Chromosome organization in Corynebacterium glutamicum: Information storage in 3D

DNA链的长度至少比细胞大小大三个数量级，因此，细菌基因组的组织高度适合细胞隔间。类核组织是一个高度复杂的过程，需要与DNA复制、分离和转录相兼容。近年来揭示了蛋白质-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，包括细胞质分裂的定位(Donovan等人，2013年，Treuner-Lange等人，2013年)。我们对染色体组织的大部分了解来自于与几个模式物种的工作，如枯草杆菌、新月弯杆菌和大肠杆菌(Badrinarayanan等人，2015，Toro&Shapiro，2010)。在第一个资助期内，我们已经提出了解决谷氨酸棒杆菌中类核是如何组织的问题，从而将谷氨酸棒杆菌确立为顶部生长放线杆菌的模式物种。我们建议对染色体结构域组织、染色体紧凑和染色体分离进行分析。在过去的三年里，我们已经能够展示ParABS系统如何分离染色体的起始区。我们揭开了谷氨酸杆菌的细胞周期，并在细菌中发现了一种新的染色体组织模式，具有稳定的染色体两极附着。一个直接的后果是谷氨酸杆菌细胞总是至少是二倍体的。我们继续展示了DNA复制可以在前一轮结束之前启动(重叠的C期/多叉式复制)。HIC接触图显示，染色体臂通过凝集素复合体SMC-ScpAB在该生物体中缠绕在一起。有趣的是，SMC的缺失释放了手臂凝聚力，对染色体分离、复制和生长速度没有显著影响，这给我们留下了手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ARM凝聚力所必需的，但可以确定在距离PARS约13kb的SMC加载位置。在新的资金期限内，我们建议对SMC的负载进行详细分析。我们将进一步分析PARB组织起源的超微结构。我们已经构建了一个缺少所有10个PARS位点的活菌株，并开始将这些位点转移到不同的染色体位置。利用我们在实验室建立的光活化定位显微镜(Palm)技术，我们将分析不同菌株背景下的PARB组织，并使用双色Palm解决SMC/PARB定位问题。目前，谷氨酰胺的染色体组织是否或如何在胁迫条件下发生变化尚不清楚。为了解决这个问题，我们将应用各种压力(DNA损伤、渗透胁迫等)。并用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.