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ydek1
zbasz1
ukasz Wojtasz1aw Dziadek3
ska1,2
1 Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. Weigla 12, 53-114 Wrocaw, Poland
2 Faculty of Biotechnology, University of Wroclaw, ul. Tamka 2, 50-137 Wroclaw, Poland
3 Medical Biology Center, Polish Academy of Sciences, Lodowa 106, 93-232 ód
, Poland
Correspondence
Jolanta Zakrzewska-Czerwiska
zakrzew{at}iitd.pan.wroc.pl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-proteobacterial chromosomes) contain parS sequences and parAB genes encoding partitioning proteins, i.e. ParA (ATPase) and ParB (DNA-binding proteins) that are components of the segregation machinery. Here, mycobacterial parABS elements were characterized for the first time. parAB genes are not essential in Mycobacterium smegmatis; however, elimination or overexpression of ParB protein causes growth inhibition. Deletion of parB also leads to a rather severe chromosome segregation defect: up to 10 % of the cells were anucleate. Mycobacterial ParB protein uses three oriC-proximal parS sequences as targets to organize the origin region into a compact nucleoprotein complex. Formation of such a complex involves ParB–ParB interactions and is assisted by ParA protein.
Three supplementary figures showing the construction of the M. smegmatis 
parB mutant, purified mycobacterial ParAB proteins and protein sequence alignment of homologous ParB proteins, and a supplementary table listing the oligonucleotides used in this study are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; and see http://www.who.int/topics/tuberculosis/en/). Due to the spread of multi-drug-resistant strains of M. tuberculosis and a synergy with HIV, the TB epidemic is growing and becoming more dangerous in both developing and industrialized countries. A unique feature of M. tuberculosis is its ability to maintain a dormant, non-replicating state for extended periods of time under unfavourable conditions. The mechanism (or mechanisms) by which this bacterium shifts to a dormant state and reverts to active growth is not well understood (Hampshire et al., 2004; Gómez & Smith, 2000; Wayne & Hayes, 1996: Wayne & Sohaskey, 2001). Knowledge regarding the steps of the mycobacterial cell cycle (replication, chromosome segregation and cell division) seems to be critical for understanding the mechanisms that are responsible for the transition from an active to a non-replicative persistent state (and vice versa) of pathogenic mycobacteria, particularly M. tuberculosis. While initiation of chromosome replication (Madiraju et al., 2006; Qin et al., 1999; Rajagopalan et al., 1995; Zawilak et al., 2004) and cell division (FtsZ ring formation) (Chauhan et al., 2006; Dziadek et al., 2002, 2003; Huang et al., 2007; Rajagopalan et al., 2005) are relatively well studied in mycobacteria, nothing is known about the segregation of chromosomes in these bacteria.
Bacterial chromosome segregation has been recently found to be an active and complex process closely coupled with replication (see Bartosik & Jagura-Burdzy, 2005; Errington et al., 2005; Hayes & Barilla, 2006; Leonard et al., 2005 for reviews). In bacteria studied to date, the newly synthesized origin (oriC) regions undergo a symmetric or asymmetric segregation process; two copies of the duplicated oriC regions migrate from the cell centre toward opposite cell poles, i.e. to the 1/4 and 3/4 positions (Escherichia coli, Bacillus subtilis, Vibro cholerae chromosome II), or one copy of the newly synthesized origins remains at the pole while the other copy migrates to the opposite pole (Caulobacter crescentus, Vibrio cholerae chromosome I) (Fogel & Waldor, 2006; Lewis, 2004; Teleman et al., 1998; Viollier et al., 2004; Webb et al., 1997).
