The partitioning protein ParB of SLP2 binds palindromic repeats in its own coding sequence

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1 The partitioning protein ParB of SLP2 binds palindromic repeats in its own coding sequence Chin-Chen Hsu 1, Carton W Chen 1 1 Faculty of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan Correspondence: Carton W Chen Faculty of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan address: cwchen@ym.edu.tw Telephone number: Fax number:

2 Abstract Many low-copy-number plasmids encode a partitioning system to ensure proper segregation of the daughter molecules into the dividing cells. In action, the ATPase protein ParA is recruited by ParB protein, which binds specifically to the centromere-like sequence of the plasmid, pars. Two large circular plasmids (psv1 and SCP2*) in Streptomyces also contain such partitioning system, which has been shown to be important for the segregation stability of these plasmids (SCP2*). However, nothing is known about the partitioning systems for linear plasmids in Streptomyces. SLP2 is a 50-kb low-copy-number linear plasmid in Streptomyces. It contains homologs of para (SLP2.30c) and parb (SLP.29c). An SLP2 derivative containing a deletion of para and parb was created and introduced into S. coelicolor M145. After one round of sporulation, about 90% of the spores lost the plasmid. The same SLP2 derivative, when placed in J2538 (a chromosomal parab mutant of M145) was lost in 99% of the spores after one round of sporulation. These results indicate that the par system is important in the stable maintenance of SLP2, and the chromosomally-encoded parab proteins may partially compensate for parab mutations on SLP2. The ParB-binding (pars) sequence on SLP2 was identified by affinity chromatography and DNaseI footprinting to two ~30-bp sequences within the parb coding sequence, each containing two complete or partial 8-bp inverted repeats (CGTTAACG). Interestingly, these octamer repeats are in the same reading frame, encoding the same aa sequence. SLP2, like another linear plasmid SCP1, does not contain the chromosomal pars sequence (GTTTCACGTGAAAC), a significantly larger palindrome. These results suggest a possibly novel par system for the linear Streptomyces plasmids.

3 Introduction Partition of bacterial chromosome and low-copy number plasmids require an active and accurate positioning process that ensures proper segregation of chromosomes and plasmids at cell division. Active partition systems have been also identified in both chromosomes and many low copy number plasmids (Bignell and Thomas, 2001). This par system is composed of two proteins, ParA and ParB, and a cis-acting site, pars. ParB is a DNA binding protein, which bind specifically to one or more centromere-like sites, pars. ParB will also recruit another protein ParA, which is a membrane-associated ATPase required for the symmetric movement of ParB-parS complex. The par system also works in Streptomyces, which has the para and parb homologs on chromosome and plasmids (Bentley et al., 2004; Haug et al., 2003; Kim et al., 2000). Streptomyces is characterized by their complex morphological differentiation and their unusually large linear chromosome and plasmids (Redenbach et al., 1996). Spores germinate to form vegetative mycelia of branching multigenomic hyphi. Such coherent structure is dispersed by developing aerial branches which later form chains of unigenomic spores. Correct partitioning of chromosome and plasmids in Streptomyces is of interest because the mechanism of the mycelium organism with linear DNA is little known, comparing to the well-studied rod-shaped organisms that grow by binary fission and possess circular DNA (Gordon et al., 1997; Lewis and Errington, 1997; Mohl and Gober, 1997). In the study of S. coelicolor, it contains para and parb genes near the replication origin of the genome. Disruption of parab did not affect the viability of colony growth, but partitioning aberration could be observed in 13% chromosomeless spores (Kim et al., 2000). Twenty-four 14-bp pars sequences (GTTTCACGTGAAAC) similar to the B. subtilis pars site have been

4 identified in the S. coelicolor genome, which are densely clustered within a relatively short distance (~200 kb) around oric (Jakimowicz et al., 2002; Lin and Grossman, 1998). Partition system is also known to be important for the segregation stability of Streptomyces plasmids. SCP2*, a 31-kb circular, low-copy- number plasmid, was originally isolated from S. coelicolar A3(2) (Haug et al., 2003). A mini-scp2* plasmid harboring the SCP2* replication region and para and parb homologs was constructed to measure the contribution to plasmid stability of the on SCP2*. An increase of plasmid stability could be seen only when both genes were present, which is in agreement with the role of the two genes as an active partition system for SCP2*. However, nothing is known about the partitioning systems for linear plasmids in Streptomyces. SLP2 is a 50-kb low copy-number linear plasmid in Streptomyces (Huang et al., 2003). It contains homologs of para (SLP2.30c) and parb (SLP.29c) with more than 30% homology to the copy on S. coelicolor chromosome. However, SLP2, like another linear plasmid SCP1, does not contain the chromosomal pars sequence (GTTTCACGTGAAAC), a significantly larger palindrome. So we are interested to understand how correct plasmid partitioning is achieved for such a large, linear plasmid during the development from non-septum structure of mycelia to unigenomic spores. Here, we describe the influence of parab genes on SLP2 stability and in vitro identification and characterization of the pars sites. Our results show that the active par system is also required for linear plasmid segregation but through binding a quite different palindromic pars sequence from those found in other bacterial genomes or plasmids.

