Repression at a distance by the global regulator KorB of promiscuous IncP plasmids

Abstract

SummaryKorB protein (358 amino acids) binds to 12 highly conserved sequences on the RK2 genome and co-ordinates the expression of at least five operons encoding genes for stable inheritance and plasmid transfer. KorB represses the trfA, korA and klaA promoters where it binds 4 bp upstream of the ¹35 region (class I KorB operators, O B ). We show here that KorB on its own can also repress the trbA, trbB, kfrA and kleA promoters where O B is between 80 and 189 bp away from the transcription start point (class II operator). A C-terminal deletion of 17 amino acids resulted in the loss of KorB's ability to repress through class II operator but not through class I operator. This deletion reduced multimerization of His 6 -tailed KorB protein in vitro and greatly reduced binding specificity for fragments containing O B sequences. At the trbBp region, where O B 9 lies 189 bp upstream of the transcription start point, mutagenesis of a proposed secondary binding site overlapping the trbBp ¹35 region had no effect on the ability of KorB to repress trbBp. Nevertheless, gel retardation analysis showed that KorB binding is promoted by sequences upstream and downstream of O B 9 and that KorB can form higher order complexes on DNA. However, DNase I footprinting suggested that RNA polymerase may interact directly with KorB bound at O B 9 and implied that contacts between these proteins could be responsible for the action of KorB at a distance.

ParB ofPseudomonas aeruginosa: Interactions with Its Partner ParA and Its TargetparSand Specific Effects on Bacterial Growth

“…This dimerization function was localized to the C-terminal domain, as found in plasmid-encoded ParB proteins. This re- (21), whose crystal structure was recently reported (8). In particular, the leucines or isoleucines and acidic or basic residues, which form a vital part of the dimerization interface, show strong conservation.…”

“…KorB RK2 (IncP1␣) and KorB from R751 (IncP1␤) that the central DNA-binding domain and the C-terminal dimerization domain are separated by a variable linker region (21,34). The dimeric nature of the C-terminal 62 aa of KorB RK2 was also confirmed by crystallographic studies (8).…”

Cooperativity between KorB and TrbA Repressors of Broad-Host-Range Plasmid RK2

“…The identified KorB binding site in the trbBp region, O B 9, lies 189 bp upstream of the trbBp transcription start point, and binding of purified KorB at this site has been explored in vitro (8). In thinking about KorB-TrbA cooperativity it was important to explore the constraints on the KorB action on trbBp in vivo.…”

“…For pMZT37, the BamHI trbBp fragment from pGBT178 was subcloned into the BamHI site of pPTO1. pGBT178 carries the same fragment as pGBT78 (8), with transposon insertion 104 in position 18594 of RK2 (20,21) cloned into pGBT100. For pMZT38, the BamHI trbBp fragment from pGBT⌬66/78 was subcloned into the BamHI site of pPTO1.…”

“…2) (24), and KorB is a strong repressor of trbBp (22). It has been proposed that KorB may cause looping between O B 9 and a possible degenerate operator overlapping the trbBp Ϫ35 region (22), but this was excluded as a necessary element for KorB repression (8). This raises the possibility that KorB bound at a distance from the promoter makes direct contact with RNA polymerase through the creation of a DNA loop.…”