1 Biochemie, Fachbereich Chemie, Hans-Meerwein-Straße, Philipps-Universität Marburg, 35032 Marburg, Germany
2 Institut für Physiologische Chemie, Karl-von-Frisch-Straße 1, Philipps-Universität Marburg, 35032 Marburg, Germany
Correspondence
Peter L. Graumann
pg32{at}biologie.uni-freiburg.de
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ABSTRACT |
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Present address: Institut für Mikrobiologie, Stefan Meier Str. 19, Albert-Ludwigs Universität Freiburg, 79104 Freiburg, Germany.
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INTRODUCTION |
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SMC proteins are key players in various aspects of chromosome dynamics in most organisms. Their functions include cohesion of sister chromosomes, which is essential for stable segregation of chromosomes during mitosis, DNA double-strand break repair, chromosome compaction and maintenance of chromosome structure (Hirano, 2002). SMC proteins invariably act within protein complexes containing an SMC protein homodimer (in prokaryotic cells) or a heterodimer, and, additionally, non-SMC proteins. In prokaryotes, an SMC protein homodimer forms a complex with ScpA, belonging to the kleisin protein family, and with ScpB, both of which are widely conserved in bacteria and archaea (Mascarenhas et al., 2002
; Soppa et al., 2002
). SMC proteins have an unusual structure: an SMC protein monomer is composed of an ATPase head domain at one end, a long coiled-coil region, and a hinge domain at the other end. SMC protein dimers are formed through a strong and specific dimerization of the hinge domains, and are thus symmetrical molecules with a central flexible hinge, two coiled-coiled arms, and two ATPase-cassette head domains. DNA binding is mediated by embracing with the coiled-coil arms and most likely through ATP-dependent dimerization of the head domains, leading to ring closure around the DNA (Gruber et al., 2003
; Volkov et al., 2003
). In vitro, condensin can introduce positive writhe into DNA, that is it bends the DNA into a right-handed superhelix (Kimura et al., 1999
), through an as yet ill-defined mechanism. This tension can be relieved by Topo I, the net result being that negative supercoiling is introduced into DNA. Indeed, in vivo, condensin and the prokaryotic SMC complex appear to induce chromosome compaction through the introduction of negative supercoiling into DNA. This has been demonstrated because: (a) the reduction of Topo I activity is a suppressor of a mukB (smc in Escherichia coli) deletion (Sawitzke & Austin, 2000
); (b) smc mutant cells are hypersensitive to inhibitors of DNA gyrase (Lindow et al., 2002a
). The bacterial SMC complex localizes in two discrete centres, one within each cell half (Lindow et al., 2002b
; Mascarenhas et al., 2002
), while DNA is replicated by a centrally located replication machinery (Lemon & Grossman, 2000
). Thus, DNA moves through the central replisome, and replicated DNA strands are moved towards opposite cell poles, by an as yet unknown mechanism. Newly replicated DNA is probably locally condensed within the SMC centres, which organize chromosome layout and facilitate segregation (Graumann, 2000
).
In this work, we wished to elucidate the connection between Bacillus subtilis Topo IV and the SMC complex. We found that overproduction of Topo IV suppressed the chromosome condensation defect of an smc deletion, as well as a defect in global protein synthesis caused by the loss of the SMC complex, but not its segregation defect, revealing an intricate connection between SMC protein and Topo IV in vivo.
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METHODS |
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Construction of plasmids and bacterial strains.
To regulate expression of parE and parC, we constructed a strain with both genes under the control of the IPTG-inducible promoter Phyperspank. A 500 bp sequence containing the 5' region of parE was PCR-amplified and cloned into pJQ43 [(Quisel et al., 2001) in which the Phyperspank region had been introduced from pDR111 (kind gift of D. Rudner, Harvard Medical School)] using SphI and NheI sites, to generate pJM45. This plasmid was integrated into PY79 by single-crossover recombination to generate the clone ST16 using Cm and different concentrations of IPTG (1103 mM). Since 101 mM IPTG supported wild-type Topo IV activity, this concentration was used for strain passage and for further genetic manipulation. ST20 was generated by transforming ST16 with chromosomal DNA from PG
388 using kan.
Image acquisition.
Fluorescence microscopy was performed on an Olympus AX70 microscope. Cells were mounted on agarose gel pads containing S750 growth medium on object slides. Images were acquired with a digital charge-coupled device (CCD) camera; signal intensities and cell length were measured using the Metamorph 5.0 program (Universal Imaging Corp., USA). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, final concentration 0·2 ng ml1) and membranes were stained with the vital membrane stain FM4-64 (Molecular Probes, final concentration 1 nM).
