Adam R. Parks

Transposition into Replicating DNA Occurs through Interaction with the Processivity Factor

The bacterial transposon Tn7 directs transposition into actively replicating DNA by a mechanism involving the transposon-encoded protein TnsE. Here we show that TnsE physically and functionally interacts with the processivity factor of the DNA replication machinery in vivo and in vitro. Our work establishes an in vitro TnsABC+E transposition reaction reconstituted from purified proteins and target DNA structures. Using the in vitro reaction we confirm that the processivity factor specifically reorders TnsE-mediated transposition events on target DNAs in a way that matches the bias with active DNA replication in vivo. The TnsE interaction with an essential and conserved component of the replication machinery, and a DNA structure reveals a mechanism by which Tn7, and probably other elements, selects target sites associated with DNA replication.

Tn7 elements: engendering diversity from chromosomes to episomes.

The bacterial transposon Tn7 maintains two distinct lifestyles, one in horizontally transferred DNA and the other in bacterial chromosomes. Access to these two DNA pools is mediated by two separate target selection pathways. The proteins involved in these pathways have evolved to specifically activate transposition into their cognate target-sites using entirely different recognition mechanisms, but the same core transposition machinery. In this review we discuss how the molecular mechanisms of Tn7-like elements contribute to their diversification and how they affect the evolution of their host genomes. The analysis of over 50 Tn7-like elements provides insight into the evolution of Tn7 and Tn7 relatives. In addition to the genes required for transposition, Tn7-like elements transport a wide variety of genes that contribute to the success of diverse organisms. We propose that by decisively moving between mobile and stationary DNA pools, Tn7-like elements accumulate a broad range of genetic material, providing a selective advantage for diverse host bacteria.

Transposon Tn7 Is Widespread in Diverse Bacteria and Forms Genomic Islands

We find that relatives of the bacterial transposon Tn7 are widespread in disparate environments and phylogenetically diverse species. These elements form functionally diverse genomic islands at the specific site of Tn7 insertion adjacent to glmS. This work presents the first example of genomic island formation by a DDE type transposon.

tCRISPRi: tunable and reversible, one-step control of gene expression

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

Bacteriophage λ N protein inhibits transcription slippage by Escherichia coli RNA polymerase

Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5′ end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination.

DNA Damage Differentially Activates Regional Chromosomal Loci for Tn7 Transposition in Escherichia coli

The bacterial transposon Tn7 recognizes replicating DNA as a target with a preference for the region where DNA replication terminates in the Escherichia coli chromosome. It was previously shown that DNA double-strand breaks in the chromosome stimulate Tn7 transposition where transposition events occur broadly around the point of the DNA break. We show that individual DNA breaks actually activate a series of small regional hotspots in the chromosome for Tn7 insertion. These hotspots are fixed and become active only when a DNA break occurs in the same region of the chromosome. We find that the distribution of insertions around the break is not explained by the exonuclease activity of RecBCD moving the position of the DNA break, and stimulation of Tn7 transposition is not dependent on RecBCD. We show that other forms of DNA damage, like exposure to UV light, mitomycin C, or phleomycin, also stimulate Tn7 transposition. However, inducing the SOS response does not stimulate transposition. Tn7 transposition is not dependent on any known specific pathway of replication fork reactivation as a means of recognizing DNA break repair. Our results are consistent with the idea that Tn7 recognizes DNA replication involved in DNA repair and reveals discrete regions of the chromosome that are differentially activated as transposition targets.

Transposon Tn7 Directs Transposition into the Genome of Filamentous Bacteriophage M13 Using the Element-Encoded TnsE Protein

The bacterial transposon Tn7 has a pathway of transposition that preferentially targets conjugal plasmids. We propose that this same transposition pathway recognizes a structure or complex found during filamentous bacteriophage replication, likely by targeting negative-strand synthesis. The ability to insert into both plasmid and bacteriophage DNAs that are capable of cell-to-cell transfer would help explain the wide distribution of Tn7 relatives.