High abundance of transcription regulators compacts the nucleoid in Escherichia coli

Abstract

AbstractIn 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 macro-domain 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 of a strain expressing the mCherry-tagged HU-α subunit (HupA) for nucleoid staining. 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 case of LacI its high-level expression in presence of the gratuitous inducer IPTG 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 on transcription, replication, and cell division.ImportanceThe 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.