MksB, an alternate condensin from Mycobacterium smegmatis is involved in DNA binding and condensation

Pradhan, Suchitra; Bipinachandran, Swathy Velaparambil; Kumari, Pratibha; Suguna, M.; Prasad, M. Dharma; Kumar, Rakesh

doi:10.1016/j.biochi.2020.03.002

Cited by 3 publications

(9 citation statements)

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“…MksBEF from Pseudomonas aeruginosa has been shown to contribute efficiently in chromosome partitioning [30]. Previously, our group has reported that MksB homologues from M. smegmatis function similar to the SMC protein [15]. Based on the previous reports, we speculate that such homologues in mycobacterial genomes might have rescued the null phenotype of the smc/scpb deletion and enable it to survive under normal or stress environments.…”

Section: Discussionmentioning

confidence: 69%

“…Previously, we reported that brief rifampicin treatment to E. coli causes chromosome decondensation and that overexpression of MsMksB effectively condenses the nucleoid [15].…”

Section: Overexpression Of Smc-scpab On Chromosome Condensation In Ementioning

confidence: 99%

“…The supernatant was mixed with Ni-NTA resin (Sigma) and allowed to bind at 4 °C for 2 h. The resin was then washed with buffer A containing 10 mM imidazole and proteins were eluted with a gradient of 30-300 mM imidazole in buffer A. The peak fractions were collected and dialysed against buffer A with 100 mM NaCl and subjected to chromatographic steps for further purification as previously described [15]. For Smc holocomplex purification, the ScpAB-Duet and SMC-pRSFDuet-1 plasmids were co-transformed in E. coli BL21(DE3) pLysS cells with appropriate antibiotics.…”

Section: Cloning Gene Expression and Purificationmentioning

confidence: 99%

“…The ATPase assay was performed as described previously [15]. Briefly, SMC holocomplex (4 µg) was added to a reaction mixture containing 20 mM Tris, 2 mM ATP, 2-20 mM MgCl 2 , 6-60 mM KCl and 20 ng DNA wherever indicated.…”

Section: Atpase Assaymentioning

confidence: 99%

“…The exponentially growing wild-type E. coli and ∆mukb cells were fixed, washed and stained with DAPI (0.5 µg ml −1 ) followed by mounting on a thin layer of 1 % agar on a microscope slide and analysed as previously described [19]. The bacterial cytological profiling of rifampicin-treated cells was performed as described previously [15]. Briefly, the cells containing the plasmids ScpAB or Smc-ScpAB were grown overnight and the grown cells were diluted to 1 : 100 in fresh LB medium containing suitable antibiotics.…”

Section: Microscopic Studiesmentioning

confidence: 99%

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Biochemical and functional characterization of the SMC holocomplex from Mycobacterium smegmatis

et al. 2021

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Multi-subunit SMC complexes are required to perform essential functions, such as chromosome compaction, segregation and DNA repair, from bacteria to humans. Prokaryotic SMC proteins form complexes with two non-SMC subunits, ScpA and ScpB, to condense the chromosome. The mutants of both scpa and scpb genes in Bacillus subtilis have been shown to display characteristic phenotypes such as growth defects and increased frequency of anucleate cells. Here, we studied the function of the Smc-ScpAB complex from Mycobacterium smegmatis . We observed no significant growth difference between the scpb null mutant and wild-type M. smegmatis under both standard and stress conditions. Furthermore, we characterized the Smc-ScpAB holocomplex from M. smegmatis . The MsSMC consists of the dimerization hinge and ATPase head domains connected by long coiled-coils. The MsSMC interacts with two non-SMC proteins, ScpA and ScpB, and the resulting holocomplex binds to different DNA substrates independent of ATP. The Smc-ScpAB complex showed DNA-stimulated ATPase activity in the presence of ssDNA. A cytological profiling assay revealed that upon overexpression the Smc-ScpAB ternary complex compacts the decondensed nucleoid of rifampicin-treated wild-type and null mukb mutant of Escherichia coli in vivo. Together, our study suggests that M. smegmatis has a functional Smc-ScpAB complex capable of DNA binding and condensation. Based on our observations, we speculate that the presence of alternative SMCs such as MksB or other SMC homologues might have rescued the scpb mutant phenotype in M. smegmatis .

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Section: Discussionmentioning

confidence: 69%

“…Previously, we reported that brief rifampicin treatment to E. coli causes chromosome decondensation and that overexpression of MsMksB effectively condenses the nucleoid [15].…”

Section: Overexpression Of Smc-scpab On Chromosome Condensation In Ementioning

confidence: 99%

Section: Cloning Gene Expression and Purificationmentioning

confidence: 99%

Section: Atpase Assaymentioning

confidence: 99%

Section: Microscopic Studiesmentioning

confidence: 99%

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