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It has been demonstrated that plasmids are not randomly distributed but are located symmetrically in mid-cell, or (1/4), (3/4) positions in bacterial cells. In this work we compared the localization of broad-host-range plasmid RK2 mini-replicons, which lack an active partitioning system, in Escherichia coli and Pseudomonas putida cells. In E. coli...
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Context 1
... and bacterial strains. The plasmids and bacterial strains used in this work are listed in Table 1. Plasmids were introduced into E. coli and Pseudomonas cells by transformation (Sambrook et al., 1989). ...
Context 2
... of ParB. The P. putida ParB protein was expressed from plasmid pETK1 ( Table 1). The bacterial strain, the conditions of parB expression and the protein purification were the same as described for the P1 plasmid ParB homologue (Davis & Austin, 1988) with the exception that only a phosphocellulose column was used. ...
Context 3
... and pLAK15 are vectors for expressing P. putida parAB. pKO10 is a pLAK15 derivative which does not contain P. putida parAB (see Table 1). Lanes 1, 2 and 6, control of immunoprecipitation with anti-ParB antibodies and lysates of P. putida was determined by tagging with TetR-EYFP in E. coli S17-1 (A-F), P. putida KT2440 (G-I, N) and P. putida KT2440 DparABVkan (J-L, P). ...
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Background
Plasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacteri...
Citations
... Plasmids designed for recombineering in E. coli (e.g. the pSIM set of vectors and plasmid pKD46) exploit temperature-sensitive mutants of oriV(RK2) and oriV(pSC101) (Datsenko and Wanner, 2000;Thomason et al., 2014). While the pSC101 replicon has a very narrow host range and does not replicate in pseudomonads (Barth et al., 1981), the broad-host-range RK2 replicon is known to be functional in P. putida (Kolatka et al., 2008). The RK2 replication mechanism depends on the plasmid-encoded replication initiator protein (TrfA) and other elements necessary for replication encoded in the host genome (e.g. ...
Genome engineering of non-conventional microorganisms calls for the development of dedicated synthetic biology tools. Pseudomonas putida is a Gram-negative, non-pathogenic soil bacterium widely used for metabolic engineering owing to its versatile metabolism and high levels of tolerance to different types of stress. Genome editing of P. putida largely relies on homologous recombination events, assisted by helper plasmid-based expression of genes encoding DNA modifying enzymes. Plasmid curing from selected isolates is the most tedious and time-consuming step of this procedure, and implementing commonly used methods to this end in P. putida (e.g. temperature-sensitive replicons) is often impractical. To tackle this issue, we have developed a toolbox for both target- and self-curing of plasmid DNA in Pseudomonas species. Our method enables plasmid-curing in a simple cultivation step by combining in vivo digestion of vectors by the I-SceI homing nuclease with synthetic control of plasmid replication, triggered by the addition of a cheap chemical inducer (3-methylbenzoate) to the medium. The system displays an efficiency of vector curing >90% and the screening of plasmid-free clones is greatly facilitated by the use of fluorescent markers that can be selected according to the application intended. Furthermore, quick genome engineering of P. putida using self-curing plasmids is demonstrated through genome reduction of the platform strain EM42 by eliminating all genes encoding β-lactamases, the catabolic ben gene cluster, and the pyoverdine synthesis machinery. Physiological characterization of the resulting streamlined strain, P. putida SEM10, revealed advantageous features that could be exploited for metabolic engineering.
... Several reports show that RK2 plasmid is present in bacterial cell at five to eight copies per chromosome (32,49,50). Furthermore, the published data indicates that the number of plasmid foci per cell does not correspond to the number of plasmid copies per cell, indicating that each focus consists of more than one plasmid and that the RK2 plasmid molecules are grouped into a few clusters (44,51). To test if proteases might affect the formation of the plasmid clusters we used FROS system (see Materials and methods), allowing the localization of mini-RK2 in the E. coli strains -C600 and its protease-deficient derivatives lon(−), clpP(−) and lon(−)clpP(−). ...
Specific nucleoprotein complexes are formed strictly to prevent over-initiation of DNA replication. An example of those is the so-called handcuff complex, in which two plasmid molecules are coupled together with plasmid-encoded replication initiation protein (Rep). In this work, we elucidate the mechanism of the handcuff complex disruption. In vitro tests, including dissociation progress analysis, demonstrate that the dimeric variants of plasmid RK2 replication initiation protein TrfA are involved in assembling the plasmid handcuff complex which, as we found, reveals high stability. Particular proteases, namely Lon and ClpAP, disrupt the handcuff by degrading TrfA, thus affecting plasmid stability. Moreover, our data demonstrate that TrfA monomers are able to dissociate handcuffed plasmid molecules. Those monomers displace TrfA molecules, which are involved in handcuff formation, and through interaction with the uncoupled plasmid replication origins they re-initiate DNA synthesis. We discuss the relevance of both Rep monomers and host proteases for plasmid maintenance under vegetative and stress conditions.
