Biology subjects

Bock, F. P.

Publications and source records attributed to Bock, F. P..

3 recordsLinked to original sources

bioRxiv2023

Direct observation of a crescent-shape chromosome in Bacillus subtilis

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genomic information. Direct visualization of the chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the intrinsic shape of the bacterial chromosome in real-time in live Bacillus subtilis cells. We show that the chromosome exhibits a crescent shape with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

microbiology↗

bioRxiv2021

The ParB clamp docks onto Smc for DNA loading via a joint-ParB interface

Chromosomes readily unlink from one another and segregate to daughter cells during cell division highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes mediate chromosome folding by DNA loop extrusion. In most bacteria, SMC complexes start loop extrusion at the ParB/parS partition complex formed near the replication origin. Whether they are recruited by recognizing a specific DNA structure in the partition complex or a protein component is unknown. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate chromosomes when transplanted into another organism. Using chimeric proteins and chemical cross-linking, we find that ParB binds to the Smc subunit directly. We map a binding interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex to initiate DNA loop extrusion.

biochemistry↗

bioRxiv2021

Relief of ParB autoinhibition by parS DNA catalysis and ParB recycling by CTP hydrolysis promote bacterial centromere assembly.

Three-component ParABS systems are widely distributed factors for plasmid partitioning and chromosome segregation in bacteria. ParB protein acts as an adaptor between the 16 bp centromeric parS DNA sequences and the DNA segregation ATPase ParA. It accumulates at high concentrations at and near a parS site by assembling a partition complex. ParB dimers form a DNA sliding clamp whose closure at parS requires CTP binding. The mechanism underlying ParB loading and the role of CTP hydrolysis however remain unclear. We show that CTP hydrolysis is dispensable for Smc recruitment to parS sites in Bacillus subtilis but is essential for chromosome segregation by ParABS in the absence of Smc. Our results suggest that CTP hydrolysis contributes to partition complex assembly via two mechanisms. It recycles off-target ParB clamps to allow for new attempts at parS targeting and it limits the extent of spreading from parS by promoting DNA unloading. We also propose a model for how parS DNA catalyzes ParB clamp closure involving a steric clash between ParB protomers binding to opposing parS half sites.

biochemistry↗