Gyrase inhibitors and thymine starvation disrupt the normal pattern of plasmid RK2 localization in Escherichia coli

Multicopy plasmids in Escherichia coli are not randomly distributed throughout the cell but exist as defined clusters that are localized at the mid-cell, or at the 1/4 and 3/4 cell length positions. To explore the factors that contribute to plasmid clustering and localization, E. coli cells carrying a plasmid RK2 derivative that can be tagged with a green fluorescent protein-LacI fusion protein were subjected to various conditions that interfere with plasmid superhelicity and/or DNA replication. The various treatments included thymine starvation and the addition of the gyrase inhibitors nalidixic acid and novobiocin. In each case, localization of plasmid clusters at the preferred positions was disrupted but the plasmids remained in clusters, suggesting that normal plasmid superhelicity and DNA synthesis in elongating cells are not required for the clustering of individual plasmid molecules. It was also observed that the inhibition of DNA replication by these treatments produced filaments in which the plasmid clusters were confined to one or two nucleoid bodies, which were located near the midline of the filament and were not evenly spaced throughout the filament, as is found in cells treated with cephalexin. Finally, the enhanced yellow fluorescent protein-RarA fusion protein was used to localize the replication complex in individual E. coli cells. Novobiocin and nalidixic acid treatment both resulted in rapid loss of RarA foci. Under these conditions the RK2 plasmid clusters were not disassembled, suggesting that a completely intact replication complex is not required for plasmid clustering.     

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.     

Localization of the naturally occurring plasmid ColE1 at the cell pole

The naturally occurring plasmid ColE1 was found to localize as a cluster in one or both of the cell poles of Escherichia coli. In addition to the polar localization of ColE1 in most cells, movement of the plasmid to the midcell position was observed in time-lapse studies. ColE1 could be displaced from its polar location by the p15A replicon, pBAD33, but not by plasmid RK2. The displacement of ColE1 by pBAD33 resulted in an almost random positioning of ColE1 foci in the cell and also in a loss of segregational stability, as evidenced by the large number of cells carrying pBAD33 with no visible ColE1 focus and as confirmed by ColE1 stability studies. The addition of the active partitioning systems of the F plasmid (sopABC) or RK2 (O(B1) incC korB) resulted in movement of the ColE1 replicon from the cell pole to within the nucleoid region. This repositioning did not result in destabilization but did result in an increase in the number of plasmid foci, most likely due to partial declustering. These results are consistent with the importance of par regions to the localization of plasmids to specific regions of the cell and demonstrate both localization and dynamic movement for a naturally occurring plasmid that does not encode a replication initiation protein or a partitioning system that is required for plasmid stability.     

Subcellular positioning of F plasmid mediated by dynamic localization of SopA and SopB

SopA, SopB proteins and the cis-acting sopC DNA region of F plasmid are essential for partitioning of the plasmid, ensuring proper subcellular positioning of the plasmid DNA molecules. We have analyzed by immunofluorescence microscopy the subcellular localization of SopA and SopB. The majority of SopB molecules formed foci, which localized frequently with F plasmid DNA molecules. The foci increased in number in proportion to the cell length. Interestingly, beside the foci formation, SopB formed a spiral structure that was dependent on SopA, which also formed a spiral structure, independent of the presence of SopB, and these two structures partially overlapped. On the basis of these results and previous biochemical studies together with our simulations, we propose a theoretical model named "the reaction-diffusion partitioning model", using reaction-diffusion equations that explain the dynamic subcellular localization of SopA and SopB proteins and the subcellular positioning of F plasmid. We hypothesized that sister copies of plasmid DNA compete with each other for sites at which SopB multimer is at the optimum concentration. The plasmid incompatibility mediated by the Sop system might be explained clearly by this hypothesis.     

Multicopy plasmids affect replisome positioning in Bacillus subtilis

The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.     

Bacterial mitosis: partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells with a single plasmid focus, the focus located preferentially at mid-cell. In cells with two foci, these located at quarter-cell positions. In the absence of ParB and parC1/parC2, ParA-GFP formed stationary helices extending from one end of the nucleoid to the other. In the presence of ParB and parC1/parC2, ParA-GFP oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid and subsequently separate them into daughter cells.     

