Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins.

BACKGROUND: DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. RESULTS: This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1ß and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as "threads" by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. CONCLUSION: KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Global transcriptional regulator KorC coordinates expression of three backbone modules of the broad-host-range RA3 plasmid from IncU incompatibility group.

The broad-host-range conjugative RA3 plasmid from IncU incompatibility group has been isolated from the fish pathogen Aeromonas hydrophila. DNA sequencing has revealed a mosaic modular structure of RA3 with the stabilization module showing some similarity to IncP-1 genes and the conjugative transfer module highly similar to that from PromA plasmids. The integrity of the mosaic plasmid genome seems to be specified by its regulatory network. In this paper the transcriptional regulator KorC was analyzed. KorCRA3 (98 amino acids) is encoded in the stabilization region and represses four strong promoters by binding to a conserved palindrome sequence, designated OC on the basis of homology to the KorC operator sequences in IncP-1 plasmids. Two of the KorCRA3-regulated promoters precede the first two cistrons in the stabilization module, one fires towards replication module, remaining one controls a tricistronic operon, whose products are involved in the conjugative transfer process. Despite the similarity between the binding sites in IncU and IncP-1 plasmids, no cross-reactivity between their KorC proteins has been detected. KorC emerges as a global regulator of RA3, coordinating all its backbone functions: replication, stable maintenance and conjugative transfer.

Identification of C-terminal hydrophobic residues important for dimerization and all known functions of ParB of Pseudomonas aeruginosa.

The ParB protein of Pseudomonas aeruginosa is important for growth, cell division, nucleoid segregation and different types of motility. To further understand its function we have demonstrated a vital role of the hydrophobic residues in the C terminus of ParB(P.a.). By in silico modelling of the C-terminal domain (amino acids 242-290) the hydrophobic residues L282, V285 and I289 (but not L286) are engaged in leucine-zipper-like structure formation, whereas the charged residues R290 and Q266 are implicated in forming a salt bridge involved in protein stabilization. Five parB mutant alleles were constructed and their functionality was defined in vivo and in vitro. In agreement with model predictions, the substitution L286A had no effect on mutant protein activities. Two ParBs with single substitutions L282A or V285A and deletions of two or seven C-terminal amino acids were impaired in both dimerization and DNA binding and were not able to silence genes adjacent to parS, suggesting that dimerization through the C terminus is a prerequisite for spreading on DNA. The defect in dimerization also correlated with loss of ability to interact with partner protein ParA. Reverse genetics demonstrated that a parB mutant producing ParB lacking the two C-terminal amino acids as well as mutants producing ParB with single substitution L282A or V285A had defects similar to those of a parB null mutant. Thus so far all the properties of ParB seem to depend on dimerization.

Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2.

Active partitioning of low-copy number plasmids requires two proteins belonging to the ParA and ParB families and a cis-acting site which ParB acts upon. Active separation of clusters of plasmid molecules to the defined locations in the cell before cell division ensures stable inheritance of the plasmids. The central control operon of IncP-1 plasmids codes for regulatory proteins involved in the global transcriptional control of operons for vegetative replication, stable maintenance and conjugative transfer. Two of these proteins, IncC and KorB, also play a role in active partitioning, as the ParA and ParB homologues, respectively. Here we describe mapping the regions in KorB responsible for four of its different functions: dimerisation, DNA binding, repression of transcription and interaction with IncC. For DNA binding, amino acids E151 to T218 are essential, while repression depends not only on DNA binding but, additionally, on the adjacent region amino acids T218 to R255. The C-terminus of KorB is the main dimerisation domain but a secondary oligomerisation region is located centrally in the region from amino acid I174 to T218. Using three different methods (potentiation of transcriptional repression, potentiation of DNA binding and activation in the yeast two-hybrid system) we identify this region as also responsible for interactions with IncC. This IncC-KorB contact differs in location from the ParA-ParB/SopA-SopB interactions in P1/F but is similar to these systems in lying close to a masked oligomerisation determinant.

kfrA gene of broad host range plasmid RK2 encodes a novel DNA-binding protein.

