Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli
2002; Springer Nature; Volume: 21; Issue: 7 Linguagem: Inglês
10.1093/emboj/21.7.1864
ISSN1460-2075
Autores Tópico(s)Bacterial biofilms and quorum sensing
ResumoArticle1 April 2002free access Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli Thanh Quoc Ho Thanh Quoc Ho Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA Search for more papers by this author Zhenping Zhong Zhenping Zhong Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA Search for more papers by this author Stefan Aung Stefan Aung Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA Search for more papers by this author Joe Pogliano Corresponding Author Joe Pogliano Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA Search for more papers by this author Thanh Quoc Ho Thanh Quoc Ho Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA Search for more papers by this author Zhenping Zhong Zhenping Zhong Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA Search for more papers by this author Stefan Aung Stefan Aung Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA Search for more papers by this author Joe Pogliano Corresponding Author Joe Pogliano Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA Search for more papers by this author Author Information Thanh Quoc Ho1, Zhenping Zhong1, Stefan Aung1 and Joe Pogliano 1 1Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0322 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1864-1872https://doi.org/10.1093/emboj/21.7.1864 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Targeting of DNA molecules to specific subcellular positions is essential for efficient segregation, but the mechanisms underlying these processes are poorly understood. In Escherichia coli, several plasmids belonging to different incompatibility groups (F, P1 and RK2) localize preferentially near the midcell and quartercell positions. Here we compare the relative positions of these three plasmids using fluorescence in situ hybridization. When plasmids F and P1 were localized simultaneously using differentially labeled probes, the majority of foci (∼75%) were well separated from each other. Similar results were found when we compared the subcellular localization of F with RK2, and RK2 with P1: regardless of the number of foci per cell or growth conditions, most of the foci (70–80%) were not in close proximity to one another. We also localized RK2 in Pseudomonas aeruginosa and Vibrio cholerae, and found that plasmid RK2 localization is conserved across bacterial species. Our results suggest that each plasmid has its own unique subcellular address, implying a mechanism for the stable co-existence of plasmids in which subcelluar targeting plays a major role. Introduction Segregation of DNA to daughter cells prior to cell division is important in all organisms, but the mechanisms underlying these processes in bacteria are poorly understood. Many DNA molecules once assumed to be randomly distributed in bacterial cells are now known to display a remarkable degree of previously unappreciated organizational complexity (Webb et al., 1997; Jensen and Shapiro, 1999b; Sharpe and Errington, 1999; Dasgupta et al., 2000; Gordon and Wright, 2000; Hiraga, 2000; Shapiro and Losick, 2000; Donachie, 2001). Individual regions of bacterial chromosomes are positioned specifically within the cell and can move rapidly from one location to another (Gordon et al., 1997; Lewis and Errington, 1997; Webb et al., 1997, 1998; Sharpe and Errington, 1998; Jensen and Shapiro, 1999a; Roos et al., 2001), despite the fact that bacteria lack obvious homologs of motor proteins or the proteins comprising the spindle apparatus. For example, in Bacillus subtilis and Escherichia coli, the origins of replication are anchored temporarily near the midcell and, after duplication, migrate rapidly to positions near the cell poles (Gordon et al., 1997; Webb et al., 1997, 1998; Niki and Hiraga, 1998). In B.subtilis, DNA replication occurs in relatively stationary complexes located either near midcell or near the future midpoints of the daughter cells (one-quarter and three-quarters cell positions) (Lemon and Grossman, 1998, 2000). A stationary replisome situated near midcell might act as a biosynthetic motor that extrudes newly duplicated chromosomes in opposite directions (Dingman, 1974; Losick and Shapiro, 1998; Cook, 1999; Lemon and Grossman, 2001). The Bacillus origins duplicated near midcell are directed away from the centrally located replisome and are captured by unknown