Grazyna Jagura-Burdzy

Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication

The kilB locus (which is unclonable in the absence of korB) of broad‐host‐range plasmid RK2 (60kb) lies between the trfA operon (co‐ordinates 16.4 to 18.2kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co‐ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBl) of the region analysed results in a Tra‐ phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBi DNA sequence KilBl is predicted to be 34995Da, In line with Mr= 36000 observed by sodium dodecyl sulphate/potyacrylamide gel electrophoresis, and contains a type I ATP‐binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tume‐ faciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI

Prokaryotic ParA–ParB–parS system links bacterial chromosome segregation with the cell cycle

While the essential role of episomal par loci in plasmid DNA partitioning has long been appreciated, the function of chromosomally encoded par loci is less clear. The chromosomal parA-parB genes are conserved throughout the bacterial kingdom and encode proteins homologous to those of the plasmidic Type I active partitioning systems. The third conserved element, the centromere-like sequence called parS, occurs in several copies in the chromosome. Recent studies show that the ParA-ParB-parS system is a key player of a mitosis-like process ensuring proper intracellular localization of certain chromosomal regions such as oriC domain and their active and directed segregation. Moreover, the chromosomal par systems link chromosome segregation with initiation of DNA replication and the cell cycle.

Regulation of transfer genes of promiscuous IncPα 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.

REPRESSION AT A DISTANCE BY THE GLOBAL REGULATOR KORB OF PROMISCUOUS INCP PLASMIDS

KorB 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 −35 region (class I KorB operators, OB). We show here that KorB on its own can also repress the trbA, trbB, kfrA and kleA promoters where OB 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 KorBs ability to repress through class II operator but not through class I operator. This deletion reduced multimerization of His6‐tailed KorB protein in vitro and greatly reduced binding specificity for fragments containing OB sequences. At the trbBp region, where OB9 lies 189 bp upstream of the transcription start point, mutagenesis of a proposed secondary binding site overlapping the trbBp−35 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 OB9 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 OB9 and implied that contacts between these proteins could be responsible for the action of KorB at a distance.

Deletion of the parA (soj) Homologue in Pseudomonas aeruginosa Causes ParB Instability and Affects Growth Rate, Chromosome Segregation, and Motility

The parA and parB genes of Pseudomonas aeruginosa are located approximately 8 kb anticlockwise from oriC. ParA is a cytosolic protein present at a level of around 600 molecules per cell in exponential phase, but the level drops about fivefold in stationary phase. Overproduction of full-length ParA or the N-terminal 85 amino acids severely inhibits growth of P. aeruginosa and P. putida. Both inactivation of parA and overexpression of parA in trans in P. aeruginosa also lead to accumulation of anucleate cells and changes in motility. Inactivation of parA also increases the turnover rate (degradation) of ParB. This may provide a mechanism for controlling the level of ParB in response to the growth rate and expression of the parAB operon.

Genomic and Functional Characterization of the Modular Broad-Host-Range RA3 Plasmid, the Archetype of the IncU Group

ABSTRACT IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

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.

Binding and Spreading of ParB on DNA Determine Its Biological Function in Pseudomonas aeruginosa

ParB protein of Pseudomonas aeruginosa belongs to a widely represented ParB family of chromosomally and plasmid-encoded partitioning type IA proteins. Ten putative parS sites are dispersed in the P. aeruginosa chromosome, with eight of them localizing in the oriC domain. After binding to parS, ParB spreads on the DNA, causing transcriptional silencing of nearby genes (A. A. Bartosik et al., J. Bacteriol. 186:6983-6998, 2004). We have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomerization. We defined specific determinants outside of the helix-turn-helix motif responsible for DNA binding. Analysis confirmed the localization of the main dimerization domain in the C terminus of ParB but also mapped another self-interactive domain in the N-terminal domain. Reverse genetics were used to introduce five parB alleles impaired in spreading into the P. aeruginosa chromosome. The single amino acid substitutions in ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect, slowed the growth rate, and impaired motilities, similarly to the pleiotropic phenotype of parB-null mutants, indicating that the ability to spread is vital for ParB function in the cell. The toxicity of ParB overproduction in Pseudomonas spp. is not due to the spreading since several ParB derivatives defective in oligomerization were still toxic for P. aeruginosa when provided in excess.

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.

The Centromere Site of the Segregation Cassette of Broad-Host-Range Plasmid RA3 Is Located at the Border of the Maintenance and Conjugative Transfer Modules

ABSTRACT RA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group isolated originally from Aeromonas spp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable replicon in Escherichia coli. This fragment specifies the korA-incC-korB-orf11 operon coding for an active partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation. IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative −35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set of unrelated repeat motifs distributed over ∼300 bp. Dissection of this region identified the centromere sequence that is vital for partitioning. The ∼300-bp fragment also encompasses the origin of conjugative transfer, oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set of cis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary origin of segregation and conjugative transfer modules in these plasmids and members of the IncU group.