E.coli Cell-cycle Regulation by Bacteriophage Lambda

The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.target location | establishment of lysogeny | viral transduction T he search for specific sequences along genomic DNA plays a key role in the location of specific sites by transcription factors (1), the repair of DNA lesions (2), and horizontal gene transfer (3). Common to these processes is a search through a very large number of possible sequences because of the long genomes involved. A fundamental question is how specific target sequences can be located with high efficiency, within physiologically relevant times. This question is crucial to understand viral transduction, one of the fundamental mechanisms of horizontal gene transfer driving the evolution of prokaryotes (3, 4). In transduction, a viral genome integrates at a unique site on a bacterial genome following infection, conferring new traits such as pathogenicity (5). A classic example of transduction is furnished by the infection of Escherichia coli cells by bacteriophage λ.Infection of an E. coli host by the temperate bacteriophage λ begins with the binding of the phage to the E. coli maltose pore LamB (6, 7). The phage then injects its DNA into the cell, a process that lasts about 5 min (8). Infection can lead to two possible outcomes, lysis or lysogeny, which reflect alternative pathways of gene expression (9-11). In the lytic pathway, execution of a viral gene expression cascade leads to the replication of the viral DNA, resulting in cell death and lysis to release about 100 phage progeny (12). Alternatively, by establishing lysogeny, the phage shuts off the lytic cycle and locates with high efficiency (13) a unique sequence along the cellular genome where it integrates i...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo

Tal

Arbel-Goren

Costantino

et al. 2014

“…No pure solid red colonies were observed. Because these cells are grown in rich medium before recombination, multiple DNA replication forks result in eight galK copies being present in each cell under our conditions (35). Although saturating levels of oligo were used, when recombination occurs in a cell, it is unlikely to occur at every copy of galK, and as cells in a colony continue to replicate their DNA and divide, segregation of the recombinant and parental allele occurs, generating mixed or sectored colonies for the Gal phenotype.…”

Section: Mismatch Repair Functions Are Important In Correcting Galktymentioning

confidence: 99%

Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

Costantino

Court

2003

263

359

Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage -encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20 -70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In E. coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence. H omologous recombination mediated by Red has been used as a genetic tool to modify the bacterial chromosome with linear double-stranded DNA (dsDNA) (1-6). In addition to linear dsDNA, single-stranded DNA (ssDNA) oligonucleotides (oligos) have been used to modify the chromosomes of both yeast and Escherichia coli (7-9). In yeast, the functions are not yet defined that allow oligo recombination; however, in E. coli, the bacteriophage Red recombination system is involved (9).The Red system includes the Gam, Exo, and Beta proteins. Whereas Red-mediated recombination between linear duplex DNA and the bacterial chromosome requires all three functions (1, 3, 10), recombination with ssDNA oligos requires only the Beta protein (9). Beta protein binds stably to ssDNA (11) Ͼ35 nt in length (12), protects it from single-strand nuclease attack (13,14), and promotes annealing to complementary ssDNA (13-15).Beta binds ssDNA from 3Ј to 5Ј (13, 16) but does not bind directly to dsDNA (13,14). However, after Beta generates dsDNA by annealing two complementary ssDNAs, it remains tightly bound to the annealed dsDNA (13,14,16). As it anneals strands to form the dsDNA, it generates DNA filaments similar to those formed by . This annealed dsDNA-Beta complex is resistant to DNase I and is much more stable than the ssDNA-Beta complex (16).Although Beta can catalyze single-strand annealing, it cannot promote strand invasion of a duplex DNA with a homologous ssDNA during recombination (16,18). Therefore, Betamediated recombination with ssDNA is likely to occur by annealing with transiently single-stranded regions of the chromosome. Initial results suggest that Beta-dependent ssDNA recombination may occur at the DNA replication fork. When either of the two complementary ssDNA oligos...

“…TraR shares 29% sequence identity with the Cterminal half of DksA (4), a 151-residue protein that binds to and regulates transcription initiation by E. coli RNA polymerase (RNAP) at specific promoters (5)(6)(7). TraR also shares homology with predicted proteins of similar length elsewhere in the bacterial sequence database that are encoded by conjugative plasmids and bacteriophages (e.g., phages 186, P2, and lambda) (8,9).…”

mentioning

confidence: 99%

TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

Gopalkrishnan

Ross

Chen

et al. 2017

The Escherichia coli F element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.