The korABF operon of broad host range IncP plasmid RK2 encodes proteins that coordinate expression of many other operons and that aid plasmid stability by providing at least part of a partitioning apparatus. The kfrA gene lies downstream from this operon and its transcription is repressed by all except one of the proteins encoded by this operon (KorA, KorFI, KorFII and KorB). We report here that transcription from the kfrA promoter is autoregulated by the kfrA gene product. We have purified KfrA, which is an acidic polypeptide of 308 amino acid residues, and show that it is a site-specific DNA-binding protein whose operator overlaps the primary kfrA promoter. Deletion analysis suggests that this activity is critically dependent on the N-terminal section of KfrA, which appears to contain an alpha-helix-beta-turn-alpha-helix motif. Circular dichroism confirmed the structural prediction that KfrA is almost entirely alpha-helical. The position of predicted turns suggests that, while amino acid residues 1 to 80 may form a globular domain of four or five helices, residues 80 to 280 of KfrA may adopt an extended coiled-coil domain containing a heptad repeat segment, which is probably responsible for formation of the multimers detected by crosslinking. The possibility that this unusual structure serves a second function, for example in providing a bridge to host structures required for plasmid partitioning, is discussed.

Purification of KorA protein from broad host range plasmid RK2: definition of a hierarchy of KorA operators.

The KorA protein of promiscuous plasmid RK2 is encoded in the central control operon, which coordinates expression of genes for replication, transfer and partitioning. KorA is known to repress transcription from seven promoters on the plasmid genome but at the trfA promoter, for the vegetative replication genes, it also causes derepression of trbAp, the first promoter for the operon whose genes are required primarily for mating pair formation prior to conjugative transfer. We have overproduced and purified KorA (101 amino acid residues). Crosslinking indicates that it exists largely as a dimer in solution. Western blotting with specific antibodies suggests that there are approximately 4000 monomers per cell in exponential phase and about 600 in stationary phase. Footprinting confirmed the expected location of the KorA operator, and indicated that KorA and RNA polymerase can bind simultaneously in promoter regions. Gel retardation experiments with DNA fragments carrying each of the seven KorA operators revealed that there is a hierarchy of binding affinities. Highest affinity (Kapp = 12 to 20 nM) occurs with operators containing the 12 bp inverted repeat GTTTAGCTAAAC (klaAp, korAp and trfAp), while lower affinities (Kapp = 136 to 272 nM) occur with less perfect repeats (klcA, kleA, kleC, kfrA). In addition, specific DNA sequences flanking the 12 bp are necessary for the characteristic type I KorA binding affinity. This hierarchy may be important in providing a graded response in expression of the operons controlled by KorA as its concentration varies, as for example on transition from exponential to stationary phase.

Dissection of the switch between genes for replication and transfer of promiscuous plasmid RK2: basis of the dominance of trfAp over trbAp and specificity for KorA in controlling the switch.

The trfA and trb operons of broad host range plasmid RK2 are required for replication and conjugative transfer, respectively. Transcription of the trb operon can be initiated at one of two promoters: trbAp or trbBp. trbBp provides a burst of trb transcription on first entry into the cell. trbAp appears to be responsible for steady-state transcription of the trb operon as well as trbA, encoding a repressor which helps to shut down trbBp. The promoters trfAp and trbAp are arranged as face-to-face divergent promoters. trfAp is very strong and shuts off trbAp activity until trfAp is inhibited by KorA, one of the plasmid-encoded global regulators. Although trfAp is also repressed by KorB, a second global regulator encoded along with KorA in the central control operon, trbAp activation only occurs when KorA is present. KorB did not activate trbAp and indeed had a significant inhibitory effect on KorA activation. In vitro trfAp binds RNA polymerease (RNAP) approximately ten times more strongly than trbAp. Comparison of single and multiple rounds of in vitro run-off transcription suggested that the inhibitory effect of trfAp is due to elongating transcription complexes. In vitro studies with purified KorA and KorB on RNAP binding, isomerization and in vitro transcription suggested that both proteins can displace RNAP from trfAp, but that once open complexes have formed at either promoter they have a good chance of generating a transcript even if they encounter an opposing RNAP. In vivo KorB repressed trbAp even when trbAp was derepressed by a trfAp-1 mutation, removing the need for KorA. This suggested that KorB not only fails to derepress but actually represses trbAp despite the KorB operator being located 90 bp downstream of the transcription start point (tsp). By contrast KorA still activated trbAp when the two promoters were moved further apart or were brought so close that RNAP binding to the two promoters was mutually exclusive. Thus, KorA plays the dominant role in achieving the balance of expression of genes for alternate modes of plasmid propagation but its action is modulated by KorB.