mechanisms near the one-quarter and three-quarters cell positions. The factory model of replication is likely to apply to many organisms, including Caulobacter crescentus and E.coli, where evidence suggests that organization and directionality of newly extruded DNA may be provided by proteins involved in nucleoid folding/condensation (Jensen and Shapiro, 1999a; Koppes et al., 1999; Onogi et al., 1999; Brendler et al., 2000; Dasgupta et al., 2000; Hiraga, 2000; Holmes and Cozzarelli, 2000; Sawitzke and Austin, 2000, 2001). The replication proteins of C.crescentus are also localized in replication factories; however, in contrast to the stationary factories of B.subtilis, the C.crescentus replisomes migrate during the cell cycle from the cell pole to midcell (Jensen et al., 2001). Despite these advances in our understanding of the in vivo dynamics of chromosomal DNA, little is known about the mechanisms by which DNA molecules are targeted to specific positions within the cell or how they move from one location to another. Several plasmids have been localized in E.coli by either fluorescence in situ hybridization (FISH) or by tagging with green fluorescent protein (GFP)–LacI after insertion of a lacO array (Niki and Hiraga, 1997; Jensen and Gerdes, 1999; Jensen and Shapiro, 1999b; Gordon and Wright, 2000; Hiraga, 2000; Weitao et al., 2000; Pogliano et al., 2001). Plasmids F, P1 and RK2 localize near midcell and, after duplication, migrate with rapid kinetics to the one-quarter and three-quarters cell positions (Gordon et al., 1997; Niki and Hiraga, 1997; Pogliano et al., 2001). Localization of F and RK2 is coordinated with the host cell cycle such that the newborn cells contain a single midcell focus, whereas cells that have progressed further through the cell cycle contain two foci located near the one-quarter and three-quarters cell positions (Gordon et al., 1997; Niki and Hiraga, 1997; Pogliano et al., 2001). RK2 localization is also coordinated with the rate of growth, so that the number of foci per cell increases with increasing growth rate, and decreases when cells enter stationary phase (Pogliano et al., 2001). These highly organized localization patterns suggest that physiological cues about the host cell cycle feed into the regulatory networks controlling plasmid copy number and distribution (Bingle and Thomas, 2001). Partitioning systems encoded by these plasmids have been proposed to play a role in mediating localization to these positions (Niki and Hiraga, 1997; Bignell et al., 1999; Erdmann et al., 1999). A single bacterial strain can stably maintain many different plasmids only if each belongs to a different incompatibility group, but the relationship between incompatibility groups and subcellular localization has never been fully understood (Novick, 1987; Nordstrom and Austin, 1989). One model proposed that each compatible plasmid is tethered to a different receptor encoded by the host cell, but an alternative model suggested that a single host protein or structure serves as a common receptor with which many plasmids interact (Novick, 1987; Nordstrom and Austin, 1989; Austin and Nordstrom, 1990). Therefore, the subcellular positioning of different plasmid molecules in the same cell could have profound implications for how plasmids interact with each other, including how they compete with one another for replication or how frequently they undergo recombination. Yet, such models of in vivo plasmid dynamics remain speculative, partly because of our limited understanding of the underlying basis of plasmid localization and movement in bacteria. Even less is known about plasmid localization in organisms besides E.coli. Here we simultaneously localize pairs of plasmids (F, P1 and RK2) by FISH to address two questions. First, do different plasmids recognize the same, conserved structure at midcell (and therefore co-localize) or does each target to separate structures located in the vicinity of the cell midpoint? Secondly, do different plasmids move from midcell to quartercell simultaneously? We show that plasmids belonging to different incompatibility groups are not targeted to the same receptor inside the cell, and that they segregate at different times relative to each other. We also show that RK2 localization is conserved between the Gram-negative bacteria Pseudomonas aeruginosa, Vibrio cholerae and E.coli. Our results provide visual evidence for another mechanism of plasmid incompatibility in which compatible plasmids are tethered to unique receptors in the cell, that are separated both spatially