Chromosome organization in Corynebacterium glutamicum: Information storage in 3D

DNA链的长度超过细胞的大小至少三个数量级，因此，细菌基因组高度组织以适应细胞区室。类核组织是一个高度复杂的过程，需要与DNA复制，分离和转录兼容。近年来已经揭示了蛋白质-DNA相互作用如何构建类核以及类核折叠如何影响重要的细胞过程，包括胞质分裂的定位（Donovan等人，2013，Treuner-Lange等人，2013年）。我们所知道的关于染色体组织的大部分知识源于对一些模式物种的研究，例如枯草芽孢杆菌、新月柄杆菌和大肠杆菌（Badrinarayanan等人，2015，Toro & Shapiro，2010）。在第一个资助期内，我们提出解决谷氨酸棒杆菌中类核是如何组织的问题，从而建立谷氨酸棒杆菌。glutamicum作为顶端生长放线菌的模式种。我们建议分析染色体结构域组织，染色体压实和染色体分离。在过去的三年中，我们已经能够显示ParABS系统如何分离染色体的起源区域。我们解开了C.谷氨酸和确定了一种新的染色体组织模式的细菌与稳定的双极附着的染色体。一个直接的结果是C.谷氨酸细胞总是至少为二倍体。我们继续表明，DNA复制可以在前一轮终止之前启动（重叠C期/多叉复制）。如HiC接触图所示，染色体臂通过凝聚素复合物Smc-ScpAB缠绕在该生物体中。有趣的是，Smc的缺失释放了臂的凝聚力，对染色体分离、复制和生长速率没有显著影响，这给我们留下了臂凝聚力的分子功能是什么的问题。我们可以证明ParB是凝聚素介导的臂凝聚所必需的，但被鉴定为距离parS位点约13 kb的推定Smc加载位点。在新的资助期内，我们建议详细分析SMC负载。我们将进一步分析起源处ParB组织的超微结构。我们已经构建了一个缺乏所有10个parS位点的可行菌株，并开始将这些位点转移到不同的染色体位置。使用光激活定位显微镜（PALM），一种我们在实验室中已经建立的技术，我们将分析ParB组织在各种应变背景和地址Smc/ParB定位使用双色PALM。目前，尚不清楚C.谷氨酸在应激条件下发生变化。为了解决这个问题，我们将施加各种压力（DNA损伤，渗透压等）。并利用HiC和成像技术分析染色体构象。

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.