5 Results and discussion Both the parab loci on chromosome and SLP2 affect the stability of SLP2 Like other low-copy number plasmids in Streptomyces, SLP2 contains homologs of para (SLP2.30c) and parb (SLP.29c). In order to evaluate the importance of these two genes on SLP2 stability, the parab copy on SLP2 was disrupted by using aac(3)iv cassette to replace the whole para and parb genes (Fig. 1). After cloned parab cluster was replaced by aac(3)iv cassette in E. coli, the plasmid was used to transform S. lividans and selected apramycin resistant colonies. Southern blotting was used to verify the colonies with single- or double-crossover recombination versions. This SLP2 derivative containing a deletion of para and parb was created and introduced into S. coelicolor M145. The stabilities of SLP2 and its parab-ko derivative were determined by measuring the rate at which plasmid-lost cells on each rounds of sporulation during growth in the absence of antibiotic selection. In contrast to the stable maintenance of intact SLP2 in M145, about 90% of the spores lost the parab SLP2 after one round of sporulation (Table 1). On the other hand, the parab genes on SLP2 have more than 30% identity to the S.coelicolor chromosomal and SCP1 homologs at the amino-acid level, and Bently et al. previously reported that the parab homologues on the other Streptomyces plasmid SCP1 can largely suppress the chromosome partitioning defects of chromosomal parab mutants(bentley et al., 2004). We further test whether the chromosomal homologs also play a part in stabilization of SLP2. The same SLP2 derivative was transferred to J2538 (a chromosomal parab mutant of M145) by conjugation. After one round of sporulation, it was lost in 99% of the spores (Table 1). Therefore, these results indicate that the par system is important in the stable maintenance of SLP2, and the chromosomally- encoded ParAB proteins may partially

6 compensate for parab mutations on SLP2. Expression and purification of ParB protein A search of the SLP2 genome sequence shows no pars-like sites by comparing the known pars sequences found in some bacterial chromosome and plasmids. In order to find out the pars sequence of SLP2, we first express ParB protein of SLP2 in E. coli. parb gene was amplified by polymerase chain reaction and fused to the His-tag coding sequence in N-terminal to overexpression in E.coli BL21. The His-ParB fusion protein was purified by affinity chromatography on Ni-agarose resin. An approximate 40 kda protein similar in size to the deduced parb gene product was detected by Western blotting using anti-polyhistidine antibody. Identification of pars sites localization using affinity chromatography Because there may be more than one pars sites distributed along SLP2, it was not possible to study the interaction of ParB with the entire 50-bp SLP2 using DNase I footprinting or gel retardation assay. Instead, affinity chromatography was performed to isolated specific binding sequences as described by Jakimowicz et al. in 2002, when they probing multiple pars sites in the vicinity of the oric region (Jakimowicz et al., 2002). In this assay, the His-ParB fusion protein bound to Ni-agarose resin was used as an affinity reagent to evaluate binding of DNA fragments containing pars sites. For our studies, five SLP2 subclones (done by Lin, 1993) cover 43 kb in the left arm of SLP2 were used (Fig. 3C). SLP2 DNA fragments cleaved from subclones and pbluescript (KS-) DNA fragments (as negative control) were load to the Ni-agarose column and incubated with the immobilized ParB protein. The specificity of DNA-ParB interaction was evaluated by washing with different salt buffer (Fig. 3A and 3B). At low salt, all DNA fragments were non-specifically bound,