... It is likely that handcuffing of plasmids is the basis for the amyloidogenic patches found in the nucleoid of bacteria carrying pPS10 (Fig. 3b). Plasmids lacking its own partition module but carrying sequences that match the centromere-like parS locus still become associated to the bacterial nucleoid for stable segregation by the ParAB proteins encoded at the chromosome 28 . This is likely the case for pPS10, because its replicon includes sequences (e.g.: C 521 CTTCCATGGGGAAGG 536 ) 2 bearing similarity to the consensus parS in P. aeruginosa 29 . ...
DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is ‘handcuffing’, i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation.
... It is likely that handcuffing of plasmids is the basis for the amyloidogenic patches found in the nucleoid of bacteria carrying pPS10 (Fig. 3b). Plasmids lacking its own partition module but carrying sequences that match the centromere-like parS locus still become associated to the bacterial nucleoid for stable segregation by the ParAB proteins encoded at the chromosome 28 . This is likely the case for pPS10, because its replicon includes sequences (e.g.: C 521 CTTCCATGGGGAAGG 536 ) 2 bearing similarity to the consensus parS in P. aeruginosa 29 . ...
... DNA probes were prepared by DNA labeling with Alexa555-dCTP (Invitrogen) and Terminal deoxynucleotideyl Transferase (Promega). DNA labeling and purification were performed as previously described for DNA fragments labeled with Cy3-dCTP (43,44). dsDNA fragments, containing minimal origin region (consisting of the AT-rich region, the iterons and the DnaA-boxes), were prepared in polymerase chain reaction (PCR) reactions with primers oriV1 and oriV2 or oriS1 and oriS2 (see Supplementary Table S1 for oligonulecotide sequences). ...
The DNA unwinding element (DUE) is a sequence rich in adenine and thymine residues present within the origin region of both
prokaryotic and eukaryotic replicons. Recently, it has been shown that this is the site where bacterial DnaA proteins, the
chromosomal replication initiators, form a specific nucleoprotein filament. DnaA proteins contain a DNA binding domain (DBD)
and belong to the family of origin binding proteins (OBPs). To date there has been no data on whether OBPs structurally different
from DnaA can form nucleoprotein complexes within the DUE. In this work we demonstrate that plasmid Rep proteins, composed
of two Winged Helix domains, distinct from the DBD, specifically bind to one of the strands of ssDNA within the DUE. We observed
nucleoprotein complexes formed by these Rep proteins, involving both dsDNA containing the Rep-binding sites (iterons) and
the strand-specific ssDNA of the DUE. Formation of these complexes required the presence of all repeated sequence elements
located within the DUE. Any changes in these repeated sequences resulted in the disturbance in Rep-ssDNA DUE complex formation
and the lack of origin replication activity in vivo or in vitro.
... In the absence of the par system the position of plasmids is altered. However, according to recent studies the P. putida chromosomal partitioning system is homologous to that of RK2 and as a result of the interaction of chromosomally-encoded ParB protein with sequences of the RK2 mini-replicon, these RK2derivatives were clustered in midcell and quarter positions similar to that seen with intact RK2 [131]. Moreover, replication of RK2 plasmids is associated with the membrane through anchoring of plasmid to membrane-bound replication initiator protein TrfA [75]. ...
Homologous recombination (HR) has a major impact in bacterial evolution. Most of the knowledge about the mechanisms and control of HR in bacteria has been obtained in fast growing bacteria. However, in their natural environment bacteria frequently meet adverse conditions which restrict the growth of cells. We have constructed a test system to investigate HR between a plasmid and a chromosome in carbon-starved populations of the soil bacterium Pseudomonas putida restoring the expression of phenol monooxygenase gene pheA. Our results show that prolonged starvation of P. putida in the presence of phenol stimulates HR. The emergence of recombinants on selective plates containing phenol as an only carbon source for the growth of recombinants is facilitated by reactive oxygen species and suppressed by DNA mismatch repair enzymes. Importantly, the chromosomal location of the HR target influences the frequency and dynamics of HR events. In silico analysis of binding sites of nucleoid-associated proteins (NAPs) revealed that chromosomal DNA regions which flank the test system in bacteria exhibiting a lower HR frequency are enriched in binding sites for a subset of NAPs compared to those which express a higher frequency of HR. We hypothesize that the binding of these proteins imposes differences in local structural organization of the genome that could affect the accessibility of the chromosomal DNA to HR processes and thereby the frequency of HR.
... So far, there is another example that one partition system interferes with the other. The chromosomal ParAB proteins of KT2440 interacts with and successfully segregates the miniplasmid of an IncP-1 plasmid RK2 that lacks partitioning proteins but contains its centromere-like site (Kolatka et al., 2008), suggesting an interaction between different partitioning systems via homologous centromeric sites. The KT2440 ParB shows only 27% identity with KorB of RK2 but can bind specifically to the centromere site of RK2. ...