and temporally from one another. Results Plasmids F and P1 are low-copy plasmids, present at one or two copies per chromosome equivalent, while RK2 is a multicopy plasmid present at five to eight copies per chromosome equivalent. When each plasmid is localized individually in cells grown in minimal media, most cells contain a single focus located near midcell or two foci located near the one-quarter and three-quarters cell positions (Gordon et al., 1997; Niki and Hiraga, 1997; Pogliano et al., 2001; Figure 1). The similarities in F, P1 and RK2 localization could indicate that each plasmid is tethered to a common structure or receptor within the cell. If this is the case, then signals from two different plasmids that are labeled with different fluorescent probes should co-localize. Alternatively, each plasmid may be targeted independently and separately from one another. In this case, signals from two differently labeled plasmids will not fully overlap, except by chance due to the small size of the cells and the limited resolution of light microscopy. To determine if plasmids F, P1 and RK2 are localized in close proximity to one another within the cell, we performed dual labeling FISH experiments. We compared the localization properties of a mini-P1 plasmid (λ-P1:5R), a mini-F plasmid (pOX38Kan) and the 60 kb RK2 plasmid or a 20 kb RK2 derivative (pRK290). Figure 1.Fluorescence micrographs showing dual labeling of plasmids F, P1 and RK2 in E.coli by FISH. Individual red (left columns) and green (middle columns) foci are shown with blue membranes, or merged together without membrane staining (right columns). Overlapping green and red foci appear yellow. Cell membranes were stained with MTG and are shown in blue false-color. White bars = 1 μm. (A–D) Plasmids F (red) and RK2 (green) showing cells (JP941) containing one fluorescent focus for each plasmid (A and B), a single F focus flanked by two RK2 foci (C) and two fluorescent foci for each plasmid (D). (E–H) Plasmids F (red) and P1 (green) showing cells (JP821) containing two overlapping fluorescent foci located near midcell (E), two non-overlapping foci (F), a single P1 focus and two F foci (G), and four non-overlapping foci (H). (I–K) Plasmids RK2 (red) and P1 (green) showing cells (JP869) containing two foci that overlap (I), two separated foci (J) and a single midcell P1 focus flanked by two RK2 foci (K). Download figure Download PowerPoint Simultaneous localization of F and RK2 We localized F and RK2 replicons simultaneously in fixed cells of strain JP941 (containing pOX38Kan and pRK290), grown in minimal glucose media at 30°C, using FISH. The RK2 probe was labeled with Cy5 (green), the F probe was labeled with Cy3 (red) and the cell membranes were stained with Mitotracker Green FM (MTG) (blue; Figure 1A–D). As expected under these growth conditions, most of the cells contain one or two foci for each plasmid (Gordon et al., 1997; Niki and Hiraga, 1997; Pogliano et al., 2001). Even though RK2 is a multicopy plasmid, many copies of the plasmid cluster together at a few positions (midcell and quartercell) inside the cell (Pogliano et al., 2001). In cells containing one fluorescent signal for each plasmid, we could readily detect two foci that were overlapping (Figure 1A; yellow), as well as two foci that were not overlapping (Figure 1B). We quantitated the percentage of overlapping foci for cells containing a total of two, three or four foci in the same cell (Figure 2). In the majority (73%) of cells containing two foci (one focus for each plasmid), the signals from the two probes did not overlap (Figures 1B and 2), even though each focus was positioned near midcell. When four foci per cell were present (two for each plasmid), they often occurred near the quartercell positions as expected but, in most cases, either one (49%) or both (29%) pairs were well separated from each other (Figures 1D and 2). Identical results were obtained if the two fluorophores of the probes were reversed (not shown). These results demonstrate that F and RK2 replicons generally are not co-localized within the cell. Figure 2.Percentage of coincident plasmid foci from dual-labeling FISH experiments. Cells containing either one or two signals for each plasmid were categorized into groups containing a total of two, three or four foci, and the percentage of cells containing coincident (purple dot) or non-overlapping foci (red and green dots) was determined. A total of 578 cells were scored with n = 249 for P1 and RK2, n = 174 for P1 and F, and n = 155 for F and RK2. Download