Subcellular localization of plasmids containing the oriC region of the Escherichia coli chromosome, with or without the sopABC partitioning system

Fluorescence in situ hybridization (FISH) analysis has revealed the subcellular localization of specific chromosomal segments and plasmid molecules during the cell division cycle in Escherichia coli: the replication origin (oriC) segments on the chromosome are localized at nucleoid borders, and actively partitioning mini-F plasmid molecules are localized at the 1/4 and 3/4 positions of the cell. In contrast, mini-F plasmid molecules lacking the sopABC segment are randomly localized in cytoplasmic areas at cell poles. In this study, we analysed the subcellular localization of an oriC plasmid that contains the minimum E. coli chromosomal replication origin and its flanking regions. These oriC plasmid molecules were mainly localized in cytosolic areas at cell poles. On the other hand, oriC plasmid DNA molecules carrying the sopABC segment of F plasmid were localized at cell quarter sites, as were actively partitioning mini-F plasmid DNA molecules. Therefore, we conclude that oriC itself and its flanking regions are not sufficient for positioning the replication origin domain of the E. coli chromosome within the cell.     

The incC korB region of RK2 repositions a mini-RK2 replicon in Escherichia coli

Analysis by fluorescence microscopy has established that plasmid RK2 in Escherichia coli and other gram-negative bacteria is present as discrete clusters that are located inside the nucleoid at the mid- or quarter-cell positions. A mini-RK2 replicon containing an array of tetO repeats was visualized in E. coli cells that express a TetR-EYFP fusion protein. Unlike intact RK2, the RK2 mini-replicon (pCV1) was localized as a cluster at the cell poles outside of the nucleoid. Insertion of the O(B1)incC korB partitioning (par) region of RK2 into pCV1 resulted in a shift of the mini-replicon to within the nucleoid region at the mid- and quarter-cell positions. Despite the repositioning of the mini-RK2 replicon to the cellular positions where intact RK2 is normally located, the insertion of the intact O(B1) incC korB region did not significantly stabilize the mini-RK2 plasmid during cell growth. Deletions within the O(B1)incC or the korB region resulted in a failure of this par region to move pCV1 out of its polar position. The insertion of the par system of plasmid F into pCV1 resulted in a similar shift in the location of pCV1 to the nucleoid region. Unlike O(B1)incC korB, the insertion of the RK2 parABC resolvase system into pCV1 did not affect the polar positioning of pCV1. This effect of O(B1)incC korB on the location of pCV1 provides additional evidence for a partitioning role of this region of plasmid RK2. However, the failure of this region to significantly increase the stability of the mini-RK2 plasmid indicates that the localization of the plasmid to the mid- and quarter cell positions in E. coli is not in itself sufficient for the stable maintenance of plasmid RK2.     

Kinetics of plasmid segregation in Escherichia coli

Low copy-number bacterial replicons occupy specific locations in their host cells. Production of a GFP-Lac repressor hybrid protein in cells carrying F or P1 plasmids tagged with a lac operator array reveals that in smaller (younger) cells these plasmids are seen mainly as a single fluorescent focus at mid-cell, whereas larger cells tend to have two foci, one at each quarter-cell position. Duplication of the central focus is presumed to represent active partition of plasmid copies. We report here our investigation by time-lapse microscopy of the subsequent movement of these copies to the quarter positions. Following duplication of the central focus, the new foci migrated rapidly and directly to their quarter-cell destinations, where they remained until the next cell cycle. The speed of movement was about five times faster than poleward migration of oriC and 50 times faster than cell elongation. Aberrant positioning of mini-F lacking its sopC centromere demonstrated the requirement for the partition system in this localization process. From the measured number of F plasmid copies per cell it appears that each migrating focus contains two or more plasmid molecules. The molecular basis of this clustering, and evidence for phasing of the partition event in the cell cycle, are discussed.     

Localization of replication forks in wild-type and <Emphasis Type="Italic">mukB</Emphasis> mutant cells of <Emphasis Type="Italic">Escherichia coli</Emphasis>

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.     

