Iteron DNA Research Articles

Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

BackgroundStreptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described.ResultsWe report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex.ConclusionsThis work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins.

Characterization of the Partitioning System of Myxococcus Plasmid pMF1

pMF1 is the only autonomously replicating plasmid that has been recently identified in myxobacteria. This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMF1.21-pMF1.23 (parCAB). The pMF1.22 ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that the pMF1.23 (parB) product is a DNA-binding protein of iteron DNA sequences, while the product of pMF1.21 (parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ib par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1 par system has novel structural and functional characteristics.

Negative regulation of pPS10 plasmid replication: origin pairing by zipping‐up DNA‐bound RepA monomers

In many plasmid replicons of gram-negative bacteria, Rep protein dimers are transcriptional self-repressors of their genes, whereas monomers are initiators of DNA replication. Switching between both functions implies conformational remodelling of Rep, and is promoted by Rep binding to the origin DNA repeats (iterons) or chaperones. Rep proteins play another key role: they bridge together two iteron DNA stretches, found either on the same or on different plasmid molecules. These so-called, respectively, 'looped' and 'handcuffed' complexes are thought to be negative regulators of plasmid replication. Although evidence for Rep-dependent plasmid handcuffing has been found in a number of replicons, the structure of these Rep-DNA assemblies is still unknown. Here, by a combination of proteomics, electron microscopy, genetic analysis and modelling, we provide insight on a possible three-dimensional structure for two handcuffed arrays of the iterons found at the origin of pPS10 replicon. These are brought together in parallel register by zipping-up DNA-bound RepA monomers. We also present evidence for a distinct role of RepA dimers in DNA looping. This work defines a new regulatory interface in Rep proteins.

Crystal structure of pi initiator protein-iteron complex of plasmid R6K: implications for initiation of plasmid DNA replication.

We have determined the crystal structure of a monomeric biologically active form of the pi initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-A resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal alpha4' and the N-terminal alpha4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in alpha4', whereas the alpha4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely alpha2 and alpha5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of pi revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.

Small deletion variants of the replication protein, π, and their potential for over-replication-based antimicrobial activity

The emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. To address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. In the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. Our focus was on the pir-encoded pi protein of plasmid R6K that possesses both positive and negative functions in controlling gamma origin-based replication. We observed that three of four pir mutations examined, including two in-frame deletions, severely impaired negative plasmid-replication control. The resulting over-replication phenotype was particularly strong when a pir mutant was placed in cis to gamma origin. In conjugative mating experiments with several representatives of the family Enterobacteriaceae, the plasmids expressed postconjugational antimicrobial activity. The potential utility of a conjugation-based antimicrobial approach is discussed. Additionally, we describe the replication inhibitory function of a novel and useful Rep protein variant, pi*M36A;M38A, which binds iteron DNA exclusively as dimers.

Isolation and sequencing of the replication region of plasmid pBFp1 isolated from a marine biofilm

A 24 kb plasmid, pBFp1, encoding mercury resistance was previously isolated from a marine biofilm. Isolation and sequencing of a 4280 bp DNA fragment containing the plasmid replicon (rep-pBFp1) revealed a putative open reading frame encoding a RepA protein and an oriV-like region containing an A + T rich sub-region, iterons, and DnaA boxes. Sequence comparisons showed significant similarities to the incW plasmid pSa both for the RepA amino acid sequence and in the iteron DNA sequence. Plasmid pBFp1 was also shown to be incompatible with pSa in standard incompatibility testing. A probe from the repA gene of pBFp1 was further made and tested on a collection of plasmids exogenously isolated from marine habitats in a previous study.

Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.

Structure and Function of DNA Replication Initiator Protein, RepE.

The RepE initiator protein of F plasmid plays an essential role in initiating DNA replication in mini-F origin. RepE exhibits two major functions; the RepE monomer plays as a replication initiator which binds to direct repeats (iterons) in origin, while the RepE dimer as an autogenous repressor to the repE operator. The crystal structure of RepE monomer (RepE54) as a complex with an iteron DNA was determined as the first 3-D structure for procaryotic initiator proteins. The RepE monomer consists of topologically similar N- and C-terminal domains related by an internal pseudo two-fold symmetry, which has not been expected from its amino acid sequence. Both domains bind to two major grooves of iteron (19bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has the additional role in dimerization.