Among the large number of proteins involved in chromosome segregation, ParAB homologues were the earliest to be identified and are the best studied, particularly in B. subtilis (Lewis & Errington, 1997; Lin et al., 1997; Webb et al., 1997), C. crescentus (Mohl & Gober, 1997; Mohl et al., 2001), Pseudomonas spp. (Bartosik et al., 2004; Godfrin-Estevenon et al., 2002; Lewis et al., 2002), and Streptomyces coelicolor (Jakimowicz et al., 2002; Kim et al., 2000). Homologues of ParAB proteins are encoded in most bacterial chromosomes (the E. coli chromosome is one of the few exceptions) (Bartosik & Jagura-Burdzy, 2005; Bignell & Thomas, 2001). Only in C. crescentus have the parA and parB genes been proved to be essential (Mohl & Gober, 1997). Recent library screening (transposon mutagenesis) suggested that parAB genes may also be indispensable for M. tuberculosis H37Rv (Sassetti et al., 2003). parAB mutants of the other organisms display perceptible defects in chromosome segregation, including the formation of anucleate cells. In all studied bacteria, ParB (Spo0J in B. subtilis) proteins interact with parS sites (14–16-mer palindromic sequences), usually located around the oriC region (Jakimowicz et al., 2002; Lin & Grossman, 1998). Binding of ParB to these sites leads to the formation of large nucleoprotein complexes (segrosomes). ParA (Soj in B. subtilis) homologues have a weak ATPase activity and interact with the segrosome (via direct contact with ParB), but their function in segregation is not clear. Recently, Waldor's group have demonstrated that ParAI forms a dynamic structure that pulls the ParBI-bound V. cholerae chromosome I to the opposite pole (Fogel & Waldor, 2006).
Besides pathogenic, slow-growing species (M. tuberculosis and Mycobacterium leprae), the genus Mycobacterium also includes saprophytic, ‘fast-growing’ species such as Mycobacterium smegmatis, which would be useful for studying some aspects of mycobacterial biology, including chromosome segregation. Detailed studies of mycobacterial chromosome segregation and characterization of the key molecules involved in this process may also be helpful in identifying novel drug targets.
In this study, we tested whether the parB gene in M. smegmatis is essential and we have demonstrated that alteration of its expression affects the growth of M. smegmatis. We have also characterized interactions of mycobacterial ParB with its target, parS sequences.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Enzymes were supplied by Fermentas, New England BioLabs or Roche; isotopes were from Amersham–Pharmacia Biotech, and oligonucleotides were from the Institute of Biochemistry and Biophysics (Warsaw, Poland). E. coli BL21 [B F– dcm ompT hsdS(
) gal] was the host for overproduction of the GST–ParA fusion proteins. E. coli strains were grown in Luria–Bertani (LB) medium at 37 °C. M. smegmatis mc2155 and its derivatives were grown in Middlebrook 7H9 medium, on 7H10 agar plates supplemented with oleic acid albumin/glucose/sodium chloride (OADC) or in NB broth [8.0 g l–1 nutrient broth (Difco), 10.0 g l–1 glucose, supplemented with 0.2 % Tween 80, pH 6.0–6.2]. For selection, kanamycin (25 µg ml–1) was used if required.
Construction of ParB overproduction strains.
The M. smegmatis parB gene was PCR amplified using the primers MsparB_fw and MsparBrv-2 (see Supplementary Table S1, available with the online version of this paper) and cloned into the BamHI/XbaI sites of the pJam2 shuttle vector (Triccas et al., 1998) downstream of the pami promoter. The resulting construct, carrying parB controlled by pami, was introduced into M. smegmatis by electroporation. The presence of plasmid DNA was confirmed by recovery as described previously (Dziadek et al., 2003).
Targeted gene replacement.
To perform unmarked deletions in the parB (MSMEG6938) gene of M. smegmatis, a suicide recombination delivery vector was constructed. In the first step, the 5' end of the parB gene (28 bp) and its upstream region (1319 bp) were amplified using the primers MsParGR1 and MsParGR2 (see Supplementary Table S1) and cloned into the HindIII/BamHI sites of p2NIL, creating pABX. Subsequently, the 3' end of the parB gene (529 bp) and its downstream region (773 bp) was amplified using the primers MsParGR3 and MsParGR4 (see Supplementary Table S1) and cloned into the BamHI/KpnI sites of pABX. The ligated 5' and 3' fragments of parB gene in the resulting vector (pABY) were out of frame. Finally, a 6 kb PacI marker cassette from pGOAL17 carrying the lacZ and sacB genes was cloned into the PacI site of pABY to create pABZ.
