Facilitated Dissociation of Nucleoid Associated Proteins from DNA in the Bacterial Confinement

Transcription machinery ultimately depends on the temporal formation of protein-DNA complexes. Recent experimental studies demonstrate that residence time (i.e., inverse off-rate) of a transcription factor protein can be a contributor to the functional diversity of the protein. In the meantime, single-molecule experiments showed that the off-rates of a wide array of DNA-binding proteins accelerate as the bulk concentration of the protein increases via a concentration-dependent mechanism (i.e., facilitated dissociation, FD). In this study, inspired by the previous single-molecule studies on the factor for inversion stimulation (Fis) protein of E. coli, which is a dual-purpose protein with a diverse functionality, we model the unbinding of Fis from specific bindings sites along a high-molecular-weight circular DNA in a cylindrical structure mimicking the cellular confinement of chromosome. Our simulations show that FD of Fis can well occur in confinement at physiological concentrations. Particularly, when nutrient-rich conditions are emulated with Fis concentrations around micromolar levels, the off-rates increase one order of magnitude compared to the lower Fis levels. However, Fis significantly changes the chromosome structure at higher concentrations by forming dense protein clusters bridging specific sites and juxtaposing remote DNA segments. As a result, at the physiologically observed maximum levels of Fis, the off-rates significantly slow down. Overall, our results indicate that cellular-concentration levels of a structural DNA-binding protein is intermingled with the genome architecture and DNA residence times, thereby providing a basis for understanding the complex effects of dynamic protein-DNA interactions on gene regulation.

Crystal structure of the nucleoid-associated protein Fis (PA4853) from Pseudomonas aeruginosa

Factor for inversion stimulation (Fis) is a versatile bacterial nucleoid-associated protein that can directly bind and bend DNA to influence DNA topology. It also plays crucial roles in regulating bacterial virulence factors and in optimizing bacterial adaptation to various environments. Fis from Pseudomonas aeruginosa (PA4853, referred to as PaFis) has recently been found to be required for virulence by regulating the expression of type III secretion system (T3SS) genes. PaFis can specifically bind to the promoter region of exsA, which functions as a T3SS master regulator, to regulate its expression and plays an essential role in transcription elongation from exsB to exsA. Here, the crystal structure of PaFis, which is composed of a four-helix bundle and forms a homodimer, is reported. PaFis shows remarkable structural similarities to the well studied Escherichia coli Fis (EcFis), including an N-terminal flexible loop and a C-terminal helix–turn–helix (HTH) motif. However, the critical residues for Hin-catalyzed DNA inversion in the N-terminal loop of EcFis are not conserved in PaFis and further studies are required to investigate its exact role. A gel-electrophoresis mobility-shift assay showed that PaFis can efficiently bind to the promoter region of exsA. Structure-based mutagenesis revealed that several conserved basic residues in the HTH motif play essential roles in DNA binding. These structural and biochemical studies may help in understanding the role of PaFis in the regulation of T3SS expression and in virulence.

Fis negatively affects binding of Tn4652 transposase by out-competing IHF from the left end of Tn4652

Transposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.