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Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland
Correspondence
Igor Konieczny
igor{at}biotech.univ.gda.pl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 
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 the location of the plasmid mini-replicon cluster was at the cell poles. In contrast, in Pseudomonas cells, as a result of the interaction of chromosomally encoded ParB protein with RK2 centromere-like sequences, these mini-derivatives were localized in the proximity of mid-cell, or , positions. The expression of the Pseudomonas parAB genes in E. coli resulted in a positional change in the RK2 mini-derivative to the mid-cell or , positions. Moreover, in a P. putida parAB mutant, both RK2 mini-derivatives and the entire RK2 plasmid exhibited disturbances of subcellular localization. These observations raise the possibility that in certain bacteria chromosomally encoded partitioning machinery could affect subcellular plasmid positioning.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Li & Austin, 2002) and F (Gordon et al., 1997, 2004; Niki & Hiraga, 1997) as well as Salmonella enterica plasmid R27 (Lawley & Taylor, 2003) are found at the mid-cell of newborn cells or symmetrically at the and positions in older, pre-divisional cells. For some plasmids, it was proposed that plasmid particles could form clusters. Plasmid R1, with a copy number of four to five per cell, also forms symmetrically located clusters, although in older cells, R1 clusters are shifted towards the cell poles (Jensen & Gerdes, 1999; Weitao et al., 2000). The mid-cell, and cellular positioning of plasmid clusters has been observed for the broad-host-range plasmid RK2 (60 kb in size) in E. coli, Pseudomonas aeruginosa and Vibrio cholerae (Pogliano et al., 2001; Ho et al., 2002). This plasmid belongs to the IncP-1 incompatibility group, is present at five to eight copies per chromosome, and is stably maintained in almost all Gram-negative bacteria (Rosche et al., 2000; Thomas et al., 1980, 1984; Wilson et al., 1997). When plasmids RK2, F and P1 were localized simultaneously, the majority of plasmid foci were close to but separated from each other, demonstrating that compatible bacterial plasmids are in independent cellular locations in E. coli (Ho et al., 2002). The subcellular localization of these plasmids is thought to be mediated by the partitioning (Par) systems they encode. Plasmid partitioning loci function as a cassette containing genes for two trans-acting proteins and a cis-acting centromere-like site. The first gene of a par operon encodes a ParA-like protein, which is a Walker box or actin-like ATPase (Ebersbach & Gerdes, 2005; Funnell, 2005). The product of the second gene, a ParB-like protein, binds as a dimer to specific sequences within the cis site (Schumacher & Funnell, 2005). The formation of a partitioning complex and oscillations of the filament-forming ParA ATPase were proposed to result in localization of the plasmid to the mid-cell or the and positions, which represent the middle of the next cell generation (Ebersbach & Gerdes, 2004; Ebersbach et al., 2006). For plasmid RK2, the partitioning cassette consists of the genes encoding IncC, a Walker box ATPase, and KorB, a protein that interacts with a palindromic operator (OB) found 12 times in RK2 DNA (Kostelidou & Thomas, 2000; Rosche et al., 2000; Williams et al., 1998). The rules governing plasmid positioning in bacterial cells are not fully understood. It is not known, for example, what bacterial factors affect plasmid cluster formation, localization and partitioning. In this work we demonstrate that chromosomal partitioning genes affect the subcellular location of a plasmid mini-replicon. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Plasmids were introduced into E. coli and Pseudomonas cells by transformation (Sambrook et al., 1989).
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Fluorescence in situ hybridization (FISH).
Mini-RK2 specific Cy3-labelled probes for FISH were prepared essentially as described by Pogliano et al. (2001). The procedure was modified as follows. After incubation with the blocking solution, the cells were treated with lysozyme (1 mg ml–1 in PBS) for 10 min at room temperature. The next step was the same as described by Jensen & Shapiro (1999). Chromosomal DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) at a concentration of 0.5 µg ml–1.
Fluorescence microscopy.
E. coli S17-1 and Pseudomonas strains were prepared for microscopy as described previously (Ho et al., 2002; Pogliano et al., 2001). The expression of tetR-eyfp from pKO10 and pKO15 was induced with 0.4 % arabinose for 1 h. The expression of parA, parB and tetR-eyfp from pLAK15 in E. coli cells was induced with 0.4 % arabinose for 1.5 h. The expression of tetR-eyfp in E. coli MGTRY was induced as described by Verheust & Helinski (2007). The cells were stained with FM4-64 (0.5 µg ml–1), immobilized on a poly-L-lysine-treated slide coverslip and observed using an Olympus BX51 fluorescence microscope with F-View-II CCD camera. Measurements and image analysis were conducted with AnalySIS software.
Gel filtration assay.