Intracellular mobility of plasmid DNA is limited by the ParA family of partitioning systems

The highly conserved ParA family of partitioning systems is responsible for positioning DNA and protein complexes in bacteria. In Escherichia coli , plasmids that rely upon these systems are positioned at mid-cell and are repositioned at the quarter-cell positions after replication. How they remain fixed at these positions throughout the cell cycle is unknown. We use fluorescence recovery after photobleaching and time-lapse microscopy to measure plasmid mobility in living E. coli cells. We find that a minimalized version of plasmid RK2 that lacks its Par system is highly mobile, that the intact RK2 plasmid is relatively immobile, and that the addition of a Par system to the minimalized RK2 plasmid limits its mobility to that of the intact RK2. Mobility is thus the default state, and Par systems are required not only to position plasmids, but also to hold them at these positions. The intervention of Par systems is required continuously throughout the cell cycle to restrict plasmid movement that would, if unrestricted, subvert the segregation process. Our results reveal an important function for Par systems in plasmid DNA segregation that is likely to be conserved in bacteria.     

Mechanism of DNA segregation in prokaryotes: ParM partitioning protein of plasmid R1 co-localizes with its replicon during the cell cycle.

The parA locus of plasmid R1 encodes a prokaryotic centromere-like system that mediates genetic stabilization of plasmids by an unknown mechanism. The locus codes for two proteins, ParM and ParR, and a centromere-like DNA region (parC) to which the ParR protein binds. We showed recently that ParR mediates specific pairing of parC-containing DNA molecules in vitro. To obtain further insight into the mechanism of plasmid stabilization, we examined the intracellular localization of the components of the parA system. We found that ParM forms discrete foci that localize to specific cellular regions in a simple, yet dynamic pattern. In newborn cells, ParM foci were present close to both cell poles. Concomitant with cell growth, new foci formed at mid-cell. A point mutation that abolished the ATPase activity of ParM simultaneously prevented cellular localization and plasmid partitioning. A parA-containing plasmid localized to similar sites, i.e. close to the poles and at mid-cell, thus indicating that the plasmid co-localizes with ParM. Double labelling of single cells showed that plasmid DNA and ParM indeed co-localize. Thus, our data indicate that parA is a true partitioning system that mediates pairing of plasmids at mid-cell and subsequently moves them to the cell poles before cell division.     

Plasmids isolated from marine sediment microbial communities contain replication and incompatibility regions unrelated to those of known plasmid groups.

Two hundred ninety-seven bacteria carrying plasmids that range in size from 5 to 250 kb were identified from more than 1,000 aerobic heterotrophic bacteria isolated from coastal California marine sediments. While some isolates contained numerous (three to five) small (5- to 10-kb) plasmids, the majority of the natural isolates typically contained one large (40- to 100-kb) plasmid. By the method of plasmid isolation used in this study, the frequency of plasmid incidence ranged from 24 to 28% depending on the samples examined. Diversity of the plasmids occurring in the marine sediment bacterial populations was examined at the molecular level by hybridization with 14 different DNA probes specific for the incompatibility and replication (inc/rep) regions of a number of well-characterized plasmid incompatibility groups (repB/O, FIA, FII, FIB, HI1, HI2, I1, L/M, X, N, P, Q, W, and U). Interestingly, we found no DNA homology between the plasmids isolated from the culturable bacterial population of marine sediments and the replicon probes specific for numerous incompatibility groups developed by Couturier et al. (M. F. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Our findings suggest that plasmids in marine sediment microbial communities contain novel, as-yet-uncharacterized, incompatibility and replication regions and that the present replicon typing system, based primarily on plasmids derived from clinical isolates, may not be representative of the plasmid diversity occurring in some marine environments. Since the vast majority of marine bacteria are not culturable under laboratory conditions, we also screened microbial community DNA for the presence of broad- and narrow-host-range plasmid replication sequences. Although the replication origin of the conjugally promiscuous broad-host-range plasmid RK2 (incP) was not detectable in any of the plasmid-containing culturable marine isolates, DNA extracted from the microbial community and amplified by PCR yielded a positive signal for RK2 oriV replication sequences. The strength of the signal suggests the presence of a low level of the incP replicon within the marine microbial community. In contrast, replication sequences specific for the narrow-host-range plasmid F were not detectable in DNA extracted from marine sediment microbial communities. With the possible exception of mercuric chloride, phenotypic analysis of the 297 plasmid-bearing isolates did not demonstrate a correlation between plasmid content and antibiotic or heavy metal resistance traits.     

