Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family)

Abstract

The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nicoperon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions in Escherichia coli, since its overexpression in transnegatively interferes with mini-RA3 replication and stable maintenance of RA3.

FIG 1

Circular map of plasmid RA3 All identified ORFs are represented by solid arrows indicating the direction of transcription. Functional gene clusters of the plasmid backbone are highlighted with different colors. Experimentally confirmed promoters in the backbone modules are marked with thin gray arrows. The locations of three KorB-binding sites, the IR-SnaBI motifs, are indicated with green arrows.

FIG 2

Regulation of RA3 promoters by KorB. (A) Schematic presentation of promoter regions potentially regulated by KorB. The −35 and −10 motifs are boxed. Predicted transcription start sites (TSS) are indicated by thin black arrows. KorB operators (IR-SnaBI motifs) and other defined binding sites, O_M for MobC and O_C for KorC, are also marked. The RA3 parS sequence as defined previously (18) is indicated with a dashed line, while the nick site (nic) in the oriT (10) is indicated by a triangle. The scheme is not drawn to scale, but RA3 nucleotide sequence coordinates are given at both ends of fragments, at the nick site, at the start of −35 motifs, and in the centers of palindromic regulatory sequences. (B) Transcriptional activities of RA3 DNA fragments cloned into promoter-probe vector. Expression of the reporter gene xylE was analyzed by measuring the catechol 2,3-oxygenase activity in cell extracts from exponentially growing cultures of DH5α(pPDB11.18) (orf02p-xylE), DH5α(pMWB6.6) (orf02prev-xylE), DH5α(pJSB7.9) (mobCp-xylE), and DH5α(pAKB4.90) (orf23p-xylE) transformants. Mean values with standard deviations for at least three assays are shown. (C) Activities of promoters in the presence of KorB. DH5α strains with promoter-probe vectors as in panel B, harboring DNA fragments as indicated on the left, were transformed with empty expression vector pGBT30 (control) or with its derivative pMMB2.50 tacp-korB. XylE activity was assayed in extracts from exponentially growing cultures and is shown as the proportion between control (taken as 100%, gray bars) and korB-expressing (black bars) strains. Mean values with standard deviations for at least three assays are shown. A parametric t test performed for the data before normalization showed that all the differences were statistically significant, with P values in a range from 0.00001 to 0.03.

FIG 3

Regulatory effect of KorB_RA3 on orf02p and orf23p with modified IR-SnaBI motifs. (A) Activity of orf02p IR-SnaBI* (disrupted by a 12-bp insertion) in the presence of KorB in trans. Promoter activity was measured in E. coli DH5α(pAKB4.20 orf02p IR-SnaBI*-xylE)(pMMB2.50 tacp-korB) and E. coli DH5α(pAKB4.20 orf02p IR-SnaBI*-xylE)(pGBT30) as a control, grown in the presence of 0.5 mM IPTG (dark gray bars) or without IPTG added (light gray bars). The results are presented relative to the control; mean values with standard deviations for at least three assays are shown. A parametric t test performed for the data obtained from IPTG-induced variants yielded a P value of 0.01. (B) Activity of orf23p IR-SnaBI* (disrupted by a 12-bp insertion) in the presence of KorB in trans. Promoter activity was measured in E. coli DH5α(pAKB4.81 orf23p IR-SnaBI*-xylE)(pMMB2.50 tacp-korB) and E. coli DH5α(pAKB4.81 orf23p IR-SnaBI*-xylE)(pGBT30) as a control, grown in the presence of 0.5 mM IPTG (dark gray bars) or without IPTG added (light gray bars). The results are shown relative to the control; mean values with standard deviations for at least three assays are shown. A parametric t test performed for the data obtained from IPTG-induced variants yielded a P value of 0.01. (C) Activity of orf23pΔIR-SnaBI with deletion of one arm of IR-SnaBI in the presence of KorB in trans. Promoter activity was measured in E. coli DH5α(pAKB4.82 orf23pΔIR-SnaBI-xylE)(pMMB2.50 tacp-korB) and E. coli DH5α(pAKB4.82 orf23pΔIR-SnaBI-xylE)(pGBT30) as a control, grown in the presence of 0.5 mM IPTG (dark gray bars) or without IPTG added (light gray bars). The results are shown relative to the control; mean values with standard deviations for at least three assays are shown.

