A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

/pdf/an-efficient-recombination-system-for-chromosome-engineering-4acmootzcr.pdf

An efficient recombination system for chromosome engineering in Escherichia coli

We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJb8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosporylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1 microgram of target Drosophila DNA gives about 5 x 10(5) clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.

Rapid and efficient cosmid cloning.

A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.

/pdf/structural-analysis-of-substrate-binding-by-the-molecular-2a1dx17hgt.pdf

Structural Analysis of Substrate Binding by the Molecular Chaperone DnaK

The accessory genes of Staphylococcus aureus, including those involved in pathogenesis, are controlled by a complex regulatory network that includes at least four two-component systems, one of which, agr, is a quorum sensor, an alternative sigma factor and a large set of transcription factors, including at least two of the superantigen genes, tst and seb. These regulatory genes are hypothesized to act in a time- and population density-dependent manner to integrate signals received from the external environment with the internal metabolic machinery of the cell, in order to achieve the production of particular subsets of accessory/virulence factors at the time and in quantities that are appropriate to the needs of the organism at any given location. From the standpoint of pathogenesis, the regulatory agenda is presumably tuned to particular sites in the host organism. To address this hypothesis, it will be necessary to understand in considerable detail the regulatory interactions among the organism's numerous controlling systems. This review is an attempt to integrate a large body of data into the beginnings of a model that will hopefully help to guide research towards a full-scale test.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2003.03526.x

Autoinduction and signal transduction in the regulation of staphylococcal virulence

An ATP-powered DNA translocation machine encapsidates the viral genome in the large dsDNA bacteriophages. The essential components include the empty shell, prohead, and the packaging enzyme, terminase. During translocation, terminase is docked on the prohead's portal protein. The translocation ATPase and the concatemer-cutting endonuclease reside in terminase. Remarkably, terminases, portal proteins, and shells of tailed bacteriophages and herpes viruses show conserved features. These DNA viruses may have descended from a common ancestor. Terminase's ATPase consists of a classic nucleotide binding fold, most closely resembling that of monomeric helicases. Intriguing models have been proposed for the mechanism of dsDNA translocation, invoking ATP hydrolysis-driven conformational changes of portal or terminase powering DNA motion. Single-molecule studies show that the packaging motor is fast and powerful. Recent advances permit experiments that can critically test the packaging models. The viral genome tran...

The bacteriophage DNA packaging motor.

Decision Making at a Subcellular Level Determines the Outcome of Bacteriophage Infection

Phage lambda, like a number of other large DNA bacteriophages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is co-ordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the lambda DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric lambda DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN to complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endonuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNu1, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in lambda has aspects that differ from other lambda DNA transactions. Unlike the site-specific recombination system of lambda, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complex, and unlike the DNA replication system of lambda, the same protein machinery is used for both initiation and translocation during lambda DNA packaging.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1365-2958.1995.tb02333.x

Virus DNA packaging: the strategy used by phage lambda.

We show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr--dnaK--dnaJ--leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

Genetic analysis of two genes, dnaJ and dnaK , necessary for Escherichia coli and bacteriophage lambda DNA replication

Large dsDNA bacteriophages and herpesviruses encode a powerful ATP-driven DNA-translocating machine that encapsidates a viral genome into a preformed capsid shell or prohead. The key components of the packaging machine are the packaging enzyme (terminase, motor) and the portal protein that forms the unique DNA entrance vertex of prohead. The terminase complex, comprised of a recognition subunit (small terminase) and an endonuclease/translocase subunit (large terminase), cuts viral genome concatemers. The terminase–viral DNA complex docks on the portal vertex, assembling a motor complex containing five large terminase subunits. The pentameric motor processively translocates DNA until the head shell is full with one viral genome. The motor cuts the DNA again and dissociates from the full head, allowing head-finishing proteins to assemble on the portal, sealing the portal, and constructing a platform for tail attachment. A body of evidence from molecular genetics and biochemical, structural, and biophysical approaches suggests that ATP hydrolysis–driven conformational changes in the packaging motor (large terminase) power DNA motion. Various parts of the motor subunit, such as the ATPase, arginine finger, transmission domain, hinge, and DNA groove, work in concert to translocate about 2 bp of DNA per ATP hydrolyzed. Powerful single-molecule approaches are providing precise delineation of steps during each translocation event in a motor that has a speed as high as a millisecond/step. The phage packaging machine has emerged as an excellent model for understanding the molecular machines, given the mechanistic parallels between terminases, helicases, and numerous motor proteins.

Michael Feiss

Papers

The bacteriophage DNA packaging motor.

Decision Making at a Subcellular Level Determines the Outcome of Bacteriophage Infection

Virus DNA packaging: the strategy used by phage lambda.

Genetic analysis of two genes, dnaJ and dnaK , necessary for Escherichia coli and bacteriophage lambda DNA replication

The bacteriophage DNA packaging machine.