7 whereas at medium salt, only the fragments containing pars sequences were selectively retained. In our case, non-specific binding DNA fragments were first released from the resin by washing with 200 to 300 mm NaCl. Specific- binding DNA fragments, candidate pars sites, were released later by eluting with 500 to 700 mm NaCl. Comparing the relative amounts of bound and unbound DNA, we narrowed down the localization of the pars sites to a 106-bp fragment on the C-terminal region of the ParB coding sequence (Fig. 3C). Identification of the pars-parb interaction sequence The specific binding sequence of ParB was further defined precisely by DNase I footprinting. The 106-bp DNA fragment was analyzed by incubation with increasing amounts of ParB. The protection assay was performed on both of the strands. The results showed that there were two ~30-bp regions protected, which were separated by a distance about forty nucleotides (Fig. 4A and 4B). Further analysis of their sequences showed they are nearly identical. Each of them contains two complete or partial 8-bp inverted repeats (CGTTAACG). Interestingly, these octamer repeats are in the same reading frame, encoding the same aa sequence in the more diverse C- terminal region of the ParB protein family (Fig.5). We could not find this octamer sequence on S. coelicolor chromosome and another linear plasmid SCP1. On the opposite, SLP2, like SCP1, does not contain the chromosomal pars sequence (GTTTCACGTGAAAC), a significantly larger palindrome. These results suggest a possibly novel par system for the linear Streptomyces plasmids.

8 Experimental procedures Disruption of the parab locus The PCR-targeting procedure described by Yu et al. (2000) was used to replace parab gene locus on a parab containing E. coli plasmid with a resistance marker. Primers are designed to amplify the aac(3)iv apramycin-resistance cassette, whereas the 5 -ends were 50-bp tails that would be homologous to sequences flanking parab. The PCR product was transformed into E. coli strain carrying λred system induced as described previously (Yu et al., 2000). Apramycin-resistant plasmid was passed through E. coli strain ET12567 and then the non-methylated DNA was used to transform TK64 SLP2 as the method described by Kieser et al., To confirm the fully disruption of parab genes, Km s and Apra r candidate colonies were analyzed by Southern blotting. ParB purification The ParB protein of SLP2 was expressed as a fusion to the N-terminus of His-tag. The parb gene was amplified by PCR and cloned into pet15(b). The His-ParB fusion protein was overexpressed in E. coli BL21 and purified using Ni-agarose columns, as the protocol of the manufacturer. The purified protein was analyzed by SDS- PAGE. Isolation of pars fragments using affinity chromatography E. coli BL21 cells containing pet15(b)-parb were grown for 3 h at 37 to an optical density about 0.6~0.8 at 600 nm in the presence of ampicillin (50 µg ml -1 ) and then induced with IPTG (1 mm) for 2 h. After centrifugation (5000 g, 4, 10 min), cell were suspended in binding buffer [0.5 M NaCl, 20 mm Tris-HCl, 5 mm imidazole, ph 7.9] and lyses by sonication. After centrifugation (20000g, 4, 30 min), the crude

9 extract containing His-ParB fusion protein was bound directly to Ni-agarose column. The column was washed with 10 bed volumes of binding buffer and 2 bed volumes of 1M NaCl buffer to remove the DNA in the bacterial extract. The DNA fragments purified from SLP2 subclones was loaded onto the column in 100 mm NaCl buffer (20 mm Tris-HCl, 100 mm NaCl, ph 8.0). After 30 min of incubation at 30, the column was washed with two column volumes of different concentration NaCl buffer (20 mm Tris-HCl, 200 mm~1m NaCl, ph 8.0) to separate DNA fragments bound to His-ParB fusion protein with different binding affinity. The DNA was followed by isopropanol precipitation and resuspended in TE buffer (10 mm Tris-HCl, 1 mm EDTA, ph 8.0) and analyzed in an agarose gel. DNase I footprinting The 5 end radiolabelled DNA fragments were incubated with different amounts of ParB protein in binding buffer [20 mm HEPES, ph 7.6, 100 mm NaCl, 5 mm Mg-acetate, 4 mm dithiothreitol (DTT), 1 mm EDTA, 0.2% Triton X-100, 5% glycerol] at 30 for 30 min. Then, the reaction mixture was treated with DNase I (5 µg ml -1, 25, 30 s). The DNase I cleavage products were separated in an 8% poly- acrylamide-urea sequencing gel. Gels were dried and analyzed by autoradiography.

