Conserved C-terminal region of global repressor KorA of broad-host-range plasmid RK2 is required for co-operativity between KorA and a second RK2 global regulator, KorB.

KorA and KorB proteins of IncP1 plasmid RK2 are encoded in the central control region (ccr) of the plasmid and act as global regulators of plasmid genes for replication, transfer and stable inheritance. KorA represses seven promoters on RK2, by binding to a defined operator site, OA, which always occurs in promoter regions. KorB recognises another operator, OB, which is found 12 times on the RK2 genome, but not always in promoter regions. At five of the KorA-regulated promoters, an OBsequence is also present. The presence of both KorA and KorB leads to severely decreased promoter activity. By measuring repression at different levels of KorA and KorB alone and in combination, we showed that there is at least 3. 4-fold co-operativity between them at korApin vivo. Testing the ability of previously isolated KorA mutants to act in a co-operative way in the presence of KorB in vivo or in vitro showed that the C-terminal part of KorA between amino acid positions 68 and 83 is required for this co-operativity. This region is part of a segment that is highly conserved between KorA and two other RK2 proteins, TrbA and KlcB. We propose that this conserved region may provide the basis for co-operativity with KorB either indirectly, by modulating DNA structure near the KorB binding site, or directly by serving as the "recognition" patch of each protein by KorB. It may thus serve as a key domain in allowing a sensitive response of the global circuits to changes in repressor concentration and thus modulation of replication, transfer and maintenance.     

The hierarchy of KorB binding at its 12 binding sites on the broad-host-range plasmid RK2 and modulation of this binding by IncC1 protein

IncC and KorB proteins of broad-host-range plasmid RK2 are members of the ParA-ParB families of proteins needed for stable partitioning of bacterial chromosomes and plasmids. KorB also functions as a global regulator of expression of RK2 genes. It recognises and binds to a palindromic operator, O(B), found 12 times on RK2 DNA (O(B)1-O(B)12). We performed detailed studies on the binding of KorB to the 12 operators and showed that they fall into three groups (A, B, C) based on the binding strength of KorB. The highest affinity site is O(B)10, which occurs in the promoter transcribing genes for replication, trfAp. Purified IncC1 potentiated KorB binding to all O(B) sites except O(B)3, a site involved in partitioning. Using O(B)10 as a test system, we showed that IncC1 increases the stability of the KorB-DNA complex. The 5 bp sequences flanking the 13mer O(B) site were found to affect KorB binding and IncC1 potentiation activity. Study of hybrid operators indicated that flanking sequences on one side only were sufficient to specify the difference between O(B)10 and O(B)3. Replacement of adenine by guanine at positions -8 and -10 from the O(B)10 centre of symmetry was needed to convert it from the highest-affinity group (A) to the medium-affinity group (B) on the basis of KorB binding. These changes also eliminated potentiation by IncC1. The -8 and -10 positions from the centre of O(B)3 symmetry are occupied by guanines and this may provide part of the specificity of IncC1 behaviour on KorB binding. Studies on a series of synthetic operators suggested that KorB contacts O(B) flanking sequences, and that IncC1 may alter the conformation of multimeric KorB so that it is better able to make these contacts, thus stabilising the complexes once formed.     

Distribution of the partitioning protein KorB on the genome of IncP-1 plasmid RK2

The ParB family partitioning protein, KorB, of plasmid RK2 is central to a regulatory network coordinating replication, maintenance and transfer genes. Previous immunofluorescence microscopy indicated that the majority of KorB is localized in plasmid foci. The 12 identified KorB binding sites on RK2 are differentiated by: position relative to promoters; binding strength; and cooperativity with other repressors and so the distribution of KorB may be sequestered around a sub-set of sites. However, chromatin immunoprecipitation analysis showed that while RK2 DNA molecules appear to sequester KorB to create a higher local concentration, cooperativity between DNA binding proteins does not result in major differences in binding site occupancy. Thus under steady state conditions all operators are close to fully occupied and this correlates with gene expression on the plasmid being highly repressed.     

