Georgi Muskhelishvili

A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli

In Escherichia coli, the transcriptional activity of many promoters is strongly dependent on the negative superhelical density of chromosomal DNA. This, in turn, varies with the growth phase, and is correlated with the overall activity of DNA gyrase, the major topoisomerase involved in the elevation of negative superhelicity. The DNA architectural protein FIS is a regulator of the metabolic reorganization of the cell during early exponential growth phase. We have previously shown that FIS modulates the superhelical density of plasmid DNA in vivo, and on binding reshapes the supercoiled DNA in vitro. Here, we show that, in addition, FIS represses the gyrA and gyrB promoters and reduces DNA gyrase activity. Our results indicate that FIS determines DNA topology both by regulation of topoisomerase activity and, as previously inferred, by directly reshaping DNA. We propose that FIS is involved in coupling cellular physiology to the topology of the bacterial chromosome.

Gene order and chromosome dynamics coordinate spatiotemporal gene expression during the bacterial growth cycle

In Escherichia coli crosstalk between DNA supercoiling, nucleoid-associated proteins and major RNA polymerase σ initiation factors regulates growth phase-dependent gene transcription. We show that the highly conserved spatial ordering of relevant genes along the chromosomal replichores largely corresponds both to their temporal expression patterns during growth and to an inferred gradient of DNA superhelical density from the origin to the terminus. Genes implicated in similar functions are related mainly in trans across the chromosomal replichores, whereas DNA-binding transcriptional regulators interact predominantly with targets in cis along the replichores. We also demonstrate that macrodomains (the individual structural partitions of the chromosome) are regulated differently. We infer that spatial and temporal variation of DNA superhelicity during the growth cycle coordinates oxygen and nutrient availability with global chromosome structure, thus providing a mechanistic insight into how the organization of a complete bacterial chromosome encodes a spatiotemporal program integrating DNA replication and global gene expression.

The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in ‘fine‐tuning’ the homeostatic control mechanism of DNA supercoiling in E. coli.

DNA supercoiling and transcription in Escherichia coli. The FIS connection

The nucleoid-associated protein FIS modulates the topology of DNA in a growth-phase dependent manner functioning homeostatically to counteract excessive levels of negative superhelicity. We propose that this is achieved by at least two mechanisms: the physical constraint of low levels of negative superhelicity by FIS binding to DNA and by a reduction in the expression and effectiveness of DNA gyrase. In addition, high levels of expression of the fis gene do themselves require a high negative superhelical density. On DNA substrates containing phased high affinity binding sites, as exemplified by the upstream activating sequence of the tyrT promoter, FIS forms tightly bent DNA structures, or microloops, that are necessary for the optimal expression of the promoter. We suggest that these microloops compensate in part for the FIS-induced lowering of the superhelical density.

Regulation of crp transcription by oscillation between distinct nucleoprotein complexes

FIS belongs to the group of small abundant DNA‐binding proteins of Escherichia coli. We recently demonstrated that, in vivo, FIS regulates the expression of several genes needed for catabolism of sugars and nucleic acids, a majority of which are also transcriptionally regulated by cAMP–cAMP‐receptor protein (CRP) complex. Here we provide evidence that FIS represses transcription of the crp gene both in vivo and in vitro. Employing crp promoter–lacZ fusions, we demonstrate that both FIS and cAMP–CRP are required to keep the crp promoter in a repressed state. We have identified in the crp promoter other transcription initiation sites which are located 73, 79 and 80 bp downstream from the previously mapped start site. Two CRP‐ and several FIS‐binding sites with different affinities are located in the crp promoter region, one of them overlapping the downstream transcription initiation sites. We show that initiation of transcription at the crp promoter is affected by the composition of nucleoprotein complexes resulting from the outcome of competition between proteins for overlapping binding sites. Our results suggest that the control of crp transcription is achieved by oscillation in the composition of these regulatory nucleoprotein complexes in response to the physiological state of the cell.

FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter.

The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase. The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register. Neither the apparent requirement for three sites nor the mechanism of FIS‐mediated activation has been established. We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which ‘traps’ RNA polymerase (RNAP) at the promoter of the tyrT operon. This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA‐bound FIS dimers. The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein‐protein interactions. We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein ‘trap’ and recycled in successive rounds of holoenzyme assembly. Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase.

FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.

Order from the Order: How a Spatiotemporal Genetic Program Is Encoded in a 2-D Genetic Map of the Bacterial Chromosome

In this article, we sketch out a holistic methodology used for exploring how the genetic program is encoded in a 2-D genetic map of a bacterial chromosome. We argue that the major problem resides in the conceptual integration of the two logically distinct types of information encoded in the chiral double-helical DNA polymer. This integration is accomplished by mapping the genetic function on the genomic sequence organisation and therefore is potentially applicable to any chromosome. The vast generalisation achieved by this approach necessarily ignores exquisite details, yet it is fundamental in providing comprehensive methodology for exploring the role of the DNA sequence organisation in harnessing genetic information and sustaining biological order.

Chapter 3:Intrinsic In vivo Modulators: Negative Supercoiling and the Constituents of the Bacterial Nucleoid

The integration of three-dimensional structures induced by DNA superhelicity and transcriptional regulation is a fundamental mechanism facilitating the adaptation of bacteria to different environmental situations. During the bacterial growth cycle changes in DNA superhelicity are paralleled by, and ...