FIG 4

Role of IncC_RA3 and Orf11_RA3 proteins in KorB_RA3-dependent repression. (A) E. coli DH5α(pPDB11.18 orf02p-xylE) was transformed with two compatible expression vectors, pAMB9.37 and pGBT30, or their derivatives overproducing KorB (pAKB5.50), IncC (pAKB2.40), or Orf11 (pMMB2.60). orf02p activity measured by the XylE level in the presence of one or two overexpressed proteins produced in trans from the expression vectors is shown relative to the activity in DH5α(pPDB11.18)(pAMB9.37)(pGBT30). Mean values with standard deviations for at least three experiments are presented. The data obtained for KorB versus KorB/IncC or KorB/Orf11 were analyzed statistically by a parametric t test, yielding P values of 0.00009 and 0.0024, respectively. (B) E. coli DH5α(pJSB7.9 mobCp-xylE) was transformed with two compatible expression vectors, pAMB9.37 and pGBT30, or their derivatives overproducing KorB (pAKB5.50), IncC (pAKB2.40), or Orf11 (pMMB2.60). mobCp activity measured by XylE level in the presence of one or two overexpressed proteins produced in trans from the expression vectors is shown relative to the activity in the control strain DH5α(pJSB7.9)(pAMB9.37)(pGBT30). Mean values with standard deviations for at least three experiments are presented. The data obtained for KorB versus KorB/IncC or KorB/Orf11 were analyzed statistically by a parametric t test, yielding P values of 0.0009 and 0.0026, respectively.

FIG 5

Analysis of KorB_RA3 spreading. (A) Transcriptional silencing by WT KorB_RA3. E. coli DH5α strains carrying pGB2 derivative pAKB8.5I (parS/mobCp), pAKB8.10 (orf02p), or pAKB8.81 (O_B+1.9 kb) were transformed with empty vector pGBT30 as a control (c) or its derivative pMMB2.50 tacp-korB (KorB). To estimate the number of transformants, 0.1 ml of undiluted as well as serially diluted transformation mixture was grown on double-selection plates without (gray bars, −) or with (black bars, +) 0.5 mM IPTG. The DNA fragments cloned in pGB2 derivatives present in the analyzed strains are indicated above the graph. Mean numbers of transformants from at least three experiments are shown on a semilogarithmic scale; <1 indicates that no transformants were obtained on double-selection plates with 0.5 mM IPTG after plating 0.8 ml of the undiluted transformation mixture. (B) Transcriptional silencing by the KorBA111T variant. E. coli DH5α(pAKB8.5I parS/mobCp) was transformed with pGBT30 (c) or pMMM2.50 tacp-korB (KorB) as negative and positive controls or with pJSB5.67 tacp-korB111. Transformation mixtures were plated on double-selection plates without (gray bars, −) or with (black bars, +) 0.5 mM IPTG. The DNA fragment cloned in the pGB2 derivative present in the analyzed strain is indicated above the graph. Mean numbers of transformants from at least three experiments are shown on a semilogarithmic scale; <1 indicates that no transformants were obtained on double-selection plates with 0.5 mM IPTG after plating 0.8 ml of the transformation mixture. (C) Overproduction of repressor proteins in the competent cells. SDS-PAGE analysis of soluble fractions of extracts from 0.5 mM IPTG-induced cultures of E. coli DH5α(pAKB8.10 orf02p)(pJSB4.7 tacp-korC) (lane 1), E. coli DH5α(pAKB8.10 orf02p)(pAMB9.37 lacI^q, tacp) (lane 2), and E. coli DH5α(pAKB8.5I parS/mobCp)(pJSB4.1 tacp-mobC) (lane 3). Proteins were stained with Coomassie brilliant blue, and bands corresponding to overproduced proteins, MobC (20 kDa) and KorC (10 kDa migrating slightly slower), are indicated with arrows. Lane 4 contains the protein markers (GPB 260-kDa prestained multicolor protein marker). (D) Transcriptional silencing by KorB in the presence of potential “roadblocks.” Competent cells of E. coli DH5α(pAKB8.10 orf02p)(pJSB4.7 tacp-korC) and E. coli DH5α(pAKB8.5I parS/mobCp)(pJSB4.1 tacp-mobC) were prepared from cultures grown in the presence of 0.5 mM IPTG to overproduce KorC and MobC, respectively, as shown in panel C. The cells were then transformed with pGBT30 (c) or pMMB2.50 (tacp-korB) (KorB), and dilutions of transformation mixtures were plated on triple-selection plates without (gray bars, −) or with (black bars, +) 0.5 mM IPTG. The DNA fragments cloned in pGB2 derivatives and overproduced proteins in the analyzed strains are indicated above the graph. Mean numbers of transformants from at least three experiments are shown on a semilogarithmic scale; <1 indicates that no transformants were obtained on triple-selection plates with 0.5 mM IPTG after plating 0.8 ml of the transformation mixture.