Mutational analysis of the global regulator KorA of broad-host-range plasmid RK2.

KorA protein encoded in the central control region of IncP plasmid RK2 binds to seven operators on the plasmid genome and acts as a global repressor of genes for replication and stable inheritance functions. At trfAp, the promoter for plasmid replication genes, KorA also causes derepression of trbAp, the promoter for trbA, encoding another global regulator (TrbA), which controls genes required for conjugative transfer. Both KorB, a second global repressor encoded in the central control region, and TrbA also act in the trfAp-trbAp region to down-regulate trfAp, but neither of these extra repressors allows derepression of trbAp. To initiate a functional dissection of KorA, we used random mutagenesis and a positive selection system to identify korA mutants which no longer repressed trfAp. Nine single amino acid changes were obtained, which did not affect polypeptide length or apparent stability. These clustered either in the N-terminal region of the protein (region I) or in the putative HTH motif (region II). No changes were obtained in the C-terminal region (region III). Four truncated KorA proteins, with deletions either from the N-terminal or the C-terminal end, were also screened together with the single mutants. Both the band-shift assay with trfAp DNA and the in vivo promoter-probe assays with either trfAp or trbAp showed that none of the region II mutants could bind to DNA and repress the promoter. The region I mutants with a conservative amino acid substitution retained some DNA binding and repressor activity, as well as the ability to dimerise. However, an in vivo system to detect trans-dominance of the mutants indicated that one region I point mutant together with the two N-terminally truncated mutants had lost their dimerisation ability. Deletions into the basic C terminus of KorA did not abolish dimerisation. The results implicate region I in dimerisation, region II in DNA binding and region III in a yet unspecified role, possibly interaction with other proteins such as KorB.

Identification of a seventh operon on plasmid RK2 regulated by the korA gene product.

Broad-host-range IncP plasmids possess a series of operons involved in plasmid maintenance, whose expression is coordinated by a series of regulators, most of which are encoded in a central regulatory operon. The nucleotide sequence of a new monocistronic operon located between coordinates 55.0 and 56.0 kb on the genome of the IncP alpha plasmids RK2 and RP4 is presented. The operon encodes a 34 kDa protein which has a net negative charge. Transcription of the operon, designated by us kfrA (korF-regulated), is repressed not only by the product of the previously described korA gene but also by the product of a gene which we have designated korF and which has not been described previously. The korF gene is encoded downstream from korB within the key korA/korB regulatory operon. We propose that K or F binds to a novel inverted repeat overlapping the promoter for the kfrA operon.

Cloning of cysB mutant alleles of S. typhimurium.

Two cysB mutant alleles of S. typhimurium have been cloned onto pBR vectors. The product of the constitutive cysBc 1352 allele present on the plasmid was found to fulfill regulatory functions: as an activator of the cysteine regulon and as an autorepressor. CysB70 auxotrophic mutation impairs both regulatory functions cysB protein. Transfer of the clones cysBc 1352 allele from E. coli to S. typhimurium and from S. typhimurium to E. coli and biochemical analysis of transformants suggest involvement of a restriction-modification system in the constitutive expression of the cysteine regulon.

ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters.

Deletions leading to complete or partial removal of ParB were introduced into the Pseudomonas aeruginosa chromosome. Fluorescence microscopy of fixed cells showed that ParB mutants lacking the C-terminal domain or HTH motif formed multiple, less intense foci scattered irregularly, in contrast to the one to four ParB foci per cell symmetrically distributed in wild-type P. aeruginosa. All parB mutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation of parA of P. aeruginosa. As complete loss of ParA destabilized its partner ParB it was unclear deficiency of which protein is responsible for the mutant phenotypes. Analysis of four parB mutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all four parB mutants demonstrate the same defects it can be concluded that either ParB, or ParA and ParB in combination, plays an important role in nucleoid distribution, growth and motility in P. aeruginosa.