The protocol of Parish & Stoker, (2000) was used to disrupt the investigated genes at their native loci on the chromosome. The plasmid DNA (pABZ) was treated with NaOH (0.2 mM) and used for transformation in order to integrate it into the M. smegmatis chromosome by homologous recombination. The resulting SCO (single-crossover recombinant) mutant colonies were blue, KanR and sensitive to sucrose. The SCO strain was further processed to select for double-crossover (DCO) mutants that were white, KanS and resistant to sucrose (2 %). PCR and Southern hybridization were used to distinguish between the wild-type and the DCO mutant (see Supplementary Fig. S1, available with the online version of this paper). A probe that hybridizes to the parB gene was generated by PCR, with MsparB_fw and MsparB_rv as primers (Supplementary Table S1) and pABY as a template.
Characterization of the parB mutant strain: growth and doubling time determination.
Culture of the M. smegmatis wild-type and mutant parB strains was performed in rich media (NB or 7H9 Middlebrook supplemented with OADC) with an initial OD600 of 0.1. Then the cells were incubated for 24 h and samples were collected every 3 h for optical density analysis, c.f.u. determination and microscopic examination.
Microscopy.
Mycobacterial cells (48 h cultures) were permeabilized by exposure to toluene (2 %) for 10 min prior to staining of DNA. The M. smegmatis cells were washed, resuspended in phosphate-buffered saline (PBS; 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 15 mM KCl), and stained with DAPI (2 µg ml–1) for 15 min at room temperature. The samples were examined under a Zeiss Axio Imager Z1 equipped with a x100 objective.
RNA analysis.
RNA isolation was performed using a modified Kirby mix as described previously (Kieser et al., 2000). Briefly, M. smegmatis was grown in modified Sauton's broth (Mordarska et al., 1972) at 37 °C. After 48 h the cells were centrifuged and the pellet was resuspended in the modified Kirby mix. The cells were disrupted in the presence of glass beads by vortexing. Subsequent steps of RNA isolation were carried out according to Chomczynski & Sacchi (1987).
For RT-PCR, total RNA was treated with RNase-free DNase I (Roche Molecular Biochemicals), followed by phenol/chloroform extraction and precipitation. The RT-PCR reactions were carried out using a RevertAid First Strand cDNA Synthesis kit (MBI Fermentas) according to the manufacturer's protocols. RNAs (3.6 µg per reaction) were reverse transcribed with RevertAid M-MuLV reverse transcriptase with random hexanucleotides at 42 °C for 90 min A sample of 2.5 µl of the 20 µl RT solution was subjected to subsequent PCR. The amplification reaction was carried out in a 50 µl volume using the pair of appropriate primers (Supplementary Table S1) for 30 cycles. RT-PCR products were analysed by agarose gel electrophoresis.
Purification of mycobacterial ParAB proteins.
The vectors pGEX-6P-2 and pET-28a(+) were chosen to overexpress the recombinant fusion proteins GST–ParA and 6His–ParB of M. tuberculosis (Mt) and M. smegmatis (Ms). The parA and parB genes of M. smegmatis or M. tuberculosis were PCR amplified with the appropriate pairs of primers (Supplementary Table S1). The PCR products were cloned into the vector pGEM-T Easy (Promega) and later recloned as BamHI–EcoRI fragments into pGEX-6P-2 (parA genes) or into pET28a(+) (parB genes). All the PCR-derived clones were analysed by DNA sequencing to check their fidelity. The fusion proteins, glutathione S-transferase (GST)–ParA or 6His–ParB were purified on a glutathione Sepharose 4B or on a Ni2+-NTA-agarose column (Qiagen), respectively, as described previously (Majka et al., 1999; Zawilak et al., 2004). For removal of the GST part, the bound fusion proteins were treated with the PreScission protease (Amersham–Pharmacia Biotech) and the ParA proteins were released from the beads. The purified ParA proteins were more than 95 % homogeneous, as judged by SDS-PAGE analysis (see Supplementary Fig. S2, available with the online version of this paper).
Electrophoretic mobility shift assay (EMSA).