FIG 6

Properties of the spreading-deficient variant KorBA111T. (A) Oligomeric state of WT KorB_RA3 and mutant KorBA111T analyzed by SEC-MALS. The diagram presents elution profiles at 280 nm of WT KorB (upper panel) and mutant KorBA111T (lower panel). Both proteins elute at an apparent molecular mass of 100 kDa. The molecular mass of His₆-KorB monomer is 50 kDa as estimated by mass spectrometry. (B) Analysis of in vivo interaction of KorBA111T with WT KorB in the BACTH system. E. coli BTH101 cyaA was cotransformed with pJSB8.67 cyaT18-korB111 and pKT25 (empty vector) as a control or pAKB15.50 cyaT25-korB. The results obtained for self-interactions of WT KorB (pAKB15.50 and pAKB16.50 cyaT18-korB) are included. The transformants were plated on double-selection MacConkey agar plates supplemented with 1% maltose and 0.1 mM IPTG. Strains were assayed for β-galactosidase activity in liquid overnight cultures. Mean values with standard deviations for at least three experiments are presented. The statistical analysis of the data performed by a parametric t test indicated a significant difference between KorB/KorB and KorB/KorBA111T (P value of 0.00002). (C) DNA-binding activity of WT KorB and KorBA111T. Aliquots of 20 ng of Cy5-labeled DNA probes, nonspecific DNA fragment Cy5-NS (upper panel), and the specific DNA fragment Cy5-O_B (middle panel), were incubated with increasing amounts of KorB or KorBA111T (20, 40, 60, and 70 pmol) in 20 μl of binding buffer (50 mM Tris-HCl [pH 8.0], 10 mM MgCl₂, 50 mM NaCl, 0.2 mg ml⁻¹ BSA) at 37°C for 15 min. A negative control (−) comprised all reaction components without the KorB protein or KorBA111T. Samples were separated on 5% native polyacrylamide gels in 1× TBE buffer. Arrows indicate the position of unbound Cy5-labeled DNA fragments. The gel images were scanned and analyzed with ImageQuant. The bottom panel represents the percentage of free specific Cy5-labeled DNA probe plotted against the protein concentration applied. Gray, KorB; orange, KorBA111T. (D) Repressor activity of KorBA111T toward mobCp. E. coli DH5α(pJSB7.9 mobCp-xylE) was transformed with expression vector pGBT30 (control strain) or with its derivative pJSB5.67 tacp-korB111. XylE activity in the presence of KorBA111T (orange bar) was assayed in extracts from exponentially growing cultures and is shown relative to the control (dark gray bar) taken as 100%. Results obtained for WT KorB_RA3 are included for comparison (light gray bar). Mean values with standard deviations for at least three assays are shown. A parametric t test performed for the data before normalization indicated a significant difference between KorB and KorBA111T (P value of 0.009).

FIG 7

Effect of KorB overproduction on RA3 plasmid maintenance, replication, and conjugation frequency. (A) RA3 stability assay. E. coli DH5α(RA3)(pGBT30) and DH5α(RA3)(pMMB2.50 tacp-korB) were grown overnight in L broth with chloramphenicol and penicillin and then diluted in L broth with penicillin and 0.5 mM IPTG. Approximately every 20 generations (gen), the cultures were diluted into fresh medium and analyzed for RA3 retention. Representative values from one of three experiments are shown. (B) Maintenance of RA3 minireplicon in the presence of excess KorB. E. coli DH5α(pJSB18Tc^r) was transformed with pBBR1MCS derivatives (empty expression vector pAMB9.37 tacp or pAKB5.50 tacp-korB), plated on L agar with selection for incoming plasmid only (chloramphenicol) or for both resident and incoming plasmids (tetracycline and chloramphenicol) as indicated above the photographs, and grown at 37°C overnight. (C) Frequency of RA3 plasmid conjugation in the presence of excess KorB. E. coli DH5α(pJSB1.24) was transformed with pAKB5.50 tacp-korB or with empty pAMB9.37 as a control. Double transformants were grown with or without 0.5 mM IPTG and used as donors in conjugation with a DH5α Rif^r strain as the recipient. The frequency of conjugation is indicated on a semilogarithmic scale as the number of transconjugants per donor cell. Mean values with standard deviations for at least three experiments are shown. The significance of the differences between the IPTG-induced control and KorB-expressing variants were evaluated by a parametric t test on log-transformed data, giving a P value of 0.07 (nonsignificant).