Midcell Recruitment of the DNA Uptake and Virulence Nuclease, EndA, for Pneumococcal Transformation

Genetic transformation, in which cells internalize exogenous DNA and integrate it into their chromosome, is widespread in the bacterial kingdom. It involves a specialized membrane-associated machinery for binding double-stranded (ds) DNA and uptake of single-stranded (ss) fragments. In the human pathogen Streptococcus pneumoniae, this machinery is specifically assembled at competence. The EndA nuclease, a constitutively expressed virulence factor, is recruited during competence to play the key role of converting dsDNA into ssDNA for uptake. Here we use fluorescence microscopy to show that EndA is uniformly distributed in the membrane of noncompetent cells and relocalizes at midcell during competence. This recruitment requires the dsDNA receptor ComEA. We also show that under ‘static’ binding conditions, i.e., in cells impaired for uptake, EndA and ComEA colocalize at midcell, together with fluorescent end-labelled dsDNA (Cy3-dsDNA). We conclude that midcell clustering of EndA reflects its recruitment to the DNA uptake machinery rather than its sequestration away from this machinery to protect transforming DNA from extensive degradation. In contrast, a fraction of ComEA molecules were located at cell poles post-competence, suggesting the pole as the site of degradation of the dsDNA receptor. In uptake-proficient cells, we used Cy3-dsDNA molecules enabling expression of a GFP fusion upon chromosomal integration to identify transformed cells as GFP producers 60–70 min after initial contact between DNA and competent cells. Recording of images since initial cell-DNA contact allowed us to look back to the uptake period for these transformed cells. Cy3-DNA foci were thus detected at the cell surface 10–11 min post-initial contact, all exclusively found at midcell, strongly suggesting that active uptake of transforming DNA takes place at this position in pneumococci. We discuss how midcell uptake could influence homology search, and the likelihood that midcell uptake is characteristic of cocci and/or the growth phase-dependency of competence.

遗传转化（Genetic transformation）指细胞摄取外源脱氧核糖核酸（DNA）并将其整合至自身染色体的过程，广泛分布于细菌域。该过程依赖一套特化的膜相关机器，用于结合双链DNA（double-stranded DNA，dsDNA）并摄取单链DNA（single-stranded DNA，ssDNA）片段。在人类致病菌肺炎链球菌（Streptococcus pneumoniae）中，这套机器仅在感受态（competence）时期特异性组装。EndA核酸酶（EndA nuclease）是一种组成型表达的毒力因子（virulence factor），在感受态时期被招募，负责将dsDNA降解为可摄取的ssDNA，发挥关键作用。本研究通过荧光显微镜术（fluorescence microscopy）证实，EndA在非感受态细胞的细胞膜中呈均匀分布，而在感受态时期会重新定位至细胞中部。这种招募过程依赖双链DNA受体ComEA（ComEA）。我们还发现，在‘静态结合’条件下，即摄取功能受损的细胞中，EndA与ComEA会在细胞中部共定位，并与末端荧光标记的双链DNA（Cy3-dsDNA）聚集在一起。据此我们提出，EndA在细胞中部的聚集反映了其被招募至DNA摄取机器的过程，而非为保护转化DNA免于过度降解而被隔离远离该机器。与之相反，感受态结束后，一部分ComEA分子会定位于细胞极，这提示细胞极是dsDNA受体的降解位点。在摄取功能正常的细胞中，我们利用可在染色体整合后表达绿色荧光蛋白融合蛋白（GFP fusion）的Cy3-dsDNA分子，将转化细胞鉴定为可表达绿色荧光蛋白（GFP）的细胞，该过程发生在DNA与感受态细胞初始接触后的60~70分钟。通过记录从细胞与DNA初始接触时刻起的成像序列，我们能够回溯这些转化细胞的DNA摄取阶段。结果显示，在初始接触后的10~11分钟，可在细胞表面检测到Cy3标记DNA的荧光位点，且所有位点均仅定位于细胞中部，这强烈表明肺炎链球菌的转化DNA主动摄取过程发生于此位置。最后我们讨论了细胞中部的摄取过程如何影响同源搜索（homology search），以及细胞中部摄取是否为球菌（cocci）的特征，抑或是感受态的生长阶段依赖性（growth phase-dependency）的可能性。