Column gel filtration was used to fractionate the DNA–ParB complexes as described previously (Konieczny & Helinski, 1997). The reaction mixture (100 µl), containing P. putida ParB (4 µg) and the indicated supercoiled plasmid DNA (2 µg) in 40 mM HEPES/KOH, pH 8.0, 40 mM potassium glutamate, 10 mM magnesium acetate, 4 % sucrose, 4 mM dithiothreitol, and 2 mM ATP, was incubated for 20 min at 32 °C and then run through a Sepharose CL-4B (0.5x12 cm) column, equilibrated at room temperature with the incubation buffer and 0.01 % Brij 58. Fractions (80 µl) were collected and a portion of each (35 µl) was analysed by SDS-PAGE followed by silver staining, while another portion (35 µl) was analysed on agarose gel stained with ethidium bromide.
Formaldehyde cross-linking and chromatin immunoprecipitation.
Cells were grown at 30 °C in LB medium. Samples were taken during exponential growth (OD600 0.6) and prepared as described by Lin & Grossman (1998). Protein–DNA complexes were immunoprecipitated with polyclonal anti-ParB antibodies (C. M. Thomas, University of Birghmingam, UK), followed by incubation with 30 µl of a 50 % Protein A-Sepharose slurry (Amersham Pharmacia). PCR was performed with Taq DNA polymerase (Fermentas) and oligonucleotide primers (OB3, 5'-TCGCCGTTGCGAACCACCTTCGG-3', 5'AAATCGGGAGTGCGAAAAGCATCACC-3'; OB10, 5'-GATTATGGCTCATATCGAAAGTCTC-3', 5'-GAGCACACGAAGGATGTTGGTG-3'; oriV, 5'-AAGCCGTGTGCGAGACACCGC-3', 5'-AAAGACAGGTTAGCGGTGGC-3'; IR-2, 5'-CCGCTTCAGCTCATCAACCCAGAC-3', 5'-CTTGCGAAGCTGGGTCAAGTGTACC-3'). PCR products were separated on agarose gels and stained with ethidium bromide.
Purification 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. Peak fractions were pooled and dialysed against P buffer (150 mM NaCl). The final product yielded 8 µg µl–1 of >95 % homogeneous protein.
Plasmid stabilization assays.
The cells carrying a mini-RK2 (pCVI) or RK2 (pZZ15) plasmid were diluted from overnight cultures into LB medium containing appropriate antibiotics (see above, Media and growth conditions) and incubated to OD600 0.5. The cells were then diluted into medium without antibiotics to allow free growth of RK2 and mini-RK2 segregants and maintained in exponential growth for at least 120 generations by sequential dilutions. The samples removed during this period were plated on agar plates and, from each plating, 
400 colonies were patched onto agar with antibiotics to score retention or loss of plasmids. The number of generations was estimated from optical density measurements.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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i, ii, iii; D). Fluorescent foci were observed in only approximately 50 % of the cells, the majority of which had only one focus. A small fraction of E. coli cells had two foci (Fig. 1A iv, E). A similar subcellular distribution of pCVI was obtained when the TetR-EYFP protein was expressed from the tetR-eyfp gene on the chromosome of E. coli MGTRY (data not shown). Polar positioning of pCVI was confirmed by FISH in E. coli cells containing only this plasmid (Fig. 1B). Plasmid fluorescent signals were observed on the edge of DAPI-stained E. coli nucleoids, suggesting that the plasmid was not located in the mid-cell or , positions but at the cell poles. Similarly as we observed for pCVI, it was previously reported that F and R1 mini-derivatives lacking the partitioning segments are distributed at the cell poles (Ebersbach & Gerdes, 2004; Niki & Hiraga, 1997). Polar localization of RK2 mini-replicons in E. coli cells has also been reported recently by Verheust & Helinski (2007), who showed that insertion of the RK2 par locus into pCVI restores the mid-cell, or , positions of plasmid foci.
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, positions (Fig. 1F i, J). Also FISH and DAPI co-staining demonstrated plasmid foci inside DAPI-stained P. putida nucleoids (Fig. 1G). Overall, the location of pCVI foci in Pseudomonas cells was similar to the location of intact RK2 (data not shown), although with a broader distribution in the case of the mini-derivative clusters. Also, as observed in E. coli, foci of the mini-derivative were detected in only a fraction of the Pseudomonas cells (60 %). Our experiments were carried out in both LB and M63/glucose media and no substantial difference was observed regarding plasmid positioning and plasmid foci number. We have also analysed RK2 mini-derivative localization in P. aeruginosa (data not shown). Using either TetR-EYFP tagging or FISH experiments we demonstrated that pCVI localization in P. aeruginosa is similar to that in P. putida.