For the binding assays, 5'-end-32P-labelled DNA fragments (5 fmol) were incubated with ParB protein in the presence of the non-specific competitor poly[(dA-dC)(dT-dG)] (100 ng) at 20 °C for 20 min in binding Marians' buffer (1x Marians' buffer: 20 mM HEPES/KOH, pH 7.6, 5 mM magnesium acetate, 1 mM EDTA, 4 mM DTT, 0.2 % Triton X-100, 1 mM ATP, 0.5 µg BSA µl–1) (Parada & Marinas, 1991). The bound complexes were separated by electrophoresis in 4 % polyacrylamide gels [0.25xTBE (Sambrook et al., 1989), at 4 V cm–1, 4 °C]. The gels were dried and analysed with a Typhoon 8600 Variable Mode Imager.
Electron microscopy.
Electron microscopic analysis of ParB–DNA interactions was performed according to a previously described method (Szalewska-Palasz et al., 1998). Briefly, the DNA fragments harbouring parS sequences were PCR-amplified (Supplementary Table S1). The M. smegmatis ParB protein was incubated at room temperature with the DNA fragment in Marians' binding buffer (as described above, but without BSA). After fixation in 0.2 % glutaraldehyde (30 min, room temperature), samples were prepared for electron microscopy by adsorption to mica (Spiess & Lurz, 1988). They were analysed with a transmission electron microscope (Philips CM100) at 100 kV.
Preparation of antisera.
Antisera were obtained by immunization of rabbits with purified M. smegmatis ParB protein mixed with monophosphoryl lipid A (MPL) adjuvant (a derivative of lipid A from Hafnia alvei PCM 1200) (Chodaczek et al., 2006). The first booster injection was given at 3 weeks and the second booster after a further 2 weeks. Serum samples were taken 10 days after the second booster injection. Cellular particles were removed by centrifugation (500 g), and the serum was stored at –20 °C. IgG was purified by ammonium sulfate precipitation (to 40 % saturation) and then dialysed against 5 mM Tris/HCl. Anti-ParB antibodies were purified by affinity chromatography. The affinity column was prepared by immobilization of 5 mg purified recombinant ParB protein on CNBr-activated Sepharose 4B, according to the manufacturer's recommendations. Antibodies in PBS (pH 7.3), were applied to the affinity column, washed with PBS, eluted with 1 mM propionic acid, and dialysed against PBS. The recombinant MtParB was also recognized by antibodies (72 % identity between MtParB and MsParB proteins; data not shown).
SDS-PAGE and Western blotting.
SDS-PAGE was performed according to the method established by Laemmli (1970). Proteins were separated by SDS-PAGE (10 % or 12 % acrylamide) and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h at room temperature with TBST (10 mM Tris/HCl, pH 8.0, 100 mM NaCl, 0.05 % Tween 20) containing 3 % BSA and subsequently incubated with polyclonal anti-ParB antibody. Afterwards the membrane was incubated with a goat anti-rabbit secondary antibody conjugated with alkaline phosphatase. The membrane was stained with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. The amount of cellular ParB protein was quantified (ImageQuant software, Molecular Dynamics) by scanning an immunoblot containing different amounts of the 6His–MsParB fusion protein.
GST pull-down.
Affinity chromatography was performed as described previously (Majka et al., 1997). Briefly, 25 µg ParA protein fused to the C terminus of GST was bound to glutathione–Sepharose beads and then used as an affinity reagent to evaluate its interaction with ParB. Beads bound to GST–ParA were incubated with the purified ParB protein in buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM MgCl2, 0.1 % Tween and 1 mM ATP and then were extensively washed with buffer containing 150 mM NaCl and 0.1 % Tween. The bound protein was eluted with increasing salt concentrations [20 mM Tris, (pH 7.5) 300, 500, 700 or 1000 mM NaCl] and analysed by Western blotting with anti-MsParB antibodies.
Immunoprecipitation assay.
