Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins.

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10-14-10-17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays.

Rates of gyrase supercoiling and transcription elongation control supercoil density in a bacterial chromosome.

Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound "nucleoid." The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σ(D)) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σ(C)) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σ(C)+σ(D). Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb "supercoil sensor" inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (-) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σ(D) at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species.

Twenty Years of Collaboration to Sort out Phage Mu Replication and Its Dependence on the Mu Central Gyrase Binding Site.

For 20 years, the intricacies in bacteriophage Mu replication and its regulation were elucidated in collaboration between Ariane Toussaint and her co-workers in the Laboratory of Genetics at the Université Libre de Bruxelles, and the groups of Martin Pato and N. Patrick Higgins in the US. Here, to honor Martin Pato's scientific passion and rigor, we tell the history of this long-term sharing of results, ideas and experiments between the three groups, and Martin's final discovery of a very unexpected step in the initiation of Mu replication, the joining of Mu DNA ends separated by 38 kB with the assistance of the host DNA gyrase.

DNA topology of highly transcribed operons in Salmonella enterica serovar Typhimurium.

Bacteria differ from eukaryotes by having the enzyme DNA gyrase, which catalyses the ATP-dependent negative supercoiling of DNA. Negative supercoils are essential for condensing chromosomes into an interwound (plectonemic) and branched structure known as the nucleoid. Topo-1 removes excess supercoiling in an ATP-independent reaction and works with gyrase to establish a topological equilibrium where supercoils move within 10 kb domains bounded by stochastic barriers along the sequence. However, transcription changes the stochastic pattern by generating supercoil diffusion barriers near the sites of gene expression. Using supercoil-dependent Tn3 and γδ resolution assays, we studied DNA topology upstream, downstream and across highly transcribed operons. Whenever two Res sites flanked efficiently transcribed genes, resolution was inhibited and the loss in recombination efficiency was proportional to transcription level. Ribosomal RNA operons have the highest transcription rates, and resolution assays at the rrnG and rrnH operons showed inhibitory levels 40-100 times those measured in low-transcription zones. Yet, immediately upstream and downstream of RNA polymerase (RNAP) initiation and termination sites, supercoiling characteristics were similar to poorly transcribed zones. We present a model that explains why RNAP blocks plectonemic supercoil movement in the transcribed track and suggests how gyrase and TopA control upstream and downstream transcription-driven supercoiling.

Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35⁻38 Amino Acids of GyrA.

Prokaryotes have an essential gene-gyrase-that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35⁻38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to ß-pinwheel elements is presented.

Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts.

The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes.

Species-specific supercoil dynamics of the bacterial nucleoid.

Bacteria organize DNA into self-adherent conglomerates called nucleoids that are replicated, transcribed, and partitioned within the cytoplasm during growth and cell division. Three classes of proteins help condense nucleoids: (1) DNA gyrase generates diffusible negative supercoils that help compact DNA into a dynamic interwound and multiply branched structure; (2) RNA polymerase and abundant small basic nucleoid-associated proteins (NAPs) create constrained supercoils by binding, bending, and forming cooperative protein-DNA complexes; (3) a multi-protein DNA condensin organizes chromosome structure to assist sister chromosome segregation after replication. Most bacteria have four topoisomerases that participate in DNA dynamics during replication and transcription. Gyrase and topoisomerase I (Topo I) are intimately involved in transcription; Topo III and Topo IV play critical roles in decatenating and unknotting DNA during and immediately after replication. RNA polymerase generates positive (+) supercoils downstream and negative (-) supercoils upstream of highly transcribed operons. Supercoil levels vary under fast versus slow growth conditions, but what surprises many investigators is that it also varies significantly between different bacterial species. The MukFEB condensin is dispensable in the high supercoil density (σ) organism Escherichia coli but is essential in Salmonella spp. which has 15 % fewer supercoils. These observations raise two questions: (1) How do different species regulate supercoil density? (2) Why do closely related species evolve different optimal supercoil levels? Control of supercoil density in E. coli and Salmonella is largely determined by differences encoded within the gyrase subunits. Supercoil differences may arise to minimalize toxicity of mobile DNA elements in the genome.

Expression of the gene encoding the major bacterial nucleoid protein H-NS is subject to transcriptional auto-repression.

Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at -313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild-type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid-log cultures and negligible in cells entering the stationary phase. The level of repression in early-log phase was lower than in mid-log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H-NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H-NS and Fis protein to transcriptional systems programmed with the same hns caf fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation get analyses revealed that H-NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around -150. When these sites are filled by H-NS, an additional site between approximately -20 and -65, which partly overlaps the promoter, is also occupied. Binding of H-NS to this site is probably the ultimate cause of transcriptional auto-repression.

Topological Behavior of Plasmid DNA.

The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells.

RNA polymerase: chromosome domain boundary maker and regulator of supercoil density.

Most bacterial chromosomes and plasmids are covalently closed circular molecules that are maintained in a dynamic supercoiled state. Average supercoil density differs significantly between Escherichia coli and Salmonella. Two related questions are: What protein(s) create supercoil domain boundaries in a bacterial chromosome? and How is supercoil density regulated in different bacterial species? RNA polymerase plays pivotal roles in both of these topological phenomena.

Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites: what's the Dif?

In this issue of Molecular Microbiology, Hendrickson and Lawrence analyse the sequence of bacterial genomes to map the historical traffic pattern of chromosome replication. Their surprising conclusion is that most forks terminate at the dif site rather than at the Tus/Ter sites where most investigators have concluded termination occurs most frequently. What make this analysis novel are the methods and the revisionist hypotheses for how and why forks might stop at dif.

Growth rate toxicity phenotypes and homeostatic supercoil control differentiate Escherichia coli from Salmonella enterica serovar Typhimurium.

Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella, showing disruption of supercoiling near the terminus and replicon failure at 42 degrees C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella. Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo.

Microarray analysis of Mu transposition in Salmonella enterica, serovar Typhimurium: transposon exclusion by high-density DNA binding proteins.

All organisms contain transposons with the potential to disrupt and rearrange genes. Despite the presence of these destabilizing sequences, some genomes show remarkable stability over evolutionary time. Do bacteria defend the genome against disruption by transposons? Phage Mu replicates by transposition and virtually all genes are potential insertion targets. To test whether bacteria limit Mu transposition to specific parts of the chromosome, DNA arrays of Salmonella enterica were used to quantitatively measure target site preference and compare the data with Escherichia coli. Essential genes were as susceptible to transposon disruption as non-essential ones in both organisms, but the correlation of transposition hot spots among homologous genes was poor. Genes in highly transcribed operons were insulated from transposon mutagenesis in both organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid cold spot, and an ectopic parS site placed in the Salmonella chromosome created a new cold spot in the presence of ParB. Our data show that competition between cellular proteins and transposition proteins on plasmids and the chromosome is a dominant factor controlling the genetic footprint of transposons in living cells.

A gyrase mutant with low activity disrupts supercoiling at the replication terminus.

When a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. The gyrB652 mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42 degrees C, strains with the gyrB652 allele stop DNA replication, and at 37 degrees C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42 degrees C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a low k(cat) compared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.

Organization of supercoil domains and their reorganization by transcription.

During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require specific types of DNA movement. Separation forces move replicated chromosomes into separate sister cell compartments during cell division, and the contemporaneous acts of DNA replication, RNA transcription and cotranscriptional translation of membrane proteins cause specific regions of DNA to twist, writhe and expand or contract. Recent experiments indicate that a dynamic and stochastic mechanism creates supercoil DNA domains soon after DNA replication. Domain structure is subsequently reorganized by RNA transcription. Examples of transcription-dependent chromosome remodelling are also emerging from eukaryotic cell systems.

Measuring chromosome dynamics on different time scales using resolvases with varying half-lives.

The bacterial chromosome is organized into multiple independent domains, each capable of constraining the plectonemic negative supercoil energy introduced by DNA gyrase. Different experimental approaches have estimated the number of domains to be between 40 and 150. The site-specific resolution systems of closely related transposons Tn3 and gammadelta are valuable tools for measuring supercoil diffusion and analysing bacterial chromosome dynamics in vivo. Once made, the wild-type resolvase persists in cells for time periods greater than the cell doubling time. To examine chromosome dynamics over shorter time frames that are more closely tuned to processes like inducible transcription, we constructed a set of resolvases with cellular half-lives ranging from less than 5 min to 30 min. Analysing chromosomes on different time scales shows domain structure to be dynamic. Rather than the 150 domains detected with the Tn3 resolvase, wild-type cells measured over a 10 min time span have more than 400 domains per genome equivalent, and some gyrase mutants exceed 1000.

Bacteriophage Mu targets the trinucleotide sequence CGG.

Target specificity for bacteriophage Mu was studied using a new phage derivative that enabled cloning of Mu-host junctions from phage DNA. Insertions distributed throughout the chromosome showed no orientation bias with respect to transcription or replication polarity. Genes with a high frequency of the triplet CGG were preferred targets.

Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome.

Transcription and replication both influence and are influenced by superhelical changes in DNA. Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years. Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion. Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze supercoiling dynamics of regions encoding membrane proteins. Regulation of Tn10-derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA. Strains deficient in TetR activity had 60-fold higher transcription levels (from P(A)) than TetR-positive strains. High tetA transcription caused a 10- to 80-fold decrease in the gammadelta resolution efficiency for the domain that includes the Tet module. Replacing tetA with genes encoding cytosolic proteins LacZ and Kan also caused the appearance of supercoil diffusion barriers in a defined region of the chromosome. In strains containing a functional TetR located next to a regulated lacZ reporter (P(R)tetR-P(A)lacZ), induction of transcription with chlortetracycline caused a 5-fold drop in resolution efficiency in the test domain interval. A short half-life resolvase showed that barriers appeared and disappeared over a 10- to 20-min span. These studies demonstrate the importance of transcription in chromosome structure and the plasticity of supercoil domains in bacterial chromosomes.