Bacillus subtilis Smc condenses chromosomes in a heterologous cell system, which is down-regulated by ScpAB
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MC Research Notes Open Access
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Bacillus subtilis Smc condenses chromosomes in a heterologous cell system, which is down‑regulated by ScpAB Tobias Knust1 and Peter L. Graumann2,3*
Abstract Objective: Structural maintenance of chromosomes (SMC) proteins are key players in chromosome dynamics in all types of organisms. The so-called condensin subfamily is essential for chromosome condensation in eukaryotic cells, as is the bacterial SMC complex (called MukBEF in Escherichia coli). We expressed the Bacillus subtilis Smc protein and its two complex partners ScpA and ScpB in E. coli cells, and monitored effects on chromosome compaction by DNA staining of live cells using epifluorescence microscopy. Data description: We show that expression of BsSmc leads to strong chromosome compaction, while expression of ScpAB does not show any effect. Chromosome compaction by Smc was also found for mutant versions lacking ATP binding or ability for head engagement, and was counteracted by concomitant expression of ScpAB. Our findings show that the SMC complex can act as autonomous condensation system in a heterologous bacterial host system, for which neither ATP binding nor ATP hydrolysis are required. Our investigation suggests that the negative effect on compaction activity of Smc exerted by ScpAB in vivo does not involve an effect on ATPase activity, but more likely a stabilization of the engagement of head domains, which in turn may affect ATPase activity. Keywords: Structural maintenance of chromosomes, SMC, Bacillus subtilis, Chromosome condensation Objective Deletion of smc, scpA or scpB genes in many bacteria, including B. subtilis, leads to slow, temperature sensitive growth, a defect in chromosome condensation/nucleoid compaction, and the generation of about 15% of cells lacking any chromosome (anucleate cells) [1–4]. Smc, ScpA and ScpB form a complex in vivo and in vitro [5, 6], and ScpA and ScpB form a sub-complex [7, 8] that affects ATPase activity of Smc [9]. Higher levels of Smc in B. subtilis cells result in chromosome hyper-compaction [7]. We wished to investigate if Smc and/or its complex partners ScpA and ScpB, which do not bind to DNA by *Correspondence: graumanp@uni‑marburg.de 2 SYNMIKRO, LOEWE-Zentrum Für Synthetische Mikrobiologie, HansMeerwein-Straße, 35043 Marburg, Germany Full list of author information is available at the end of the article
themselves [10], can also condense chromosomes when expressed in other bacterial species. For this purpose, we chose E. coli cells as a model system, and employed a two-plasmids expression system. The PCR products of scpA and of scpB were cloned in tandem into pETDuet-1 (Novagen). Smc was cloned into pCDFDuet-1 (Novagen). Mutants of smc were created by the QickChange-protocoll. 20 ml culture of Rosetta 2 (DE3) pLysS cells hosting the desired vector was inoculated to an OD600 of 0.05 in LB containing required antibiotics and incubated on a shaking platform at 37 °C until O D600 0.9 was reached. Expression was induced with 0.5 mM IPTG for Smc or Smc m
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