M. smegmatis and its parB deletion mutant cultivated for 48 h in modified Sauton's broth (Mordarska et al., 1972) were cross-linked by addition of formaldehyde (final concentration: 1 %) for 5 min as described previously (Solomon & Varshavsky, 1985). Cells not subjected to cross-linking and mutant strains served as negative controls and were treated subsequently in the same way as the experimental samples. Subsequent steps of the immunoprecipitation assay were carried out according to Jakimowicz et al. (2002). Briefly, cell extracts were prepared by resuspension of 
50 mg cells in 200 µl PBS buffer and sonicated (2x20 s). After centrifugation, 500 µl IMP buffer (50 mM Tris/HCl, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1 mg BSA ml–1, 2 % Nonidet P40, protease inhibitors) and 20 µl immunopurified anti-ParB antibody were added to the cell extracts. Immunoprecipitation was carried out overnight with gentle agitation at 4 °C followed by binding of immunocomplexes to proteinA–Sepharose (Sigma; 4 h at 4 °C). The Sepharose slurry was washed five times with IMP-wash buffer (50 mM Tris/HCl, pH 7.5, 300 mM NaCl, 0.5 % Nonidet P40, 0.1 % SDS), and then cross-links were reversed by overnight incubation of the slurry with proteinase K (0.2 mg ml–1), 1 % SDS at 55 °C.
Immunoprecipitated DNA released from the protein complexes was treated with phenol, ethanol precipitated and resuspended in TE buffer (Sambrook et al., 1989). Semiquantitative PCR reactions (25 cycles) on precipitated DNA were set up with six primer sets (Supplementary Table S1) amplifying DNA fragments of 400 bp. The input DNA (not immunoprecipitated), subjected to the same cross-link reversal and purification procedures as the experimental samples, served as a positive control for PCR. Following electrophoresis, the gel was analysed using a PhosphorImager and ImageQuant software (Molecular Dynamics).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The identified parAB homologues are candidates for mycobacterial chromosome segregation genes. In addition, we found a third element crucial to chromosome segregation, a set of parS sequences (see below, Fig. 4a).
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Since mycobacterial segregation genes were identified as putative ORFs we first tested whether these genes were expressed in M. smegmatis. Expression of parA and parB genes was proved by RT-PCR (Fig. 1b
) and additionally the presence of ParB protein was confirmed by Western blotting. A rabbit polyclonal antibody was raised against purified MtParB protein. The affinity-purified anti-6His MtParB rabbit antibodies specifically detected an 40 kDa protein (corresponding in size to the deduced parB gene product) in an M. smegmatis strain (Fig. 2). The antibodies were also used to determine the number of ParB molecules by Western blotting of cells of M. smegmatis. By using various defined amounts (10–100 ng) of purified 6His–MsParB protein as standards, the amount of ParB protein (at the end of the exponential phase) was found to be approximately 4000 molecules per single cell (0.1 % of total soluble cellular protein) (the intensities of bands were compared with the standards and the amount of ParB protein was determined by quantitative densitometry; data not shown).
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). To investigate whether transcription of the parAB genes is under the control of this promoter, a comprehensive RT-PCR analysis was performed (Fig. 1b). Total RNA extracted from M. smegmatis was used in RT-PCR experiments with seven sets of primers designed to amplify the regions spanning the gene junctions (Supplementary Table S1, Fig. 1b). A reaction without reverse transcriptase was carried out as a negative control in all cases to ensure that the resulting DNA fragment was not due to amplification of contaminant DNA. RT-PCR products were observed for each pair of primers, while the corresponding products in the negative controls were absent (Fig. 1b). Thus, the transcriptional analysis results suggest that parAB as well as other genes from this cluster may be co-transcribed. However, the mRNA used for transcription analysis was isolated from cells propagated under laboratory conditions; thus the existence of the other parAB promoter(s) that were not detected in this particular experimental set cannot be ruled out.
Lack or excess of ParB protein causes growth inhibition of M. smegmatis
Library screening has suggested previously that ParB protein is essential in M. tuberculosis H37Rv (Sassetti et al., 2003). To verify that parB is also essential for the viability of M. smegmatis, we attempted to construct a strain defective in the production of this protein. A two-step recombination protocol (Parish & Stoker, 2000) was used to generate an unmarked deletion within the M. smegmatis chromosome. Surprisingly, the parB gene could be easily deleted from the M. smegmatis chromosome. The resultant strain, M. smegmatis parB, was verified by PCR, Southern hybridization (see Methods and Supplementary Fig. S1), and Western blotting (Fig. 2, lane 3). The viability of this strain shows that parB is not essential in M. smegmatis (Fig. 3a). However, in rich medium (7H9/OADC), growth of the parB deletion strain was delayed and it exhibited a longer lag phase than was seen with the wild-type (Fig. 3b).