In control experiments, the expression of tetR-eyfp in E. coli and P. putida cells without pCVI resulted in yellow fluorescence of the whole cells (Fig. 1C ii, H ii). When tetR-eyfp was not expressed in the cells, no signal was detected (Fig. 1C i, H i). Also FISH and DAPI co-staining demonstrated no signal when bacterial cells did not contain pCVI (Fig. 1C iii, H iii).
P. putida ParB protein interacts with RK2 DNA by binding centromere-like sites
The fluorescence microscopy experiments indicated that the subcellular localization of an RK2 mini-derivative that lacks partitioning genes depends on the host bacterium. This raised the possibility that the subcellular position of an RK2 mini-derivative could be affected by host factors, including the host-encoded partitioning machinery. Some similarities between the RK2 and Pseudomonas partitioning systems have been described (Hayes, 2000). In addition, RK2 and Pseudomonas, but not E. coli, have a type I par locus (Mohl & Gober, 1997; Ogasawara & Yoshikawa, 1992). The ParB protein from P. putida shows high similarity to the RK2 KorB protein (Lin & Grossman, 1998): based on amino acid sequence, it is 27 % identical with KorB. It has also been demonstrated that KorB of RK2 specifically interacts with the P. putida chromosomal cis site (Chiu & Thomas, 2004). We asked if the P. putida ParB protein could interact with RK2 DNA. Size-exclusion chromatography was used to investigate this possibility. Purified ParB protein was incubated with or without plasmid DNA and then fractionated using a Sepharose CL-4B column. The results demonstrated that P. putida ParB formed nucleoprotein complexes with RK2 DNA (Fig. 2). When the entire RK2 plasmid or its mini-derivative pCVI were present in the incubation mixture, the ParB protein was detected in the column void volume, indicating the formation of a nucleoprotein complex. In the absence of DNA or in the presence of pUC18 DNA, no ParB protein was found in void volume fractions (Fig. 2). This result indicated interaction of ParB with RK2 plasmid DNA. The pCVI plasmid used in our study did not contain incC or the korB genes; however, two RK2 centromere-like sites, OB10 and OB3, were still present adjacent to the trfA and oriT sequences. To investigate whether those sites are capable of interacting with P. putida ParB protein, DNA fragments containing OB10, OB3 or P. putida centromere site IR2 were introduced into pUC18 plasmid DNA. When incubations were carried out with these plasmid constructs, ParB was detected in the void fractions (Fig. 2). Since no ParB complex formation was observed with pUC18, we concluded that the observed nucleoprotein complexes are the result of ParB protein interaction with RK2 centromere-like sites. The RK2 OB motifs differ from the Pseudomonas IR2 sequence; therefore we could not exclude the possibility that ParB possesses two domains for interaction with DNA.
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, lane 1 and Fig. 4 lane 1) was present in P. putida cells. Less intensive bands of OB10-specific PCR product detected in immunoprecipitation experiments suggest that ParB interaction with this site on RK2 mini-derivatives could be less efficient (Fig. 3A, lane 1 and Fig. 4 lane 1). When oriV-specific primers were used, we did not detect any signal, indicating that a ParB complex is not formed with the RK2 origin (Fig. 3A, lane 1 and Fig. 4 lane 1).When the RK2 mini-derivative was not present in P. putida cells, we were able to detect ParB interaction only with the chromosomal IR2 sequence (Fig. 3B, lane 2). Similar results indicating P. putida ParB interaction with both OB3 and OB10 sites were obtained when the entire RK2 plasmid was present in P. putida cells (Fig. 3C lane 2). When RK2 plasmid was present in E. coli cells, no signal was detected, indicating that anti-ParB antibodies did not interact with RK2 KorB protein (Fig. 3C lane 3). These data showed that in P. putida cells chromosomally encoded ParB protein interacts with centromere-like OB sites of plasmid RK2 DNA.
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). Approximately 40 % of the cells contained fluorescent plasmid foci and multiple signals were detected in only 3 % of the cells (Fig. 5M). In contrast, when TetR-EYFP and both ParA and ParB P. putida proteins were expressed, the distribution of pCVI in E. coli cells was more or less symmetrical, with plasmid foci located in the proximity of the mid-cell or the , positions (Fig. 5D–F). Also, the proportion of the cells containing plasmid foci increased (Fig. 5M). We detected plasmid foci in 60 % of the cells and among them approximately 30 % had more than one fluorescent signal. These data were very similar to those obtained in a control experiment where pCVI number and location were analysed in wild-type P. putida cells (Fig. 5G–I, M, N). Further, we asked if P. putida ParB protein interacted with pCVI centromere-like sites when both ParA and ParB were expressed from pLAK15 or pKO15 in E. coli cells. Chromatin immunoprecipitation demonstrated that indeed under those experimental conditions promoting the mid-cell or the , positions of pCVI in E. coli cells (Fig. 5D–F), both OB3 and OB10 sites were bound by P. putida ParB (Fig. 4, lanes 4 and 5). In a control reaction, no PCR products were observed when E. coli cells contained plasmid pKO10, without the P. putida parAB region (Fig. 4, lane 3).