figure Download PowerPoint Two fluorescent signals were overlapping ∼20–30% of the time (Figure 2). This degree of overlap could be due to the small diameter of E.coli (∼0.6 μm) and the limited resolution of light microscopy (0.2 μm). We estimate that two separate signals randomly positioned near midcell would appear to overlap by chance approximately one-third of the time, which may account for the fraction of overlapping foci observed here. Alternatively, plasmids may be targeted to the same region of the cell during a portion of their replication/segregation cycles. Simultaneous localization of F and P1 We next determined if F and P1 were in close proximity or well separated from one another using a Cy3-labeled probe for F and a Cy5-labeled probe for P1. In cells of JP821 (containing pOX38Kan and λ-P1:5R) grown in minimal glucose media at 30°C, most contained one or two foci for each plasmid, with a single focus of P1 (green) or F (red) preferentially near midcell (Figure 1E and F). Sometimes (21%), these foci at midcell were overlapping (Figures 1E and 2), suggesting that they were too close together to be resolved by light microscopy. However, in the majority of cells (79%), the single P1 and single F foci did not overlap (Figures 1F and 2). When two foci per plasmid were present, the percentage of cells in which neither, one or both pairs of foci overlapped was 45, 40 and 15%, respectively (Figure 2). Therefore, F appears to be spatially separated from both P1 and RK2 in the majority of cells. Simultaneous localization of RK2 and P1 From comparing cells containing the same number of F and RK2 or F and P1 foci, the above results suggested that these plasmids are targeted independently of one another. We next simultaneously localized P1 and RK2 plasmids in strain JP869 (containing λ-P1:5R and RK2) grown in minimal glucose media at 30°C. Using a Cy3-labeled probe for RK2 and a Cy5-labeled probe for P1, most of the cells contained one or two foci corresponding to each plasmid. When cells contained a single focus for P1 (green) and RK2 (red), some of the foci appeared to overlap (Figure 1I), but the majority (72%) did not (Figures 1J and 2). When four foci were present (two for each plasmid), the largest class of cells (52%) contained four foci that did not overlap (Figure 2), while one pair of foci did not overlap in 20% of the cells (Figure 2). Taken together, our results suggest that plasmids F, P1 and RK2 are generally not co-localized inside the cell, despite their generally similar localization patterns. Relative timing of plasmid segregation A substantial number of cells (between 40 and 60%) contained three foci, suggesting that segregation of the different plasmids is asynchronous. For F and P1, most of these cells (94%) contained two F foci flanking a single P1 focus (Figures 1G and 2), suggesting that the F plasmids segregated before P1. For RK2 and P1, 89% of the cells with three foci contained two RK2 foci flanking a single P1 focus (Figures 1K and 2). P1 segregation thus appeared to lag behind RK2 in most of the cells. In comparison, for F and RK2, 62% of the cells with three foci contained two F foci flanking a single RK2 focus. Therefore, individual plasmids may be separated temporally as well as spatially. Cell division is not required for plasmid segregation Given the location of F, P1 and RK2, a central question is whether cell division is required for plasmid localization or segregation. Previous studies found that segregation of a mini-P1 plasmid tagged with GFP–LacI is inhibited when cell division is blocked by β-lactams specific for FtsI (Gordon et al., 1997). In contrast, segregation of a mini-F plasmid tagged with GFP–LacI was unaffected, suggesting that each plasmid is targeted in the cell by a different mechanism, only one of which is dependent upon cell division. Since our co-localization results support the proposal that different plasmids are targeted to different structures, we sought to determine if plasmid RK2 is affected by inhibitors of cell division. We also re-examined the effect of cell division inhibition on F and P1, since a more recent study suggested that some P1 plasmids may not require cell division for segregation (Erdmann et al., 1999). We localized RK2 in E.coli cell filaments formed by inactivating cell division with the antibiotic cephalexin. Cell growth and chromosome segregation continue in the absence of cell division, and long, multinucleate filaments are produced. Localization of RK2 in LB at 30°C in the presence of cephalexin is shown in