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parB strain was subjected to analysis by fluorescence microscopy. Staining with DAPI to visualize DNA revealed that 10.3 % of the cells were anucleate compared with 0.8 % of the wild-type (222 of 2165 parB cells and 15 of 1915 wt cells counted) (Fig. 3c). Thus, the lack of ParB affects segregation of chromosomes and this may influence growth of M. smegmatis.
The expression of parB as well as other genes from the operon analysed is under the control of a single promoter (Salazar et al., 2003) recognized by mycobacterial housekeeping sigma factor (homologous to E. coli 
70); we therefore tested whether altering ParB expression affected growth and segregation. A M. smegmatis mutant overexpressing ParB upon acetamide induction (pami promoter) was constructed (for details see Methods). Overexpression of ParB (10 times increased protein level) was verified by Western blot analysis using purified antibodies against MsParB protein (Fig. 2, lane 4). In the presence of an inducer (0.4 % acetamide), the ParB overproducer grew more slowly than the wild-type strain as analysed by c.f.u. and growth density analysis (Fig. 3a, b). Surprisingly, in contrast to the parB strain, the ParB overproducer showed no obvious chromosome segregation defect, as demonstrated by microscopic examination including DNA visualization (data not shown).
ParB protein binds only oriC-proximal parS sequences in vivo
In all the studied bacteria, ParB fulfils its function by binding to numerous parS sites in the oriC-proximal part of the chromosome. The consensus sequences of parS determined for Streptomyces coelicolor, a bacterium closely related to Mycobacterium, is 5'-GTTTCACGTGAAAC-3' (Jakimowicz et al., 2002). A database search for this palindromic sequence allowed us to identify two perfect copies in both M. tuberculosis and M. smegmatis chromosomes. In addition, we found five (Fig. 4a) and three (data not shown) putative ParB-binding sites with one, two or three mismatches from the perfect one in M. smegmatis and M. tuberculosis, respectively. Interestingly, the identified parS sequences are not clustered in the oriC-proximal part of the chromosome as in the other bacteria (e.g. C. crescentus or B. subtilis), but scattered over the chromosomes. To address the question of whether the putative parS sequences are bound by ParB in vivo and to find out how their localization and/or sequence affects their recognition by the protein, we performed immunoprecipitation of ParB–DNA complexes. In intact M. smegmatis cells, the proteins were cross-linked to DNA and then the ParB–DNA complexes were selectively immunoprecipitated using purified antibodies against the MsParB protein (Fig. 4b; see Methods). After isolation of DNA from the immunoprecipitated complexes, the ParB-bound DNA fragments were amplified by PCR using primers designed to flank the seven potential parS sequences: parS1–parS7 (Fig. 4b) In addition, DNA was amplified with primers flanking a DNA fragment that does not contain a putative parS sequence and is distant from the other parS sequences (Fig. 4a, b, pnull). A parB deletion mutant of M. smegmatis served as a negative control. As shown in Fig. 4(b), two strong parS sequences (parS1 and parS2) were clearly detected in the ParB immunoprecipitate from the wild-type strain and none of them was detected in the equivalent samples from the deletion parB mutant. Only one sequence, parS3 (located in the vicinity of perfect parS sequences), of the remaining five putative parS sequences could be amplified, although more weakly than the perfect parS sites. Thus, our results suggest that only the oriC-proximal parS sites are engaged in the formation of the segregation complex.