Flexibility of KorA,a plasmid-encoded,global transcription regulator,in the presence and the absence of its operator

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.     

Effect of growth rate and incC mutation on symmetric plasmid distribution by the IncP-1 partitioning apparatus

The incC and korB genes of IncP-1 plasmid RK2 encode homologues of ubiquitous ParA and ParB partitioning proteins of bacterial plasmids and chromosomes. Using immunofluorescence microscopy, we found that KorB, which binds to 12 widely distributed sites on the genome, is located in symmetrically placed foci in cells containing IncP-1 plasmids. When maintained by the low-copy-number P7 replicon, an RK2 segment including incC, korB and the kla, kle and korC regions encodes an efficient partitioning system that gives a pattern of foci similar to RK2 itself. Symmetrical distribution of KorB foci correlates with segregational stability conferred by either the IncP-1 or P7 partitioning systems; KorB distribution follows plasmid distribution. In the absence of a second partitioning system, incC inactivation resulted in paired or clumped foci that were not symmetrically distributed. At a slow growth rate, position analysis of foci showed a cycle from one central focus to two foci (at one- and three-quarter positions) and back, and at a high growth rate it showed a cycle from two foci to four and back. This pattern fits with the plasmid being coupled to the replication zones in the cell and being moved to successively younger zones by active partitioning, indicating a tight association between replication and partitioning.     

Mutations that affect regulation of the korB gene of Streptomyces lividans plasmid plJ101 alter plasmid transmission

Using the catechol dehydrogenase gene as a reporter, we isolated random mutations in the plJ101 korB gene operator/promoter (OP) region that affect korB expression and regulation. We mapped these mutations to inverted repeat sequences within the promoter and studied their effects on binding of the KorB repressor protein to the OP, on expression of the korB gene, and on plasmid transmission during mating. Additionally, we investigated the biological effects of KorB binding to a locus (sti, for st rong I ncompatibility) adjacent to the korB OP and implicated in plJ101 replication. Our results identify sites that influence the synthesis and autoregulation of KorB; they also show that interaction of KorB with sti affects repression of korB and transmission of plasmids to spores of recipients.     

A small protein-protein interaction domain common to KlcB and global regulators KorA and TrbA of promiscuous IncP plasmids.

The kor regulon of broad host-range, incompatibility group P (IncP) plasmids uses the KorA, KorB, and KorC repressors to regulate expression of genes for replication, conjugation, segregation, and host range. One operon, kilC, encodes the KorC repressor and two genes of unknown function (klcA and klcB). The predicted sequences of the 51.1 kDa KlcB protein, the 11.3 kDa KorA repressor, and another small (13.5 kDa) regulatory protein, TrbA, show a highly related 35 amino acid residue segment (V-L-P domain). We found that induction of the klcB gene is toxic to Escherichia coli host cells harboring an IncP plasmid. We confirmed a model in which the V-L-P domain of KlcB interacts directly with the V-L-P domain of KorA to derepress KorA-regulated operons, thereby allowing expression of toxic genes. First, a lacZ reporter fused to the kleA promoter, which is regulated by KorA and KorC, revealed that klcB induction specifically releases KorA-repression but has no effect on KorC repression. Second, induced expression of the V-L-P domains from KorA or KlcB is sufficient to release KorA-repression at the kleA promoter. Third, purified GST-KlcB fusion protein interacts specifically with His-tagged KorA. Fourth, fusion of the V-L-P domains of KorA and TrbA and full-length KlcB polypeptide to the DNA-binding domain of bacteriophage lambda repressor leads to the formation of functional, dimeric repressors, and mutations that alter conserved residues of the V-L-P domain adversely affect dimerization. Fifth, crosslinking experiments demonstrated that the V-L-P domain of KorA is able to dimerize in solution and form heterodimers in mixtures with full-length KorA polypeptide. These findings show that the V-L-P domain is a protein-protein interaction module that is likely to be responsible for dimerization of KorA and TrbA, and important for KlcB dimerization. We speculate on the possible significance of KlcB-KorA heterodimers in IncP plasmid maintenance.     

Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA.

Plasmid RK2 encodes several kil determinants whose lethal action on Escherichia coli host cells is prevented by RK2 kor genes. Here we show that the mini-RK2 plasmid, pRK248, specifies a kilB component (kilB1) in the region of the replication gene trfA. kilB1 is different from trfA and is completely encoded within the pRK248 HaeII A fragment. Transformation of E. coli cells with hybrid plasmids containing the cloned kilB1 determinant is very inefficient and results in the selection of variant kil- plasmids, many of which show genetic and physical evidence of deletions. If another pRK248 gene (korB1) is present in the cells, kilB1+ plasmids can be established at high efficiency and without any detectable changes. KorB1 is encoded by the trfB region of pRK248 because recombinant plasmids with this region are able to control kilB1 in trans. These results substantiate our earlier explanation for the structure of pRK248 and for the perplexing requirement of the trfB region in this plasmid.     

Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB

The KorB protein of the broad-host-range plasmid RP4 acts as a multifunctional regulator of plasmid housekeeping genes, including those responsible for replication, maintenance and conjugation. Additionally, KorB functions as the ParB analog of the plasmid's partitioning system. The protein structure consists of eight helices, two of which belong to a predicted helix-turn-helix motif. Each half-site of the palindromic operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based mainly on two side chain interactions outside the helix-turn-helix motif with two bases next to the central base pair of the 13-base pair operator sequence. The surface of the KorB DNA-binding domain mirrors the overall acidity of KorB, whereas DNA binding occurs via a basic interaction surface. We present a model of KorB, including the structure of its dimerization domain, and discuss its interactions with the highly basic ParA homolog IncC.     

RP4 repressor protein KorB binds to the major groove of the operator DNA: a Raman study

KorB is a member of the ParB family of bacterial partitioning proteins. The protein encoded by the conjugative plasmid RP4 is part of the global control circuit and regulates the expression of plasmid genes, the products of which are involved in replication, transfer, and stable inheritance. KorB is a homodimeric protein which binds to palindromic 13 bp DNA sequences [5'-TTTAGC((G)/(C))GCTAAA-3'] present 12 times in the 60 kb plasmid. Each KorB subunit is composed of two domains; the C-domain is responsible for the dimerization of the protein, whereas the N-terminal domain recognizes and binds to the operator sequence (O(B)). Here we describe results of a Raman spectroscopic study of the interaction of the N-domain with a double-stranded model oligonucleotide composed of the palindromic binding sequence and terminal 5'-A(Br)U and AG-3' bases. Comparison of the Raman spectra of the free KorB N-domain and O(B) DNA with the spectrum of the complex reveals large differences. KorB-N binds in the major groove of the O(B) DNA, and the interactions induce changes in the DNA backbone and in the secondary structure of the protein.     

Yeast alpha 2 repressor positions nucleosomes in TRP1/ARS1 chromatin.

The yeast alpha 2 repressor suppresses expression of a-mating-type-specific genes in haploid alpha and diploid a/alpha cell types. We inserted the alpha 2-binding site into the multicopy TRP1/ARS1 yeast plasmid and examined the effects of alpha 2 on the chromatin structure of the derivative plasmids in alpha cells, and a/alpha cells. Whereas no effect on nucleosome position was observed in a cells, nucleosomes were precisely and stably positioned over sequences flanking the alpha 2 operator in alpha and a/alpha cells. In addition, when the alpha 2 operator was located upstream of the TRP1 gene, an extended array of positioned nucleosomes was formed in alpha cells and a/alpha cells, with formation of a nucleosome not present in a cells, and TRP1 mRNA production was substantially reduced. These data indicate that alpha 2 causes a positioning of nucleosomes over sequences proximal to its operator in TRP1/ARS1 chromatin and suggest that changes in chromatin structure may be related to alpha 2 repression of cell-type-specific genes.