Structural Maintenance of Chromosomes Protein of Bacillus subtilis Affects Supercoiling In Vivo

Abstract

Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.

The bacterial nucleoid: A highly organized and dynamic structure

“…Inactivation of either of these complexes leads to temperaturesensitive growth due to a severe disturbance of nucleoid organization and chromosome segregation at elevated temperatures [Niki et al, 1991;Britton et al, 1998;Moriya et al, 1998;Jensen and Shapiro, 1999]. This phenotype was shown to be suppressed by mutations that cause excessive negative supercoiling, which suggests a role for these proteins in DNA compaction or folding [Sawitzke and Austin, 2000;Lindow et al, 2002a]. Such a function is supported by the global chromosome compaction phenotype observed upon overproduction of SMC in B. subtilis [Volkov et al, 2003].…”

Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation

“…The original observation that smc mutants fail to form axial filaments remains unexplained (99); it may be that RacA cannot bind to the uncondensed and disorganized chromosomes of smc mutants (25,176). racA and divIVA mutants also undergo asymmetric division even though axial filament formation is impaired (21,287,308), whereas the smc mutant is deficient in both processes (99).…”

“…Despite their name, none of the classic spo0 mutations clearly prevented axial filament formation (228): spo0H mutants formed axial filaments (29,94), whereas spo0A, spo0B, and spo0F mutants underwent an additional symmetric division during sporulation, and it was not clear if that was preceded by axial filament formation (29,53). Some insight into the process was derived from analysis of the SMC (structural maintenance of chromosomes) protein, required for chromosome compaction and partitioning (25,176). Mutants lacking this protein are able to activate Spo0A but are unable to form axial filaments (99), suggesting that an effector of axial filament formation is either missing or nonfunctional in this background.…”

Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

“…If EptC's net effect is the stabilization of negative supercoiling like the MukB proteins (39), then EptC binding to DNA would restrict topoisomerase I from accessing and relaxing negative supercoils and would be manifested as a net inhibition of topoisomerase I activity in the presence of EptC. If EptC functions like BsSMC and eukaryotic condensing/SMC by stabilizing positive supercoils (12,38), compensatory negative supercoils would be introduced in the plasmid after EptC binding, which would be relaxed by topoisomerase I. In this scenario, addition of EptC will display a net effect of stimulation in topoisomerase I activity.…”

“…In prokaryotes, chromosome segregation has mainly been studied by genetic and biochemical analyses of the low-copy number plasmid DNA (11)(12)(13). High-copy number plasmids rely primarily on passive diffusion for plasmid maintenance (14), which is inapplicable to chromosomal DNA.…”