Binding of ParB to parS sequences in vitro involves ParB–ParB interaction and is assisted by ParA
ParB homologues are known to be able to assemble into large complexes (Breier & Grossman, 2007; Jakimowicz et al., 2002), which encompass a large fragment of DNA. This may result from the formation of high-molecular-mass complexes on DNA. To study the properties of the ParB complexes formed in vitro, electron microscopy (EM) and EMSA were performed. For both types of experiments we used a PCR-amplified DNA fragment (Supplementary Table S1) harbouring two parS sequences separated by 78 bp (Fig. 5). Analysis of electron micrographs showed that at a ratio of 0.5 : 1 MsParB protein to parS, one or two nucleoprotein complexes per single DNA fragment were observed, while at a ratio of 1 : 1, two complexes were usually present (Fig. 5a). The relative positions of the analysed nucleoprotein complexes corresponded to the positions of the parS sequences (data not shown).
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). At lower protein concentrations, two nucleoprotein complexes were observed for both ParB proteins. An elevated concentration, particularly in the case of MtParB, caused the formation of higher-molecular-mass complexes, suggesting ParB–ParB interaction(s).
To investigate if ParA–ParB interactions contribute to the formation of ParB complexes, EMSA was performed in the presence of ParA. For this assay we used ‘unfavourable’ binding conditions: a low MtParB concentration, so a substantial amount of DNA was not bound by the protein, and a DNA fragment with two parS sequences separated by a long stretch of DNA (280 bp) to minimize contact between ParB molecules bound to distant parS sequences. Addition of MtParA enhanced ParB–parS interaction, particularly at low ParB concentrations (Fig. 6a), at which ParB did not bind efficiently to the DNA. Thus our data suggest that ParA contributes to nucleoprotein complex formation and presumably increases the affinity of ParB for its target (Fig. 6a). In contrast, incubation of ParA with the DNA fragment did not result in formation of any nucleoprotein complexes, indicating that ParA itself did not bind the parS sequence(s) (Fig. 6a). In order to test whether a direct ParA–ParB interaction occurs, an affinity chromatography assay was performed. In this assay, the MtParB protein was incubated with the GST–MtParA fusion protein bound to glutathione–Sepharose beads. After extensive washing, the bound protein was analysed by immunoblotting with anti-MsParB antibodies (Fig. 6b). MtParB was detected in the GST–MtParA sample, while only a tiny amount of the protein was observed with GST alone (negative control). Thus our in vitro data suggest that ParA interacts with ParB protein, enhancing its affinity for DNA.
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DISCUSSION |
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Our transcriptional analysis (Fig. 1b) suggests that the parAB genes of M. smegmatis may belong to a large operon, which, in addition to the partitioning genes, contains six other genes, including those required for basic cellular functions, encoding GidB, a subunit of RNaseP, and ribosomal protein L34. Interestingly, it has been demonstrated recently that gidB from this operon is a new streptomycin-resistance locus in M. tuberculosis (GidB functions as an rRNA methyltransferase) (Okamoto et al., 2007). This operon is under the control of a single promoter as has been recently demonstrated (Salazar et al., 2003) and is recognized by mycobacterial housekeeping sigma factor, homologous to E. coli 70 (Gómez & Smith, 2000; Salazar et al., 2003). In contrast, parAB genes of S. coelicolor are arranged in a two-gene operon whose transcription proceeds from two promoters. Streptomyces and Mycobacterium belong to the same order of Actinomycetales, but they exhibit substantial differences, particularly in their life cycle: Mycobacterium species are straight or slightly curved bacilli which divide by binary fission, while Streptomyces species are filamentous bacteria with a complex life cycle involving mycelial growth and spore formation (Chater, 2001). In S. coelicolor, one of its two parAB promoters is strongly upregulated shortly before sporulation septation at about the time of the synchronous segregation of several dozen chromosomes into prespore compartments (Kim et al., 2000). As in Streptomyces, the parAB genes of B. subtilis, a spore-forming organism, are organized as a bicistronic operon, but they are transcribed from a single promoter. In the other organisms studied so far (C. crescentus, P. aeruginosa and P. putida), parAB genes are, as in mycobacteria, not independently expressed, but are part of the gidAB operon (Bartosik et al., 2004). Thus, comparison of parAB gene expression among different bacteria suggests that their mode of expression seems to be closely coupled with life cycle.