Two-dimensional (2D) gel electrophoresis.
The different strains were grown at 23 °C in 500 ml LB broth to OD600 0·5. Cells were collected by centrifugation, washed with ice-cold sterile water, resuspended in buffer (10 mM Tris/HCl, 1 mg MgCl2 ml1, 100 µg DNase ml1, 100 µg RNase ml1, 1·5 mM PMSF) and disrupted by sonication. The supernatant was freeze-dried in liquid nitrogen and lyophilized. The freeze-dried protein sample was precipitated overnight using acetone, and dissolved in 125 µl rehydration buffer [9 M urea, 4 % (v/v) CHAPS, 2 % (v/v) ampholytes pH 47, 0·5 % (v/v) ampholytes pH 310, 10 mM DTT]. 2D gel eletrophoresis was performed using cylindrical tube gels (3x100 mm), and 15 % polyacrylamide slab gels for the second dimension, applying 50 µg total protein from the cell extract, according to the protocol described in Klein et al. (2002). The gels were stained with silver nitrate and dried between two sheets of cellophane. The gels were scanned as 16 bit images (UMAX, PowerLook 2100XL scanner) and analysed with ProteomWeaver software version 2.1 (Definiens, Munich, Germany). The pattern of all of the gels was used to generate an average, and the spots detected were compared with one another in terms of their intensity. SPSS version 11.0 software was used for analysis of the raw data obtained from ProteomWeaver. An unpaired t test was employed for testing the statistical significance of the different mutants on the 2D gels and to compare the mean protein spot intensities with one another.
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RESULTS |
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An increase in synthesis of Topo IV partially suppresses an smc deletion
The global effect of Topo IV on chromosome condensation led us to speculate that the defect in chromosome supercoiling due to an smc deletion could be suppressed by varying levels of Topo IV. To address this question, we moved the PhyperspankparE construct into an smc null background and grew the cells at permissive temperature (23 °C) with different levels of IPTG. At 0·1 mM IPTG (which would correspond to an smc null background with a wild-type level of Topo IV), cells displayed a typical smc phenotype, that is, chromosomes were decondensed and anucleate cells arose at a frequency of 1520 % (Fig. 2H, indicated by arrowhead). Lowering the Topo IV levels in smc mutant cells further exacerbated the smc phenotype: nucleoids were further decondensed and extended, and 25 % anucleate cells were counted at 0·01 mM IPTG (Fig. 2I
). Growth almost ceased at this level of inducer. In contrast, growth of mutant cells in the presence of 1 mM IPTG resulted in markedly more condensed chromosomes in 80 % of the cells (Fig. 2G
). Chromosomes were still less condensed than in wild-type cells, but the difference was rather subtle. These experiments show that overproduction of Topo IV suppresses the chromosome-condensation defect in smc mutant cells. We also measured the growth of cultures from cells of the wild-type, smc mutant and smc mutant with overexpressed Topo IV. It is apparent from Fig. 1
that increased expression of Topo IV in an smc mutant background led to a higher growth rate than that of the smc deletion strain, but the rate of growth was still considerably lower than that of wild-type cells. Smc null cells overexpressing Topo IV were able to grow at 30 °C, but not at 37 °C, whereas smc null cells were only able to grow below 23 °C (Table 1
). Additionally, even with increased levels of Topo IV, 510 % anucleate cells were observed (Table 1
). Thus, overproduction of Topo IV partially suppresses the loss of function of SMC protein, in that it almost fully rescues the condensation defect, but only partially reduces the severity of the segregation and growth defects.
Deletions of smc, scpA or scpB differentially affect global protein synthesis
The link between Topo IV and the SMC protein prompted us to investigate if the SMC complex has a global effect on supercoiling, that is, if it influences global transcription throughout the chromosome, in a similar manner to Topo I, Topo IV and DNA gyrase. We chose an indirect approach, through analysing the pattern of protein expression, which is tightly linked to rates of transcription in bacteria. We performed 2D gel analysis of cells growing exponentially at 23 °C in rich medium (which is permissive for smc, scpA and scpB mutant cells), and analysed the rate of synthesis of cytosolic proteins. We were able to detect 420 discrete protein spots, corresponding to approximately the same number of proteins, in cell extracts from wild-type and mutant cells (Fig. 3). Of these, 81 reproducibly showed different levels of expression (that is in at least two independent experiments), with 46 spots being expressed at a lower level in the smc mutant cells, and 35 having a higher expression level than that of wild-type cells (Fig. 3A, B
). Thus, the synthesis of at least 20 % of the cytosolic proteins was affected by the smc deletion. Likewise, 82 protein spots had considerably altered levels in the scpB mutant strains (Fig. 3C
), as well as a similar number in scpA mutant cells (data not shown). Strikingly, many proteins that were down-regulated in smc mutant cells had a higher level in scpB mutant cells, compared to wild-type cells, and vice versa. For example, EF-G had a lower level in smc mutant cells, but showed an increased level in scpB mutant cells, while the opposite was true for spot 11 (Table 2
). Thus, loss of different parts of the SMC complex had an effect on the synthesis of different groups of proteins.