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parAB
kan mutant. The lack of functional ParA and ParB proteins changed the localization of the pCVI foci from the approximately mid-cell and , positions to positions near the cell poles (Fig. 5J–L, P). We did not observe differences in the number of cells containing plasmid foci; however, the number of cells with a single signal was slightly increased compared to the wild-type P. putida (Fig. 5M). No OB3- and OB10-specific PCR products were observed after immunoprecipitation of P. putida parABkan(pCVI) extract with anti-ParB antibodies (Fig. 4, lane 2). In the control, when oriV-specific primers were used (Fig. 4, lane 2), no signal was detected either.
Interestingly, when the localization of the entire RK2 plasmid (pZZ15) was analysed in the P. putida parABkan mutant, dispersed signals were observed compared with the localization of pZZ15 in P. putida wild-type cells (Fig. 5O, Q). This indicates that in P. putida disturbances in the chromosomally encoded partitioning system affect the subcellular position of RK2 plasmid.
Stability of RK2 and its mini-derivative pCVI in E. coli and P. putida strains
The data presented above clearly show that the expression of P. putida parAB genes affects the position of RK2 mini-replicons. In E. coli, the expression of P. putida parAB genes compensates for the lack of the RK2 partitioning system with regard to the cellular location of the RK2 mini-derivative pCVI. In P. putida, the lack of chromosomal partitioning genes results in a shift of the position of pCVI towards the cell poles and disturbances of the localization of the entire RK2 plasmid. To test if the observed subcellular localizations of the RK2 plasmids affect their stabilities, we analysed the maintenance of pZZ15 and pCVI in the strains used in the course of this work. pZZ15 was stably maintained in both E. coli S17-1 and P. putida KT2440 (Fig. 6A, E). Although we observed disturbances in pZZ15 localization in P. putida KT2440parABkan (Fig. 5Q), stability tests revealed that the plasmid is stably maintained in this mutant (Fig. 6A). This result could be explained by the compensating effect of the dispersed plasmid localization. Stabilities of RK2 mini-derivative pCVI in E. coli S17-1 and P. putida KT2440 were significantly reduced compared to the stabilities of pZZ15 in these strains (compare Fig. 6E with Fig. 6A and B). It must be pointed out that pCVI stability in P. putida KT2440, where the plasmid was found at approximately mid-cell and the , positions (Fig. 1G), was better when compared to pCVI stability in E. coli S17-1, where it was located at the cell poles (Fig. 1B and compare Fig. 6E and B). The disruption of the P. putida parAB locus (mutant KT2440parABkan) resulted in only a very limited reduction of pCVI stability (Fig. 6B). A similar effect was observed when pCVI stability was tested with_pKO10 also present in the cell. The pCVI stability observed in P. putida KT2440parABkan was slightly reduced in comparison with its stability in P. putida KT2440 (Fig. 6C). Similarly, only a limited effect on chromosome stability was previously observed in P. putida KT2440parABkan (Godfrin-Estevenon et al., 2002). Also, during our experiments the pCVI stability loss rate tested in E. coli in the presence of pKO15 or pKO10 was very similar, regardless of the expression of P. putida parAB genes (Fig. 6D). However, at the beginning of the experiment more cells contained pCVI in the strain where P. putida parAB was expressed (Fig. 6D).
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In summary, the experiments reported here demonstrate, we believe for the first time, that the subcellular position of broad-host-range plasmid RK2 is affected by Pseudomonas partitioning machinery. The Pseudomonas partitioning module plays a role in chromosomal segregation (Godfrin-Estevenon et al., 2002; Lewis et al., 2002); however, it has also been speculated that it is a remnant of RK2-like plasmid integration (Chiu & Thomas, 2004). That may explain cross-talk between chromosomal and plasmid partitioning systems. Our results at least raise the possibility that in certain bacteria the chromosomally encoded partitioning machinery could affect the subcellular positioning of a specific plasmid element.
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ACKNOWLEDGEMENTS |
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parABkan. We also thank Donald Helinski for critically reading the manuscript. This work was supported by the Polish State Committee for Scientific Research Grant 2P04A02730, The Foundation for Polish Science and the EMBO/HHMI Young Investigator Programme. Katarzyna Kolatka and Marcin Pierechod are recipients of stipends from the Foundation for Polish Science. Edited by: L. Jannière
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Received 22 March 2008;
revised 28 May 2008;
accepted 5 June 2008.
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