The iteron regions necessary for the RepE-iteron interaction in vivo in mini-F plasmids of Escherichia coli.

We have determined the nucleotide positions of an incC iteron essential for RepE binding by analyzing mutated incC iterons defective in exerting incompatibility towards mini-F plasmids. The mutations affecting this incompatibility occurred mostly at two positions within the incC iteron, i.e. an iteron conserved position and a mini-F specific position. Most of the iterons with a base-change at either of these two positions had lost the binding affinity for RepE. This agrees with the crystallographic structure of the RepE-iteron complex which showed that the N and C terminal domains of RepE interact with the two major grooves on one face of the iteron DNA. These grooves contain the iteron conserved and mini-F specific positions necessary for RepE binding. Thus the binding mode may be common to in the case of mini-F like plasmids.

Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6 A resolution.

The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication.

Crystallization and preliminary X-ray diffraction studies of a replication initiator protein (RepE54) of the mini-F plasmid complexed with iteron DNA.

A replication initiator protein (RepE54) complexed with iteron DNA at its binding site was crystallized by the hanging drop vapor diffusion method. The crystals belong to monoclinic space group C2 with unit cell dimensions of a = 108.4 A, b = 81.9 A, c = 73.9 A, and beta = 121.5 degrees, where one molecule of the protein-DNA complex exists per asymmetric unit. They diffract X-rays up to 2.6 A resolution with synchrotron radiation.

The Plasmid RK2 Initiation Protein Binds to the Origin of Replication as a Monomer

The TrfA protein encoded by the broad host range bacterial plasmid RK2 specifically binds to eight direct repeats (iterons) present at the plasmid replication origin to initiate DNA replication. Purified TrfA protein is largely in the form of a dimer, and using a dimerization test system that involves the fusion of the amino-terminal domain of the lambda cI repressor protein to TrfA, we show that the TrfA protein forms dimers in vivo. Because of the high stability of the dimer form of TrfA, the formation of heterodimers between the wild-type and different sized TrfA proteins requires in vivo de novo folding of the primary protein sequence or in vitro denaturation and renaturation. The results of gel mobility shift assays using in vitro or in vivo formed heterodimers indicated that the TrfA protein binds to the iteron DNA as a monomer. Furthermore, when the monomeric and dimeric forms of TrfA are separated by gel filtration chromatography, only the protein in the chromatographic position of the monomeric form demonstrated significant DNA binding activity. These results indicate that only the monomer form of the TrfA protein is active for binding to the iterons at the RK2 replication origin.

Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region.

pAD1 (60 kb) is a conjugative, hemolysin/bacteriocin plasmid in Enterococcus faecalis. It confers a mating response to the peptide sex pheromone cAD1 produced by recipient (plasmid-free) cells, leading to highly efficient plasmid transfer in broth matings. Control of the physiological response to cAD1 can been overridden by a reversible phase variation event at frequencies on the order of 10(-4) to 10(-3) per cell per generation (L. T. Pontius and D. B. Clewell, Plasmid 26:172-185, 1991). The variant forms are designated Dryc and Dry+, which reflects the colony morphologies of cells whose conjugation functions are switched on and off, respectively. Here we show that Dryc variants exhibit a structural change in a region between repA and repB that contains two clusters of 8-bp iterons. The change involved a 31- or 32-bp increase in size of this region. In three or four independent variants examined, one of the iteron clusters increased in size from 13 to 17 iterons. When iteron DNA was placed on a multicopy plasmid and introduced into a wild-type pAD1 derivative, the Dryc phenotype was generated. Since traA, a key negative regulator of conjugation, bears several centrally located iteron-like sequences with the same orientation, we speculate that the protein(s) that normally binds iterons (possibly RepA and/or RepB) blocks traA transcription in Dryc variants.