As in almost all other bacteria studied so far, with the exception of C. crescentus, deletion of the parB gene is not lethal to M. smegmatis. Thus, it would be interesting to verify in vivo if the M. tuberculosis parB gene is essential, as has been proposed previously (Sassetti et al., 2003). Substantial differences in parB requirements between these closely related organisms may be related to their very distinct life styles. However, the lack of ParB in M. smegmatis does cause a quite severe phenotype: growth inhibition (Fig. 3a, b) combined with missegregation of chromosomes – a substantial fraction (10 %) of cells were anucleate. In Pseudomonas putida, deletion of the parB gene had a similar effect on growth and chromosome segregation (up 10 % anucleate cells) (Lewis et al., 2002).
Recently, Lee et al. (2003) demonstrated that Spo0J is not sufficient to recruit ectopic chromosomal parS sequences to the cell quarters of B. subtilis. Thus it was suggested that ParB, instead of being involved in origin movements, presumably plays a role in the organization and localization of this region during the last step of the cell cycle, i.e. cell division. We demonstrated, using in vivo immunoprecipitation of ParB–DNA complexes, that the three origin-proximal parS sequences distributed in an 60 kb segment (Fig. 4a, b) are bound by ParB protein, while those located far away were not. Thus, as in other organisms, binding of ParB protein only to sites close to oriC presumably facilitates proper chromosome orientation during cell division of M. smegmatis.
In contrast to M. smegmatis, ParB of S. coelicolor binds to numerous parS sites scattered over a large chromosomal segment (400 kb) that encompasses the oriC region and assembles into a massive nucleoprotein complex (Jakimowicz et al., 2002). We speculate that organisms whose life cycle includes sporulation (Streptomyces, B. subtilis) possess numerous ParB binding sites (Breier & Grossman, 2007; Jakimowicz et al., 2002; Lee & Grossman, 2006; Murray et al., 2006) that are presumably necessary for proper compaction of the oriC region and/or its localization in tiny prespore compartments.
Although the complex assembled in Mycobacterium seems to encompass a smaller chromosome fragment, it apparently still influences the organization of the nucleoid. This may be the result of the higher-order structure subsequently formed after the binding of parS by ParB. Our in vitro EMSA results indicated that ParB binding to parS sequences involves ParB–ParB interactions. Recruitment of additional ParB molecules may cause binding to DNA adjacent to parS; spreading of ParB protein along the DNA around its target has been observed in B. subtilis (Breier & Grossman, 2007) and S. coelicolor (Jakimowicz et al., 2002). Moreover, we showed that ParA, interacting directly with ParB, promotes ParB–DNA interacton in vitro; particularly binding to distant parS sequences (Fig. 6a). Thus, all the elements, ParA, ParB and the parS sequences, are believed to be engaged in the formation of the mycobacterial segregation complex in the oriC-proximal region in vivo.
Overexpression of ParB also caused a delay in growth, even more severe than that of the parB disruption mutant. However, neither chromosome segregation nor condensation defects were observed in cells overexpressing ParB protein. Although we can not exclude that ParB overexpression could be a source of non-specific toxicity in M. smegmatis we assume that excess of ParB presumably led to the formation of massive nucleoprotein complexes around the oriC region. Such complexes may prevent further rounds of replication by reducing origin accessibility for the initiator protein DnaA and consequently delay growth. A similar effect has been observed in B. subtilis (Lee & Grossman, 2006) and S. coelicolor (D. Jakimowicz, unpublished).
Recent studies showed that M. tuberculosis cells in the dormant state are blocked at the cell division stage. In macrophages, M. tuberculosis cells are filamentous and deficient in FtsZ rings (Chauhan et al., 2006). Since bacterial cell division is tightly coupled to chromosome segregation, it would be interesting to examine how chromosome segregation is coordinated with cell division in M. tuberculosis.
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ACKNOWLEDGEMENTS |
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Edited by: C. W. Chen.
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REFERENCES |
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Received 13 July 2007;
revised 20 August 2007;
accepted 23 August 2007.
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), parB deletion derivative (
), and ParB-overproducing (pami, 
) strains by c.f.u. analysis (a) and optical density analysis (b). Each culture was supplemented with 0.4 % acetamide (for induction of the pami promoter). Results are representative of two independent experiments. (c) Examples of images showing cells stained with DAPI. Anucleate cells are indicated by empty arrowheads.
