Different phenotypes of Walker-like A box mutants of ParA homolog IncC of broad-host-range IncP plasmids

The promiscuous IncPα plasmids RK2 and R995 encode a broad-host-range partition system, whose essential components include the incC and korB genes and a DNA site (O(B)) to which the korB product binds. IncC2, the smaller of the two incC products, is sufficient for stabilization of R995ΔincC. It is a member of the type Ia ParA family of partition ATPases. To better understand the role of ATP in partition, we constructed three alanine-substitution mutants of IncC2. Each mutation changed a different residue of the Walker-like ATP-binding and hydrolysis motif, including a lysine (K10) conserved solely among members of the ParA and MinD families. All three IncC2 mutants were defective in plasmid partition, but they differed from one another in other respects. The IncC2 T16A mutant, predicted to be defective in Mg2? coordination, was severely impaired in all activities tested. IncC2 K10A, predicted to be defective in ATP hydrolysis, mediated enhanced incompatibility with R995 derivatives. IncC2 K15A, predicted to be defective in ATP binding, exhibited two distinct incompatibility properties depending on the genotype of the target plasmid. When in trans to plasmids carrying a complementable incC deletion, IncC2 K15A caused dramatic plasmid loss, even at low levels of expression. In trans to wild-type R995 or to R995ΔincC carrying a functional P1 partition system, IncC2 K15A-mediated incompatibility was significantly less than that caused by wild-type IncC2. All three Walker-like A box mutants were also defective for the host toxicity that normally results from co-overexpression of incC and korB. The phenotypes of the mutants support a model in which nucleotide hydrolysis is required for separation of paired plasmid complexes and possible interaction with a host factor.     

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.     

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.     

Evidence for past integration of IncP-1 plasmids into bacterial chromosomes

Plasmids of the IncP-1 incompatibility group are self-transmissible between and stably maintained in a very broad range of Gram-negative bacteria. A characteristic feature of IncP-1 genomes is the existence of multiple binding sites (OB) for the KorB protein which plays a dual role in active partitioning of plasmid and coordinate regulation of expression of genes for replication, maintenance and transfer. A search of the available bacterial genome sequences revealed a significant number (70 out of 322) with one or more putative KorB binding sites. Binding of KorB to such a site was demonstrated by chromatin immunoprecipitation (ChIP) for Pseudomonas putida KT2440. While such a site may arise by chance, this is unlikely for Pseudomonas aeruginosa UCBPP-PA14 whose genome sequence contains four clustered OB sites and several regions have more than 80% nucleotide identity to traJ, trbJ and trbL of IncP-1 plasmids. A number of other bacterial genomes also contain integrated partial IncP-1 genomes or their remnants. These data provide evidence for multiple past integration events of IncP-1 plasmids into bacterial chromosomes and provide new evidence for IncP-1 plasmids being important elements in gene mobility.     

ParI,an orphan ParA family protein from Pseudomonas putida KT2440‐specific genomic island,interferes with the partition system of IncP‐7 plasmids

Pseudomonas putida KT2440 is an ideal soil bacterium for expanding the range of degradable compounds via the recruitment of various catabolic plasmids. In the course of our investigation of the host range of IncP‐7 catabolic plasmids pCAR1, pDK1 and pWW53, we found that the IncP‐7 miniplasmids composed of replication and partition loci were exceptionally unstable in KT2440, which is the authentic host of the archetypal IncP‐9 plasmid pWW0. This study identified ParI, a homologue of ParA family of plasmid partitioning proteins encoded on the KT2440‐specific cryptic genomic island, as a negative host factor for the maintenance of IncP‐7 plasmids. The miniplasmids were destabilized by ectopic expression of ParI, and the loss rate correlated with the copy number of ParB binding sites in the centromeric parS region. Mutations in the conserved ATPase domains of ParI abolished destabilization of miniplasmids. Furthermore, ParI destabilized miniplasmid derivatives carrying the partition‐deficient parA mutations but failed to impact the stability of miniplasmid derivatives with parB mutations in the putative arginine finger. Altogether, these results indicate that ParI interferes with the IncP‐7 plasmid partition system. This study extends canonical partition‐mediated incompatibility of plasmids beyond heterogeneous mobile genetic elements, namely incompatibility between plasmid and genomic island.     

Interaction of Pseudomonas and Enterobacteriaceae plasmids in Aeromonas salmonicida.