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Increased synthesis of Topo IV rescues the defect in protein synthesis in smc mutant cells
The link between SMC protein and Topo IV with respect to chromosome compaction prompted us to investigate if an increase in Topo IV level in smc mutant cells might influence global protein synthesis via an effect on transcription. Strikingly, at 1 mM IPTG, phyperspankparE smc mutant cells showed a pattern of protein synthesis that closely resembles that of wild-type cells (Fig. 3D). Of the 81 protein spots that showed increased or reduced intensity in smc mutant cells compared to wild-type cells, upon the overexpression of Topo IV, 74 had an intensity closely resembling that of wild-type cells. Except for S6, which had a lower expression in smc null and scpB-deleted cells, all of the marked spots in Fig. 3
had a similar intensity between wild-type and smc null cells with overexpressed Topo IV. To verify our visual observations, we employed the Proteomweaver 2.1 software, which compares and evaluates spot intensities from 2D gels. Table 3
shows that there was a highly significant difference (P< 0·05) in overall protein synthesis between wild-type (PY79) and smc mutant (P
388) cells for a large number of the spots analysed. Likewise, smc mutant cells and ST20 cells (smc null cells in which Topo IV is overproduced) showed a highly significant difference in global expression levels, while there was no significant overall difference between PY79 and ST20 cells (Table 3
, P>0·05). However, statistically, there was no major difference between smc and scpB (PG32) mutant cells (Table 3
, P>0·05), showing that although several proteins showed different expression levels between the mutant strains, most of the proteins had similar levels (i.e. similarly different levels from wild-type cells). These data analyses verify the conclusions drawn from the visual inspection of the 2D gels and show that increased production of Topo IV in smc mutant cells results in protein expression that is similar to that in wild-type cells and thus rescues the defect in protein synthesis in smc mutant cells. Because Topo IV acts at the level of transcription, these data suggest that the defect in protein synthesis in smc mutant cells is based on a defect in transcription, due to loss of supercoiling, which is compensated by an increase in Topo IV activity. Moreover, these data show that Topo IV can rescue cell growth in smc mutant cells largely by suppressing the defect in supercoiling (and thus the defect in transcription), but cannot fully rescue the growth defect, since the defect in chromosome segregation is still severe.
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DISCUSSION |
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Fourthly, our work suggests that the SMC complex has a dual function: in chromosome compaction and protein synthesis (via general supercoiling), which can be suppressed by overproduction of Topo IV; and in active chromosome segregation, which can only partially be rescued by Topo IV, because the formation of anucleate cells is reduced by only half through overproduction of Topo IV. Even if general supercoiling is brought to wild-type levels, chromosomes still fail to be separated in smc mutant cells, accompanied by slower growth and temperature sensitivity above 30 °C (compared to wild-type cells). Possibly, the special function of the SMC complex in chromosome segregation is brought about by the ability to form discrete subcellular centres on the nucleoids, predominantly one in each cell half (Mascarenhas et al., 2002), from which the complex influences compaction of the whole chromosome (Volkov et al., 2003
), and also mediates its crucial function in the segregation of chromosomes after the separation of origin regions (Graumann, 2000
). We favour the view that from the middle of the cell (where replication takes place), newly replicated DNA is somehow brought to the bipolar SMC condensation centres, where DNA from each sister chromosome is locally condensed, and thereby pulled into each cell half. We have recently found evidence that in vitro SMC protein forms rosette-like structures containing multiple SMC protein molecules (Mascarenhas et al., 2005
), which may connect many DNA loops bound by individual SMC protein dimers, and which could be the basis for the formation of the observed condensation centres in vivo.
In toto, our results suggest that the bacterial nucleoid is compacted through a combined action of Topo I, SMC complex, DNA gyrase and Topo IV, and that the latter three introduce net negative supercoiling, which is counterbalanced by Topo I. The genetic interactions with DNA gyrase and Topo IV underline the intricate interplay of the SMC complex with topoisomerases, yet reveal the distinct role of the SMC complex in active chromosome segregation. The molecular bases underlying these processes will be interesting to study in detail in future work.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 May 2005;
revised 3 August 2005;
accepted 15 August 2005.
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