Table 1.
Bacterial strains and plasmids used in this study.
Figure 1.
Localization of the chr promoter within the tnpR-chrB intergenetic region using gfp transcriptional fusions.
A) PCR fragments containing the promoter portions (indicated by the boxes) were cloned upstream of a promoterless gfp on pProbe-NT. Sequences are numbered relative to the first nucleotide of the chrB start codon. Primer pairs used in this work are indicated. B) Green fluorescence of E. coli cells harboring the different constructs. The values represent averages and standard deviations of three replicates.
Figure 2.
Mutagenesis analysis of the putative protein binding site within chr promoter region.
A) Imperfect inverted repeat of the original and mutated sequence. The mutated nucleotides are shown in bold. B) Fluorescence of E. coli carrying the mutated and no-mutated plasmid constructs. Fluorescence was measured after 3 h of growth in medium without chromate (black bars), with 1 µM (grey bars) and 10 µM (white bars) of chromate. The values represent averages and standard deviations of three replicates.
Figure 3.
Study of the functionality of ChrB (total or partial) using reporter plasmids.
A) Total or partial chrB (coding for N-terminal or C-terminal ChrB), with their promoter sequence, were cloned upstream of gfp gene in pProbe-NT (Table1). Primer pairs used in this work are indicated. B) Fluorescence of E. coli carrying the different plasmids constructs; pChrBGFPn (white bars), pChrBGFPc (grey bars) and pChrBGFP (dashed bars). Fluorescence was measured after 3 h of growth in medium without chromate and with increasing chromate concentrations. The values represent averages and standard deviations of three replicates.
Figure 4.
Purification of the ChrB protein.
A) ChrB was overexpressed with a carboxy-terminal 6-histidine tag in E. coli BL21 (DE3). Lane 1, whole-cell extract from uninduced cells containing pET30a expressing ChrB-His6; lane 2, whole-cell extract from induced cells containing pET30a expressing ChrB-His6; lane 3, purified ChrB-His6 by Ni2+ nitrilotriacetic acid affinity chromatography. B) ChrB was overexpressed without any tag in E. coli BL21 (DE3). Lane 1, whole-cell extract from uninduced cells containing pET30a expressing ChrB; lane 2, whole-cell extract from induced cells containing pET30a expressing ChrB; lane 3, purified ChrB by DEAE chromatography; lane 4, purified ChrB by Sepharose chromatography; lane 5, purified ChrB by resource chromatography.
Figure 5.
A) SDS PAGE of purified ChrB in glutaraldehyde crosslinking assays: lane 1, ChrB; lane 2, ChrB+glutaraldehyde (2 min); lane 3, ChrB+glutaraldehyde (5 min); lane 4, ChrB+glutaraldehyde (10 min); lane 5, ChrB+glutaraldehyde (15 min); lane 6, ChrB+glutaraldehyde (30 min); lane 7, ChrB+glutaraldehyde (1 hour). B) Immunoblot of purified ChrB-His6 in glutaraldehyde crosslinking assays: lane 1, ChrB-His6; lane 2, ChrB-His6+ glutaraldehyde; lane 3, ChrB-His6+ target chr promoter DNA+glutaraldehyde; lane 4, ChrB-His6+ glutaraldehyde+Cr(VI).
Figure 6.
A) Band shift assays were performed by incubating the DNA fragment (chr promoter) with increasing concentration of ChrB protein. Lane 1, competitor protein (EBNA extract from Pierce); lane 2, 0 µM ChrB; lane 3, 1 µM ChrB; lane 4, 3 µM ChrB; lane 5, 10 µM ChrB; lane 6, 30 µM ChrB. B) DNA fragment was incubated with ChrB (10 µM) in the absence and in the presence of unlabeled competitor DNA. Lane 1, without protein; lane 2; 0 µg/µl; lane 3, 1 µg/µl; lane 4, 10 µg/µl; lane 5, 50 µg/µl; lane 6, 100 µg/µl; lane 7, 250 µg/µl of competitor DNA. C) EMSA assays with or without chromate. The chr promoter was incubated with ChrB protein (10 µM) and with increasing concentrations of chromate. Lane 1, without protein (control); lane 2, without Cr(VI); lane 3, with 10 µM Cr(VI); lane 4, with 100 µM Cr(VI); lane 5, with 1 mM Cr(VI).
Figure 7.
Alignment of ChrB homologues from different organisms.
Amino acid sequences (obtained from NCBI) were aligned via CLUSTAL W. Ochrobactrum tritici 5bvl1 (ABO70326), Pseudomonas aeruginosa (AEQ93502); Cupriavidus metallidurans CH34, ChrB1 and ChrB2 (ABF13062 and ABF10734, respectively); Herminiimonas arsenicoxydans (CAL61077); Burkholderia pseudomallei 1106b (EES21856); Klebsiella pneumoniae 342 (YP_002238121); Janthinobacterium sp. Marseille (ABR91486); Azoarcus sp. BH72 (CAL95580); Chromobacterium violaceum ATCC 12472 (AAQ58595); Sphingobium japonicum UT26S (BAI96957). Highly conserved residues in ChrB homologues (black shading), similar residues in ChrB homologues (grey shading), conserved residues chosen for mutagenesis approach (arrow) and the putative HTH motif (into the box).
Figure 8.
GFP expression of pChrBGFP and mutants.
A) Green fluorescence of pChrBGFP (ChrB no mutated) and pChrBGFP mutants, in which, ChrB is mutated in R175A; R180A; R182A; R187A; R195A; R196A; C213A, H229A and R258A. B) Green fluorescence of pChrBGFP mutants, in which, ChrB is mutated in R18A; R23A; A241R; G244R and R252A. Fluorescence was measured after 3 h of growth in medium without chromate (white bars), with 1 µM (black bars) and 10 µM (dashed bars) of chromate. The values represent averages and standard deviations of three replicates.
Figure 9.
Fluorescence of E. coli BL21 co-transformed with two different plasmids (indicated in graphic).
Fluorescence was measured after 3(white bars) and with 10 µM (black bars) of chromate. The values represent averages and standard deviations of three replicates.