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Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis, by D. Jakimowicz, A. Brzostek, A. Rumijowska-Galewicz, P. Zydek, A. Dolzblasz, A. Smulczyk-Krawczyszyn, T. Zimniak, L. Wojtasz, A. Zawilak-Pawlik, A. Kois, J. Dziadek and J. Zakrzewska-Czerwinska

Microbiology vol. 153, part 12, pp. 4050 - 4060

Table S1. Oligonucleotides used in this study. [PDF] (94 kb)

Fig. S1. Construction of the M. smegmatis parB mutant The chromosomal parB (MSMEG_6938) and parA (MSMEG_6939) genes are represented by grey arrows (A). The restriction sites used for digestion of chromosomal DNA are denoted by single letters (H, HindIII; S, SmaI). The restriction fragment and the size of the internal deletion in the mutated copy of parB (black rectangle) are shown by double-headed arrows. The single-headed arrows represent PCR primers. The genomic DNA designated for PCR (B) and Southern hybridization (C) was isolated from wild-type M. smegmatis (wt); single cross-over homologous recombination mutants (parB-ΔparB); double cross-over homologous recombination mutants carrying the wild-type parB gene (parB) or the parB gene with the internal deletion (ΔparB), exclusively. [PDF] (340 kb)

Fig. S2. Purified mycobacterial ParAB proteins. The fusion proteins glutathione S-transferase-ParA or 6His-ParB were purified on a glutathione-Sepharose 4B or a Ni²⁺-NTA-agarose column (Qiagen), respectively. For removal of the glutathione S-transferase part, the fusion proteins bound to glutathione-Sepharose beads were treated with PreScission protease (Amersham-Pharmacia Biotech) and the ParA proteins were released from the beads. His-tagged proteins were eluted from the Ni²⁺-NTA-agarose column with 100 nM imidazole. Ms, Mycobacterium smegmatis; Mt, M. tuberculosis. Proteins were analysed in a 12% SDS-PAGE gel and stained with Coomassie brilliant blue. [PDF] (247 kb)

Fig. S3. Protein sequence alignment of homologous ParB proteins. Identical and similar amino acids are shown with a yellow or green background, respectively. The colours of the secondary structure elements (S, beta-sheet; H, alpha-helix) correspond to those used for Thermus thermophilus SpoOJ protein by Leonard et al. (2004). The HTH motif is coloured yellow. Identical residues in M. tuberculosis and M. smegmatis are shown in bold type. [PDF] (319 kb)

REFERENCE
Leonard, T. A., Moller-Jensen, J. & Lowe, J. (2005). Towards understanding the molecular basis of bacterial DNA segregation. Philos Trans R Soc Lond B Biol Sci 360, 523-535.

This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Citing Articles Citing Articles via CrossRef