samples contained 8.1 nM gntT DNA and the designated concentrations of GntR and gluconate.
- Fig. 6.
Formation of GntR- and CRP-DNA complexes with cleavedgntT DNA fragment. Digestion of the gntT fragment with different restriction enzymes resulted in following fragments:MaeIII (67, 142, and 240 bp), KpnI (182 and 274 bp), and HinfI (223 and 233 bp). The formation of complexes was tested with GntR (A) and CRP (B). Complexes of the DNA fragment with one GntR molecule, two GntR molecules, and Crp were given the suffixes R, R2, and C, respectively.
- Fig. 7.
Effects of mutations in the internal and external operator sites on the formation of the repressor-operator complex. Addition of different concentrations of purified GntR to the 456-bpgntT fragment gave a mixture containing free DNA (F) and 1:1 (R1) and 2:1 (R2) repressor complexes, respectively. Samples a to i and j to n contained 6 and 2.2 nM gntT DNA, respectively. Samples a to c, d to f, g to i, and j to n contained gntTfragments from strains NP105, NP206, NP1256, and NP220, respectively.
- Fig. 8.
Formation of a ternary and a tertiary complex of GntR, CRP, and gntT operator shown by electrophoretic mobility assay. The gntT promoter fragment was incubated with different amounts of CRP and GntR in the presence of 5 mM cAMP. Besides the free DNA (F), three DNA bands with reduced mobility, FC, FCR1, and FCR2, corresponding to DNA bound to one CRP, one CRP and one GntR, and one CRP and two GntR molecules, respectively, were formed.
Tables
- Table 1.
Bacterial strains, plasmids, and phage used in this study
Strain, plasmid, or phage Relevant genotype Source or reference E. coli strains DH5α lacZΔM15 recA Bethesda Research Laboratories P90C araΔ(pro-lac)thi 41 DPB271 recD::Tn10F− λ− B. Bender HT28 W3110 Δcya::Kanr 19 RH74 Δcya-851 ilv::Tn10 26 RH77 Δcya-851 Δcrp zhd-731::Tn10 26 HT216 RH74 gntR::Kanr This study M15 recA+ uvr+F−mtl gal ara lac (pREP4) 50 NP100 P90C (λRS88gntT::lacZ) 35 PR201 NP100gntR::Kanr This study Plasmids pQE-30 Expression vector; His6 affinity tag 42 pRS551 bla′ lacZ+ (operator fusion vector) 41 pRS552 bla′ lacZ+ (protein fusion vector) 41 pTC229 pTC221 Kanr in StuI This study pNP-41 gntR in pQE30 (gntRoverexpression) This study pNP-52 crp in pQE30 (crp overexpression) This study Phage λRS88 bla′ lacZ imm434ind 41 - Table 2.
Effects of the cya and crpmutations on gntT::lacZ expression
E. coli strain Relevant genotype β-Galactosidase activity (U) with indicated carbon source None Gluconate Glucose −cAMP +cAMPa −cAMP +cAMP −cAMP +cAMP NP100 Wild type 10 10 460 680 0 0 RH112 crp 20 15 105 70 0 0 HT110 cya 30 25 115 620 0 0 RH114 crp cya 0 10 90 60 0 0 RH216 crp gntR 50 60 45 35 50 55 HT216 cya gntR 75 585 55 380 60 290 ↵a Added to a final concentration of 5 mM.
- Table 3.
Effects of mutations in the regulatory region of gntT
E. colistrain β-Galactosidase activity (U) with indicated carbon source(s) None Gluconate Glucose Gluconate + glucose NP100 (wild type) 10 510 0 280 NP102 780 520 100 320 NP103 280 490 60 260 NP104 840 490 100 330 NP105 920 500 90 310 NP106 110 610 40 300 PN206 1,320 560 190 190 PN211 60 80 40 40 PN208 1,260 460 170 210 PN212 1,180 480 160 190 PN220 0 130 60 60 PN221 0 110 50 70 PN1256 1,460 640 190 280 PN400 0 590 0 290 PN401 10 10 0 0 PN404 0 140 40 60
