High Abundance of Transcription Regulators Compacts the Nucleoid in Escherichia coli.

Abstract

In enteric bacteria organization of the circular chromosomal DNA into a highly dynamic and toroidal-shaped nucleoid involves various factors, such as DNA supercoiling, nucleoid-associated proteins (NAPs), the structural maintenance of chromatin (SMC) complex, and macrodomain organizing proteins. Here, we show that ectopic expression of transcription regulators at high levels leads to nucleoid compaction. This serendipitous result was obtained by fluorescence microscopy upon ectopic expression of the transcription regulator and phosphodiesterase PdeL of Escherichia coli. Nucleoid compaction by PdeL depends on DNA-binding, but not on its enzymatic phosphodiesterase activity. Nucleoid compaction was also observed upon high-level ectopic expression of the transcription regulators LacI, RutR, RcsB, LeuO, and Cra, which range from single-target gene regulators to global regulators. In the case of LacI, its high-level expression in the presence of the gratuitous inducer IPTG (isopropyl-β-d-thiogalactopyranoside) also led to nucleoid compaction, indicating that compaction is caused by unspecific DNA-binding. In all cases nucleoid compaction correlated with misplacement of the FtsZ ring and loss of MukB foci, a subunit of the SMC complex. Thus, high levels of several transcription regulators cause nucleoid compaction with consequences for replication and cell division. IMPORTANCE The bacterial nucleoid is a highly organized and dynamic structure for simultaneous transcription, replication, and segregation of the bacterial genome. Compaction of the nucleoid and disturbance of DNA segregation and cell division by artificially high levels of transcription regulators, as described here, reveals that an excess of DNA-binding protein disturbs nucleoid structuring. The results suggest that ectopic expression levels of DNA-binding proteins for genetic studies of their function but also for their purification should be carefully controlled and adjusted.