Suppression of Co1E1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro.

Hybrid plasmids were constructed in vitro by linking the Inc P-1 broad host range plasmid RK2 to the colicinogenic plasmid ColE1 at their EcoRI endonuclease cleavage sites. These plasmids were found to be immune to colicin E1, non-colicin-producing, and to exhibit all the characteristics of RK2 including self-transmissibility. These joint replicons have a copy number of 5 to 7 per chromosome which is typical of RK2, but not ColE1. Unlike ColE1, the plasmids will not replicate in the presence of chloramphenicol and are maintained in DNA polymerase I mutants of Escherichia coli. In addition, only RK2 incompatibility is expressed, although functional ColE1 can be rescued from the hybrids by EcoRI cleavage. This suppression of ColE1 copy number and incompatibility was found to be a unique effect of plasmid size on ColE1 properties. However, the inhibition of ColE1 or ColE1-like plasmid replication in chloramphenicol-treated cells is a specific effect of RK2 or segments of RK2 (Cri⁺ phenotype). This phenomenon is not a function of plasmid size and requires covalent linkage of RK2 DNA to ColE1. A specific region of RK2 (50.4 to 56.4 × 10³ base-pairs) cloned in the ColE1-like plasmid pBR313 was shown to carry the genetic determinant(s) for expression of the Cri⁺ phenotype.     

荧光蛋白标签对gD囊膜蛋白在BHK-21细胞亚细胞定位的影响

【目的】探究荧光蛋白标签对马疱疹病毒I型(Equine herpes virus type 1,EHV-1)gD囊膜蛋白亚细胞定位的影响。【方法】以EHV-1基因组为模板利用PCR扩增gD全基因,分别克隆至pAcGFP1-C1和p Ds Red2-N1质粒,构建p Ac-GFP-gD(GFP-gD)和p Ds-gD-Red(gD-Red)重组质粒;将GFP基因插入gD基因信号肽序列之后并克隆至PVAX-1质粒,构建PVAX-S-GFP-gD’(S-GFP-gD’)重组质粒;将Flag标签序列与gD囊膜蛋白N端序列融合后并克隆至p VAX-1表达载体,构建p VAX-Flag-gD(Flag-gD)重组质粒。将4种不同重组真核表达质粒分别转染BHK-21细胞,通过激光共聚焦显微镜对不同融合蛋白gD进行亚细胞定位。【结果】成功构建4种不同的融合蛋白gD真核表达载体;在BHK-21细胞单独表达时,不同融合蛋白gD绝大部分都定位于高尔基体,极少量定位于细胞核内。【结论】不同插入位点的荧光蛋白标签对gD囊膜蛋白亚细胞定位无明显影响,这对今后研究其它蛋白亚细胞定位提供参考。     

Generation in vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease

Several non-lethal deletions of the broad host range plasmid RK2 (molecular weight of 37.6 . 10(6) have been produced in vitro. The method employed relied on the single HindIII restriction nuclease site in RK2 and the ability of phage Mu to insert and thereby add new HindIII restriction sites at various positions in the plasmid. The deleted plasmids have in each case lost kanamycin (Km) resistance, and in two cases are defective in self-transmissibility. The method used to reduce the size of the RK2 plasmid also results in the cloning of each of the two ends of the Mu phage DNA on the plasmid derivatives.     

A family of ATPases involved in active partitioning of diverse bacterial plasmids

Low copy-number bacterial plasmids F (the classical Escherichia coli sex factor) and prophage P1 encode partitioning functions which may provide fundamental insights into the active processes which ensure that bacterial genomes are segregated to both daughter cells prior to cell division. These partitioning systems involve two proteins: ParA and ParB. We report that incC from the broad host-range plasmid RK2 is a member of the family of ParA partitioning proteins and that these proteins (as well as related proteins encoded by plasmids from Agrobacterium tumefaciens and Chlamydia trachomatis) contain type I nucleotide-binding motifs. Also, we show that the cell division inhibitor MinD is homologous to members of the ParA family. Sequence comparisons of ParB proteins suggest that they may contain sites for phosphorylation. We propose that ATP hydrolysis by the ParA protein may result in phosphorylation of the ParB protein, thereby causing a conformational shift necessary to separate paired plasmid molecules at the cell division plane.     

Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.