... This raises the possibility of competition between the RK2-and chromosome-specified partitioning machineries on the plasmid centromeric site. However, as is the case of full-length IncP-7 plasmid, such presumptive competition did not result in the destabilization of RK2 itself probably due to the RK2-encoded postsegregational killing and multimer resolution systems (Kolatka et al., 2008). ...
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.
... Perhaps surprisingly, an extra-chromosomal RK-2 plasmid showed the same behavior, while smaller RNA particles had a higher subdiffusive exponent. It must be mentioned that the localization of RK-2 plasmids appears to be highly regulated (Derman et al., 2008;Kolatka et al., 2008). The underlying viscoelasticity of the bacterial cytoplasm surrounding the nucleoid is still poorly understood. ...
Recent experimental and theoretical approaches have attempted to quantify the physical organization (compaction and geometry) of the bacterial chromosome with its complement of proteins (the nucleoid). The genomic DNA exists in a complex and dynamic protein-rich state, which is highly organized at various length scales. This has implications for modulating (when not directly enabling) the core biological processes of replication, transcription and segregation. We overview the progress in this area, driven in the last few years by new scientific ideas and new interdisciplinary experimental techniques, ranging from high space- and time-resolution microscopy to high-throughput genomics employing sequencing to map different aspects of the nucleoid-related interactome. The aim of this review is to present the wide spectrum of experimental and theoretical findings coherently, from a physics viewpoint. In particular, we highlight the role that statistical and soft condensed matter physics play in describing this system of fundamental biological importance, specifically reviewing classic and more modern tools from the theory of polymers. We also discuss some attempts toward unifying interpretations of the current results, pointing to possible directions for future investigation.
... Although the endogenous promoters of RK2 plasmids are repressed during normal growth [15], and the foci formation is due to the introduction of a transcriptionally active promoter, we did observe a small number of fluorescence foci with the control plasmid, pRW901, which lacks a constitutively active promoter (Figures 2A and 2C). This is consistent with the work of others who have reported polar congregation of partition-defective plasmids [16–19]. We reason that this effect is due to the very low levels of transcription of essential plasmid functions [15]. ...
In the present paper, we report that transcription affects the location of a DNA target in Escherichia coli K-12. A strain whose chromosome had been engineered to encode a lac repressor-GFP (green fluorescent protein) fusion was used as a host for a low copy number plasmid that carries an array of five lac operator sites. Individual cells of this strain exhibited a diffuse fluorescence signal, suggesting that the plasmid is distributed throughout the cell cytoplasm. However, a derivative of this plasmid carrying a cloned constitutive promoter is targeted to a location at the edge of the nucleoid towards the pole of the host cell. We conclude that transcription from the cloned promoter is driving the location of the plasmid and that specific locations in bacterial cells may favour gene expression.
... Unfortunately, not much is known about the factors causing the plasmid to form clusters. RK2's subcellular localization is thought to be due to the plasmid partitioning system, as the deletion of incC and korB led to an alteration in the position of mini-RK2 particles in E. coli cells from midcell or 1 = 4 and 3 = 4 positions to the cell poles (Verheust and Helinski, 2007;Kolatka et al., 2008). The deletion of or mutations in par genes also altered the position of low copy number E. coli plasmids P1 (Li and Austin, 2002) and R1 (Ebersbach and Gerdes, 2004). ...
... What is very interesting is that in P. putida and P. aeruginosa, two bacteria which encode chromosomal partitioning systems that are homologous to the plasmid ones, mini-RK2 derivatives lacking incC and korB, were observed in midcell and quarter positions (Kolatka et al., 2008) similar to what is seen with intact RK2. It was shown that the P. putida ParB protein interacted with the plasmid O B sites both in vitro and in vivo and that expression of the P. putida parA and parB genes in E. coli cells restored mini-RK2 localization to midcell and quarter positions. ...
... It has also been shown that the lack of a functional RK2 partitioning system results in improper plasmid segregation and localization, thus increasing the plasmid loss rate (Williams et al., 1998;Bignell et al., 1999;Verheust and Helinski, 2007;Kolatka et al., 2008). Unfortunately, the insertion of incC, korB and O B sequences into mini-RK2 did not restore the plasmid to full stability, despite the fact that these RK2 minireplicons were observed in ½ and 1 = 4 , 3 = 4 positions (Verheust and Helinski, 2007). ...
The broad-host-range plasmid RK2 has been a model for studying DNA metabolism in bacteria for many years. It is used as a vector allowing genetic manipulations in numerous bacterial species. The RK2 genome encodes several genes providing the plasmid with diverse functions allowing for its stable maintenance in a variety of bacterial hosts. This review will focus on two processes indispensable for plasmid DNA maintenance. We will summarize recent understanding of the molecular mechanisms contributing to the RK2 DNA replication and partitioning.
