DNA-loop extruding SMC complexes can traverse one another in vivo

Chromosome organization by structural maintenance of chromosomes (SMC) complexes is vital to living organisms. SMC complexes were recently found to be motors that extrude DNA loops. However, it remains unclear what happens when multiple complexes encounter one another in vivo on the same DNA and how interactions help organize an active genome. We created a crash-course track system to study SMC complex encounters in vivo by engineering the Bacillus subtilis chromosome to have defined SMC loading sites. Chromosome conformation capture (Hi-C) analyses of over 20 engineered strains show an amazing variety of never-before-seen chromosome folding patterns. Via 3D polymer simulations and theory, we find that these patterns require SMC complexes to bypass each other in vivo, contrary to the common assumption that SMC complexes mutually block each other’s loop extrusion activity. We posit that the bypassing activity enables SMC complexes to spatially organize a functional and busy genome.

Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced β-galactosidase production using the CRISPR/Cas9 system

The ganA gene from Bacillus subtilis encoding a β-galactosidase for degradation of the galactomannan was integrated in different loci of the B. subtilis chromosome employing the CRISPR/Cas9 system. Hereby a total of five copies of ganA cassettes in which the ganA gene was fused with the glucitol-promoter were inserted in the recipient chromosome wherein hypothetical, sporulation and protease genes were deleted. The strain with five copies of ganA expression cassette showed a β-galactosidase activity similar to the one with the same gene on a pUB110 derived multi-copy plasmid and under the same regulatory control of the glucitol promoter and GutR activator. The production of β-galactosidase in the strain with the multi-copy plasmid decreased rapidly when growth was performed under induced conditions and without antibiotic selection. In contrast, the strain with the five copies of ganA in the chromosome produced β-galactosidase for at least 40 generations. This demonstrates that the CRISPR/Cas9 system is a valuable and easy tool for constructing stable producer strains. The bigger efforts that are needed for the multiple target gene integration into the chromosome compared to cloning in expression vectors were justified by the higher stability of the target genes and the lack of antibiotic resistance genes.

Dynamic Association of the Replication Initiator and Transcription Factor DnaA with the Bacillus subtilis Chromosome during Replication Stress

ABSTRACT DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when replication was inhibited, indicating that the association of DnaA with at least some of its target sites is altered after replication stress. When replication resumed from oriC after inhibition of replication initiation, these high levels of binding decreased rapidly at origin-proximal and origin-distal regions, well before a replication fork could pass through each of the regulated regions. These findings indicate that there is rapid signaling to decrease activation of DnaA during replication and that interaction between DnaA bound at each site and the replication machinery is not required for regulation of DnaA activity in response to replication stress.