Chromosome organization in Corynebacterium glutamicum: Information storage in 3D

• Applicant: Professor Dr. Marc Bramkamp
• Subject Area: Microbial Ecology and Applied Microbiology
• Term: from 2014 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 258303182

The length of the DNA strand exceeds the size of the cell by at least three orders of magnitude, and hence, the bacterial genome is highly organized to fit into the cellular compartment. Nucleoid organization is a highly complex process that needs to be compatible with DNA replication, segregation and transcription. Recent years have revealed how protein-DNA interactions structure the nucleoid and how nucleoid folding affects vital cellular processes including positioning of cytokinesis (Donovan et al., 2013, Treuner-Lange et al., 2013). Most of what we know about chromosome organization stems from work with a few model species such as Bacillus subtilis, Caulobacter crescentus, and Escherichia coli (Badrinarayanan et al., 2015, Toro & Shapiro, 2010). Within a first funding period we have proposed to address the question how the nucleoid is organized in Corynebacterium glutamicum, thereby establishing C. glutamicum as a model species for apically growing actinobacteria. We proposed to analyze chromosome domain organization, chromosome compaction and chromosome segregation. Within the last three years we have been able to show how the ParABS system segregates the origin region of the chromosome. We unraveled the cell cycle of C. glutamicum and identified a novel chromosome organization pattern in bacteria with a stable bi-polar attachment of chromosomes. A direct consequence is that C. glutamicum cells are always at least diploid. We went on to show that DNA replication can be initiated before a previous round is terminated (overlapping C-periods/multi-fork replication). Chromosome arms are entangled in this organism by the condensin complex Smc-ScpAB as shown by HiC contact maps. Interestingly, deletion of Smc, which releases arm cohesion, has no drastic effect on chromosome segregation, replication, and growth rates, leaving us with the question what the molecular function of arm cohesion is. We could show that ParB is necessary for condensin mediated arm cohesion, but be identified a putative Smc loading site some 13 kb away from parS sites. Within the new funding period we propose to analyze Smc loading in detail. We will further analyze the ultrastructure of the ParB organization at the origin. We have already constructed a viable strain lacking all 10 parS sites and started to shift these sites to various chromosomal positions. Using photo-activated localization microscopy (PALM), a technique that we have well established in the lab, we will analyze ParB organization in various strain backgrounds and address Smc/ParB localization using dual color PALM. Currently, it is unclear whether or how chromosome organization in C. glutamicum changes during stress conditions. To address this question we will apply various stresses (DNA damage, osmotic stress etc.) and analyze chromosome conformation with HiC and imaging technologies.

• DFG Programme: Research Grants