Figure 3A and B. The cell membranes are shown in green, the nucleoids are shown in blue and the foci are shown in red. RK2 foci were distributed regularly along the entire length of the filament, indicating that targeting of each cluster of RK2 plasmids was unaffected by the inactivation of cell division. Foci always occurred in association with the bacterial nucleoid and were rarely observed in the spaces between the nucleoids (Figure 3). Although cells grown in LB contained many RK2 foci as expected (Pogliano et al., 2001), the number of foci per cell increased with filament length (Figure 4A). The number of foci per micron of cell length remained unchanged after cephalexin treatment (Figure 4B), suggesting that RK2 does not require cell division for segregation. Figure 3.Fluorescence micrographs of FISH experiments showing plasmid localization in cells grown in LB at 30°C and treated with cephalexin two generations prior to collecting samples for microscopy. The outlines of the filaments were visualized by staining the cell membranes with MTG and appear green in (A), (C) and (E) and blue in (G). The nucleoids were stained with DAPI and appear blue in (B), (D) and (F). (A and B) RK2 plasmids form regularly spaced foci (red) along the length of the filaments of strain JP704. (C and D) P1 plasmids form foci (red) regularly distributed along the entire length of the filaments of strain JP857. (E and F) F plasmids form foci (red) along the entire length of the filaments of strain JP911. (G and H) Dual labeling of F (red) and P1 (green) in cephalexin-induced filaments of JP821. (G) F plasmids (red) and P1 plasmids (green) form foci that are regularly spaced along the length of the filaments (blue). (H) F and P1 alone (no membranes) showing that most of the foci do not overlap. Bar in (A) = 1 μm. All panels are at the same scale. Download figure Download PowerPoint Figure 4.Segregation of F, P1 and RK2 in the presence of the cell division inhibitor cephalexin. (A) The number of RK2 foci per cell increases with cell length in filaments of strain JP704 formed by treatment with cephalexin in LB at 30°C. (B) The number of RK2 foci per cell was normalized to cell length (microns per focus) and plotted versus the percentage of total cells. Strain JP704 was grown in LB at 30°C in the absence (white bars) or presence (black bars) of cephalexin. For (A) and (B), a total of 59 wild-type cells containing 148 foci and 47 filaments containing 540 foci were measured. (C–F) The effect of cephalexin on F and P1 distribution in filaments of strain JP821 grown in LB at 30°C. A total of 108 wild-type (untreated) cells containing 191 P1 and 287 F foci were measured. A total of 81 cephalexin-treated cells (filaments) containing 408 P1 and 583 F foci were measured. (C) The number of F foci per cell increases with cell length in cephalexin-induced filaments of JP821. The number of foci per filament is plotted versus filament length. (D) The number of F foci per cell was normalized to cell length (microns per focus) and plotted versus the percentage of total cells. Untreated cells (white bars) have approximately the same number of microns per focus as cephalexin-treated cells (black bars). (E) The number of P1 foci per cell increases with cell length in cephalexin-induced filaments of JP821. The number of foci per filament is plotted versus filament length. (F) The number of P1 foci per cell was normalized to cell length (microns per focus) and plotted versus the percentage of total cells. Untreated cells (white bars) range from 0.5 to 2.5 μm per focus, whereas a small percentage of cephalexin-treated cells (black bars) range up to 5 μm per focus. Download figure Download PowerPoint We compared F, P1 and RK2 localization in the presence of cephalexin. In our experiments, F (Figure 3E and F) formed foci that were regularly spaced along the length of the filament and were associated with the nucleoid region, in agreement with previous results (Gordon et al., 1997). However, P1 localization was more complex. Some filaments had very few foci, as seen previously (Gordon et al., 1997), but the majority contained many regularly spaced P1 foci (Figure 3C and D) similar to F. To understand better the effect of cephalexin on P1 segregation, we quantitated F and P1 co-localization after cephalexin treatment. The numbers of F and P1 foci per filament are plotted versus filament length in Figure 4C and E. For P1, the number of foci per filament ranged from one to 11, with only a small percentage of filaments having only one (5%) or two