We observed that Aeromonas salmonicida ARO200 will maintain either or both the Pseudomonas R-factor, pMG1, and Enterobacteriaceae R-factors. This bacterial strain, therefore, provides a unique background wherein the host ranges of Pseudomonas and Enterobacteriaceae plasmods overlap. Co-maintenance of these plasmids resulted in behavior of plasmid aggregates that allowed transfer of R-dterminants beyond the host range of the parent plasmid. We observed that the ARO200 genetic background facilitated the redistribution of B determinants among unrelated and conjugally noninterfertile gram-negative bacteria. Aberrant behavior resulting in the deletion of R-determinants for plasmids singly maintained in ARO200 was also observed. Plasmids studied included RP1, R702, IncP; Rs-a, IncW; R192.7, IncFII; R64-11, IncI; R390, IncN; and R6K, IncX.     

Self-transmissible IncP R995 plasmids with alternative markers and utility for Flp/FRT cloning strategies

The IncP plasmid R995 has been a useful self-transmissible, broad-host-range vector for a number of applications including the recombinase/conjugation-based cloning of large genomic DNA segments. However, R995 derivatives (or related plasmids) expressing a wide range of different resistance markers and Flp recombinase target sites do not exist in the literature. In addition, documented strategies for applying such plasmids in cloning applications that take advantage of conjugation for the convenient isolation and recovery of constructs are extremely limited. Here, we report a new series of R995 plasmids with alternative markers to increase options for applications in backgrounds already expressing resistance to a particular antibiotic(s). These R995 plasmids have been engineered to contain FRT sites that can be used for recombinase-based cloning. We demonstrate the utility of this approach by cloning 20 kb regions from the Salmonella Typhimurium and Escherichia coli genomes and by cloning DNA from an exogenous plasmid source. To our knowledge, this represents the first systematic engineering of an intact, self-transmissible IncP plasmid with a series of alternative antibiotic markers and FRT sites.     

The plasmid-maintenance functions of the P7 prophage

The region responsible for the maintenance of the prophage of bacteriophage P7 as a stable, unit-copy plasmid was isolated in a lambda att vector which lysogenizes Escherichia coli as a stable unit-copy plasmid under the control of the P7 replication origin. The P7 plasmid-maintenance region was shown to consist of adjacent replication and partition regions capable of functioning independently. The isolated replication region could support plasmid maintenance but the resulting plasmids were highly unstable unless the partition region was also included. Stable composite plasmids were isolated containing the putative P7 partition region and the origin of replication of the unrelated plasmid F, indicating that P7 encoded an active partition mechanism. The replication regions of P7 and P1 were shown to be highly homologous but the partition regions of the two plasmids appear to be unrelated in sequence. The incompatibility determinants associated with the two replication regions showed the same specificity, whereas the partition-region incompatibility determinants were different, showing no cross-specificity.     

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.     

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101–pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1β) and RK2 (IncP-1α), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.     

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.     

pHH502, a plasmid with IncP and IncI alpha characters, loses the latter by a specific recA-independent deletion event

Plasmid pHH502, of molecular weight 70 X 10(6), determined resistance to tetracycline, chloramphenicol, trimethoprim, sulphonamides and mercuric chloride and was incompatible with members of IncP and IncI alpha. It resembled other plasmids of IncI alpha in the following properties: it determined pili that were morphologically and serologically I alpha pili, whose production was repressed in established plasmid-carrying (R+) cultures; its transfer was equally efficient in liquid or on solid medium; it exerted surface exclusion against other IncI alpha plasmids; it was non-transferable to Proteus. In a reproducible, recA-independent event, pHH502 gave rise to pHH502-1, a plasmid of molecular weight 40 X 10(6), lacking determinants for resistance to tetracycline and chloramphenicol and all detectable IncI alpha characteristics. pHH502-1 was incompatible only with IncP plasmids and resembled other IncP plasmids in determining constitutive production of rigid pili, in its surface exclusion, in transferring at greater frequency on solid than in liquid medium and in being transmissible to Proteus mirabilis. It differed from other IncP plasmids in the morphology and serological type of its pili and in failing to transfer to Pseudomonas aeruginosa or Acinetobacter calcoaceticus. Small numbers of pHH502-1 rigid pili were present on bacteria carrying pHH502. Possible mechanisms for the generation of pHH502 and pHH502-1 are discussed.     

Genetic determinants of tetracycline resistance of plasmids from the bacterial species Pseudomonas

The colony hybridization and tetracycline accumulation techniques made possible to demonstrate the majority of the 29 bacteria of Pseudomonas genera to harbour the plasmids carrying tet determinants belonging to classes A-C, while one strain contained a plasmid carrying tet determinant of class B. For the first time the determinants of classes D and E were found on R plasmids in Pseudomonas aeruginosa. The determinant of G class was identified on two plasmids identical to pBS221 plasmid described previously.     

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.