Summary
To fit the chromosome inside a bacterial cell, DNA is compacted more than 1000-fold. In numerous bacterial models, nucleoid-associated proteins (NAPs) are crucial for maintaining chromosomal architecture and are actively involved in DNA replication, recombination, repair, and gene regulation. A predominant NAP in the opportunistic pathogen Streptococcus pneumoniae is the histone-like protein A (HlpA) also known as HU. By tracking HU in live cells with superresolution microscopy, we describe the nucleoid as a dynamic structure with HU binding transiently across the entire nucleoid. Whole-genome binding analysis confirms a global binding, although showing a preference for the origin of replication over the terminus. Reducing cellular HU levels impacts nucleoid maintenance and disrupts the robust nucleoid scaling with cell size. This effect is comparable to the distortion caused by fluoroquinolones, supporting earlier observations that HU is essential for maintaining DNA supercoiling. Furthermore, cells lacking HU are unable to initiate and proceed with replication rounds. Chromosome conformation capture (Hi-C) revealed that HU is required to maintain cohesion between the two chromosomal arms, in a similar way to the structural maintenance of the chromosome complex SMC. Together, we show that by promoting long-range chromosome interactions and supporting the architecture of the domain of origin, HU is a benchmark for chromosome integrity and the intimately related processes of chromosome replication and segregation.
Significance
Streptococcus pneumoniae is a major cause of fatal bacterial infections, claiming over a million lives annually. Understanding the mechanisms of pneumococcal survival is critical for the development of new antimicrobial drugs. Although the fundamental aspects of chromosome dynamics have been extensively studied in bacterial models like E. coli, C. crescentus, and B. subtilis, there remains a significant knowledge gap concerning pneumococcus, particularly regarding the impact of NAPs on chromosome conformation. Here, we provide the first detailed map of chromosome organisation in S. pneumoniae and demonstrate that HU promotes chromosomal contacts in the megabase range and maintains the conformation of the origin of replication. Overall, with this work we show that pneumococcal HU is essential for chromosome conformation and consequential DNA replication and chromosome segregation.
In this submission:
* ChIP-sequencing samples of HU-GFP and RpoB-GFP in S. pneumoniae D39V.
* Hi-C samples of WT, HU- and SMC-depleted S.pneumoniae D39V. Less...