KorA protein of promiscuous plasmid RK2 controls a transcriptional switch between divergent operons for plasmid replication and conjugative transfer.

The trfA and trb operons, encoding genes essential for replication and conjugative transfer of broad host range plasmid RK2, are transcribed divergently. Deletion analysis presented here indicates that trfAp and trbAp are arranged as face to face promoters. The presence of the korA gene, whose product is known to repress seven operons on RK2, including the trfA operon, is shown here to stimulate trbAp. The effect of korA on trbAp is mimicked by the trfAp-1 promoter down mutation, suggesting that a reduction in the activity of trfAp is required for derepression of trbAp activity. The trfAp-1 mutation reduces RNA polymerase binding and open complex formation at trfAp but does not stimulate melting at trbAp in vitro. Therefore, the inhibition of trbAp is most probably due to forward transcription initiated at trfAp. The simultaneous inhibition/stimulation by KorA is seen even in the presence of the other repressors KorB and TrbA, which act at this region, thus providing a dominant mode of coordinating plasmid replication and transfer. This may be one of the keys to understanding how the maintenance and spread of promiscuous plasmids are balanced in different environments.

Cloning and physical mapping of the cysB region of Salmonella typhimurium.

The cysB region of Salmonella typhimurium was cloned in pBR322 and localized to a 1.75-kilobase HincII fragment. Two-dimensional protein electropherograms showed levels of the cysB polypeptide chain that were several fold higher in plasmid-bearing strains than in the wild type. Fully derepressed levels of sulfite reductase and O-acetylserine sulfhydrylase in cysB plasmid-bearing strains were only 25% higher than in the wild type, suggesting that the product of this regulatory gene ordinarily is not a limiting factor in the expression of the cysteine regulon. The mapping of cysB deletions by Southern blots showed a good correlation between the genetic and the physical maps of this gene. The supX gene was initially cloned with cysB and is within 0.7 kilobase of cysB.

Use of gene fusions to study expression of cysB, the regulatory gene of the cysteine regulon.

Strains of escherichia coli were constructed in which the lacZ gene is fused to cysB, the positive regulator gene of the cysteine regulon. The fusion strains were used to study the regulation of the cysB gene by assaying the fused lacZ gene product. The introduction of a cysB allele, either on a plasmid or on an episome to the fusion strains, resulted in the decrease of beta-galactosidase activity. This implies that the cysB gene expression is autoregulated by its own product. The direction of cysB gene transcription was determined to be clockwise.

Regulation of transfer genes of promiscuous IncP alpha plasmid RK2: repression of Tra1 region transcription both by relaxosome proteins and by the Tra2 regulator TrbA.

The Tra1 region of broad host range IncP alpha plasmid RK2 encodes proteins essential for its promiscuous conjugative transfer and includes oriT, the site at which nicking occurs to initiate transfer replication. Unregulated expression of the Tra1 region genes would be likely to place a major burden on the host. To investigate the control of these genes the three transcriptional promoters from this region were cloned by PCR and inserted into xylE promoter probe vectors. The strength of traJp and traKp was estimated to be six to eightfold less than the strong trfA promoter which is required for expression of genes for vegetative replication of RK2. The traG promoter was about one-tenth the strength of the other two. These promoters are not repressed by products of the central control operon of RK2. However, traJp and traKp, which are arranged as back to back divergent promoters in the oriT region, are repressed by TraK which constitutes part of the relaxosome necessary for nicking at oriT. A second relaxosome protein, TraJ, represses traJp. traGp is not repressed by any relaxosome proteins. All three promoters are repressed by TrbA, which is encoded at the start of the trb operon containing the rest of the transfer genes (the Tra2 region). These circuits provide: (i) an autoregulatory way of ensuring production of enough relaxosome proteins without overburdening the host; and (ii) a means of coordinating expression of both blocks of transfer genes.

The korF region of broad-host-range plasmid RK2 encodes two polypeptides with transcriptional repressor activity.