10 Reference Bentley, S. D., Brown, S., Murphy, L. D., Harris, D. E., Quail, M. A., Parkhill, J., Barrell, B. G., McCormick, J. R., Santamaria, R. I., Losick, R., et al. (2004). SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2). Mol Microbiol 51, Bignell, C., and Thomas, C. M. (2001). The bacterial ParA-ParB partitioning proteins. J Biotechnol 91, Gordon, G. S., Sitnikov, D., Webb, C. D., Teleman, A., Straight, A., Losick, R., Murray, A. W., and Wright, A. (1997). Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms. Cell 90, Haug, I., Weissenborn, A., Brolle, D., Bentley, S., Kieser, T., and Altenbuchner, J. (2003). Streptomyces coelicolor A3(2) plasmid SCP2*: deductions from the complete sequence. Microbiology 149, Huang, C. H., Chen, C. Y., Tsai, H. H., Chen, C., Lin, Y. S., and Chen, C. W. (2003). Linear plasmid SLP2 of Streptomyces lividans is a composite replicon. Mol Microbiol 47, Jakimowicz, D., Chater, K., and Zakrzewska-Czerwinska, J. (2002). The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of pars sequences within the origin-proximal region of the linear chromosome. Mol Microbiol 45, Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, F.A. (2000) practical Streptomyces Genetics. Norwich, UK: The John Innes Foundation. Kim, H. J., Calcutt, M. J., Schmidt, F. J., and Chater, K. F. (2000). Partitioning of the linear chromosome during sporulation of Streptomyces coelicolor A3(2) involves an oric-linked parab locus. J Bacteriol 182, Lewis, P. J., and Errington, J. (1997). Direct evidence for active segregation of oric regions of the Bacillus subtilis chromosome and co-localization with the SpoOJ partitioning protein. Mol Microbiol 25, Lin, D. C., and Grossman, A. D. (1998). Identification and characterization of a

11 bacterial chromosome partitioning site. Cell 92, Mohl, D. A., and Gober, J. W. (1997). Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Cell 88, Redenbach, M., Kieser, H. M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H., and Hopwood, D. A. (1996). A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol Microbiol 21, Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G., and Court, D. L. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97,

12 (A) probe (B) Fig.1. Construction of the ΔparAB SLP2 derivative in Streptomyces (A) Schematic drawing of the integration and excision of aac3(iv) cassette containing plasmid that gave rise to the allelic exchange at the parab locus. (B) Southern blotting analysis of Streptomyces total DNA to confirm the disruption of parab locus. Tracks: 1, TK64; 2, TK64 SLP2; 3, TK64 SLP2[ΔparAB::aac(3)IV] Probe is indicated in (A)

13 rounds of M145 J2538 [parab c ::aac3(iv)] sporulation SLP2 Mutant SLP2 [parabslp2::aac3(iv)] SLP2 Mutant SLP2 [parabslp2::aac3(iv)] 0 (from selective medium) 1 (from nonselective medium) 2 (from nonselective medium) 101% 104% 99.5% 5.6% 89.73% 12.12% 98.08% 0.84% 99.4% 6.1% 93.4% 0.12% Table.1. Stability of SLP2 and its derivatives in S. lividans TK64

14 M M Fig.2. Expression and purification of ParB SLP2 in E. coli. (A) Fusion protein His-ParB was isolated from E. coli BL21 containing pet15(b)-parb, and the His-ParB was purified on Ni-agarose columns. Proteins were separated by 8% SDS-PAGE. Tracks: M, molecular weight markers; 1, uninduced cell extract; 2, IPTG-induced cell extract; 3, purified ParB released from column by imidazole elution buffer. (B) Western analysis of the ParB-overexpressing cell extract using anti-poly histidine antibody against the His-ParB fusion protein. Tracks: M, molecular weight markers; 1, uninduced cell extract; 2, IPTG-induced cell extract

15 (A) (B) (C) Fig 3. in vitro identification of ParB binding sites. (A) (B) Affinity chromatography by selective binding of pars-containing fragments to Ni-ParB resin. DNA fragments purified from SLP2 subclones and digested pbluescript (KS-) were bound to the resin. Fragments were eluted with different concentration salt buffer and then were analyzed by agarose gel electrophoresis. Tracks: I, Input DNA before affinity chromatography; B, precipitation from elution before loading DNA fragments; F, flow-through after DNA binding to the resin in 100mM salt buffer. And in (A) were digested pbluescript (KS-) DNA as binding control. (C) Localization of pars sequences on SLP2. Bold black arrows represent DNA fragments bound by ParB in affinity chromatography.

16 (A) (B) Fig.4. DNase I footprinting analysis of the ParB binding sequence. DNA fragments containing putative pars sites were incubated with increasing ParB concentration. (A) and (B) were assayed in the different strands. Protected regions in (A) and (B) are separated in a distance of ~40 nucleotides. Sequencing markers are shown in the left panel of figure 4A. The nucleotide sequences protected by ParB protein are shown on the left side of the figures. The solid and open bars indicate the perfect and partial palindromic 8-bp sequence. The arrows indicate the inverted repeats.

17 (A) (B) Translational direction Fig.5. Features of the ParB protecting regions from SLP2 The relative localization of the putative pars sites defined by DNase I footprinting is shown in (A). The solid and open bars indicate the perfect and partial palindromic 8-bp sequence. The arrows indicate the inverted repeats. (B) The translational codons of the pars sites within the the coding region of parb gene. Two putative pars boxes are shown. The translational direction is from the right to the left.