foci (7%), while 12% contained ≥8 foci (Figure 4E). These results appear similar to F (Figure 4C); however, differences between the two plasmids become more apparent when foci number is normalized to cell length (Figure 4D and F). For P1, most of the filaments (90%) had a micron per focus ratio of ≤2, with the remainder containing up to 5 μm per focus (Figure 4F, black bars). In comparison, when F was localized in these same filaments, 100% of the filaments had a micron per focus ratio of 30 Gram-negative bacteria (Thomas and Helinski, 1989). To determine if plasmid dynamics are conserved among Gram-negative bacteria, we localized a GFP-tagged version of RK2 in two other species: P.aeruginosa and V.cholerae. We chose P.aeruginosa because of its evolutionary distance from E.coli. Vibrio cholerae was chosen because it contains two separate circular chromosomes rather than one (Trucksis et al., 1998; Heidelberg et al., 2000). Since these two chromosomes differ in size by 2 Mb, the timing of replication initiation, termination or segregation is likely to be regulated differentially. Therefore, V.cholerae potentially represents a very different way of orchestrating DNA dynamics in bacteria. We constructed a derivative of RK2 (pZZ15) that contains a 10 kb lacO array and expresses GFP–LacI from the arabinose promoter of E.coli. Binding of GFP–LacI to the lacO array produces green fluorescent foci, indicating the subcellular position of the RK2 plasmid (Straight et al., 1996; Gordon et al., 1997; Belmont, 2001). When GFP–LacI is induced by the addition of arabinose to a culture of the V.cholerae strain JP909 (O395N1/pZZ15), green fluorescent foci are observed in 99% of the cells containing pZZ15 (Figure 6A), while green fluorescence fills the cytoplasm of the cells containing the control plasmid pZZ16, which expresses GFP–LacI in the absence of lacO sequences (Figure 6B). When grown in minimal glucose media, 95% of the cells contained from one to three foci, with a maximum of five or six foci occurring in only a small (1%) percentage of cells (Figure 6). In E.coli, the average number of foci per cell increases substantially with growth rate; however, in V.cholerae, the number of foci per cell changes only slightly between growth in LB glucose and minimal glucose (Figure 7). We quantitated the subcellular distribution of the foci by measuring the length of the cell and the position of the foci with respect to one cell pole. A single focus occurred primarily near midcell (Figure 6C), while two foci occurred primarily near the one-quarter and three-quarters cell positions (Figure 6D). Figure 6.Localization of GFP-tagged RK2 in Vibrio cholerae (A–D) and Pseudomonas. aeruginosa (E–H). Fluorescence micrographs showing a field of V.cholerae (A) or P.aeruginosa (E) in which the cell membranes were stained red with FM 4-64, and GFP–LacI was expressed from plasmid pZZ15 by induction with 0.2% L-arabinose as described in Materials and methods. Cells containing the control plasmid pZZ16 are shown in (B) and (F). White bars = 1 μm. Subcellular distribution of RK2 in V.cholerae for cells (n = 144) containing one focus (C) or two foci (D) and in P.aeruginosa for cells (n = 191) containing one focus (G) or two foci (H). the positions of GFP foci were measured with respect to one end of the cell (reported as a percentage of cell length) and plotted versus the percentage of total cells. Download figure Download PowerPoint Figure 7.Histogram showing the number of GFP-tagged RK2 foci per cell in V.cholerae strain JP909 in different growth conditions. The percentage of total cells is plotted versus the number of foci per cell during growth in M63 glucose at 30°C (black bars, 470 total cells) and LB glucose at 30°C (gray bars, 374 total cells). Download figure Download PowerPoint Similar results were found when we localized RK2 in P.aeruginosa strains JP977 (PAO1/pZZ15) and JP978 (PAO1/pZZ16) (Figure 6). When these strains were grown in the presence of arabinose, green fluorescent foci only occurred in cells of the strain (PAO1/pZZ15) containing the lacO array (Figure 6E). In LB glucose media, most of these cells (78%) contained between one and three foci, with a maximum number of four foci per cell. A single focus occurred primarily near midcell (Figure 6G), while two foci occurred preferentially near the one-quarter and three-quarters cell positions (Figure 6H). These results are strikingly similar to those obtained in V.cholerae and to those previously reported in E.coli
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