Broad-host-range IncP plasmid RK2 possesses a series of operons involved in plasmid maintenance, whose expression is coordinated by a number of regulators, most of which are encoded in the central regulatory korA-korB operon. The nucleotide sequence of two new cistrons in this operon, comprising what we have previously designated the korF locus located between coordinates 57.0 and 56.0 kb on the genome of the IncP alpha plasmid RK2, is presented. The cistrons encode polypeptides of 173 and 175 amino acids. Each can repress transcription from the promoters for the kfrA (a monocistronic operon which follows the korA-korB operon) and trfA (a polycistronic operon encoding a putative single-stranded-DNA-binding protein as well as the essential plasmid replication protein TrfA) operons. In addition, the korF loci allow korB to repress kfrA transcription. Both polypeptides contain hydrophobic segments, suggesting that they may be membrane associated. KorFI is highly basic protein whose predicted properties are similar to those of histone like proteins.

DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli.

Nucleotide sequences of the cysB region of Salmonella typhimurium and Escherichia coli have been determined and compared. A total of 1759 nucleotides were sequenced in S. typhimurium and 1840 in E. coli. Both contain a 972-nucleotide open reading frame identified as the coding region for the cysB regulatory protein on the basis of sequence homology and by comparison of the deduced amino acid sequences with known physicochemical properties of this protein. The DNA sequence identity for the cysB coding region in the two species is 80.5%. The deduced amino acid sequences are 95% identical. The predicted cysB polypeptide molecular weights are 36,013 for S. typhimurium and 36,150 for E. coli. For both proteins a helix-turn-helix region similar to that found in other DNA-binding proteins is predicted from the deduced amino acid sequence. Sequences upstream to cysB contain open reading frames which represent the carboxyl-terminal end of the topA gene product, DNA topoisomerase I. A pattern of highly conserved nucleotide sequences in the 151 nucleotides immediately preceding the cysB initiator codon in both species suggests that this region may contain multiple signals for the regulation of cysB expression.

Transcriptional and translational control of the genes for the mating pair formation apparatus of promiscuous IncP plasmids.

The trb operon of broad-host-range plasmid RK2 encodes most of the genes required for formation of mating-pair apparatus and is thus essential for the promiscuous spread of this plasmid. Only two promoters, lying upstream of trbA and trbB, have been identified for this operon. trbB encodes a protein belonging to a large family of proteins which function in the assembly of apparatuses associated with the cell surface. trbA encodes a repressor protein, one of whose targets is the trbB promoter. trbAp is arranged as a face-to-face divergent promoter with trfAp, the strongest of the three promoters in this region. trfAp completely inhibits trbAp unless it is repressed by the KorA protein, a key regulator encoded in the plasmid's central control operon. We show that when trfAp is firing constitutively, it also appears to interfere with trbBp, but that trbBp activity increases when trfAp activity is decreased by repression or mutation. A second global regulator encoded in the central control operon, KorB, represses trbBp, trfAp, and trbAp. The results presented here show that both KorB and TrbA are necessary for full repression of trbBp. The region between trbA and trbB encodes a large inverted repeat which has been proposed to modulate translation of trbB on transcripts which are initiated at trbAp but not trbBp. Using translational fusions to lacZ, we show that translation of trbB is completely blocked when transcripts incorporate the inverted repeat upstream of trbB but proceeds with reasonable efficiency when deletions remove the sequences predicted to sequester the ribosome binding site. Results from both transcriptional fusion and direct measurement of transcript size and intensity by Northern blot analysis show that most trbA transcripts are monocistronic and serve to express only trbA, although some transcription continues into trbB. The monocistronic trbA transcript appears to be the result of transcription termination downstream of trbA. Thus, trbAp and trbA appear to form an operon distinct from the trbB-trbP operon. Consequently, trbA and the switch that controls its expression help to provide the sequential steps which allow efficient expression of transfer genes during plasmid establishment but tight repression once the plasmid is established.

Cloning and characterization of the cysAMK region of Salmonella typhimurium.

A total of 30 kilobases of DNA comprising the cysAMK region of S. typhimurium was cloned as a series of fragments in phage lambda 1059. The genetic organization of this region was established through studies of gene expression from fragments subcloned in pBR322 and from blot hydridization analyses of restriction sites in chromosomal DNA from multisite deletion strains. The results give a gene order of cysA-cysM-crr-ptsl-ptsH-cysK over a distance of approximately 12 kilobases. cysM and cysA have been cloned and expressed in pBR322; attempts to obtain stable pBR322 derivatives carrying cysK were unsuccessful.