λ Recombineering Used to Engineer the Genome of Phage T7.

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of λ Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.

Effect of a point-of-care ultrasound protocol on the diagnostic performance of medical learners during simulated cardiorespiratory scenarios.

BACKGROUND: Goal-directed point-of-care ultrasound (PoCUS) protocols have been shown to improve the diagnostic accuracy of the initial clinical assessment of the critically ill patient. The diagnostic impact of the Abdominal and Cardiac Evaluation with Sonography in Shock (ACES) protocol was assessed in simulated emergency medical scenarios. METHODS: Following a focused PoCUS training program, the diagnostic accuracy, confidence, and precision of 12 medical learners participating in standardized scenarios were tested using high-fidelity clinical and ultrasound simulators. Participants were assessed during 72 simulated cardiorespiratory scenarios. Differential diagnoses were collected from participants before and after PoCUS in each scenario, and confidence surveys were completed. Data were analysed using R software. RESULTS: Prior to PoCUS, 45 (62.5%) correct primary diagnoses were made compared with 64 (88.9%) following PoCUS (χ2=14, 1df, p=0.0002). PoCUS was also shown to increase participants' confidence in their diagnoses. The mean confidence in diagnosis score pre-PoCUS was 52.2 (SD=14.7), whereas post-PoCUS it was 81.7 (SD=9.5). The estimated difference in means (-28.36) was significant (t=-7.71, p<0.0001). Using PoCUS, participants were further able to narrow their differential diagnoses. The median number of diagnoses for each patient pre-PoCUS was 3.5 (interquartile range [IQR]=3.8, 3.0) with a median of 2.3 (IQR=2.9,1.5) diagnoses post-PoCUS. The difference was significant (W=0, p<0.001). CONCLUSION: This pilot study suggests that, in medical learners newly competent in PoCUS, the addition of an ACES PoCUS protocol to standard clinical assessment improves diagnostic accuracy, confidence, and precision in simulated cardiorespiratory scenarios. This is consistent with clinical studies and supports the use of ultrasound during medical simulation.

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.

Can medical learners achieve point-of-care ultrasound competency using a high-fidelity ultrasound simulator?: a pilot study.

BACKGROUND: Point-of-care ultrasound (PoCUS) is currently not a universal component of curricula for medical undergraduate and postgraduate training. We designed and assessed a simulation-based PoCUS training program for medical learners, incorporating image acquisition and image interpretation for simulated emergency medical pathologies. We wished to see if learners could achieve competency in simulated ultrasound following focused training in a PoCUS protocol. METHODS: Twelve learners (clerks and residents) received standardized training consisting of online preparation materials, didactic teaching, and an interactive hands-on workshop using a high-fidelity ultrasound simulator (CAE Vimedix). We used the Abdominal and Cardiothoracic Evaluation by Sonography (ACES) protocol as the curriculum for PoCUS training. Participants were assessed during 72 simulated emergency cardiorespiratory scenarios. Their ability to complete an ACES scan independently was assessed. Data was analyzed using R software. RESULTS: Participants independently generated 574 (99.7%) of the 576 expected ultrasound windows during the 72 simulated scenarios and correctly interpreted 67 (93%) of the 72 goal-directed PoCUS scans. CONCLUSIONS: Following a focused training process using medical simulation, medical learners demonstrated an ability to achieve a degree of competency to both acquire and correctly interpret cardiorespiratory PoCUS findings using a high-fidelity ultrasound simulator.

Bacteriophages lytic for Salmonella rapidly reduce Salmonella contamination on glass and stainless steel surfaces.

A cocktail of six lytic bacteriophages, SalmoFresh™, significantly (p < 0.05) reduced the number of surface-applied Salmonella Kentucky and Brandenburg from stainless steel and glass surfaces by > 99% (2.1-4.3 log). Both strains were susceptible to SalmoFresh™ in the spot-test assay. Conversely, SalmoFresh™ was unable to reduce surface contamination with a Salmonella Paratyphi B strain that was not susceptible to the phage cocktail in the spot-test assay. However, by replacing two SalmoFresh™ component phages with two new phages capable of lysing the Paratyphi B strain in the spot-test assay, the target range of the cocktail was shifted to include the Salmonella Paratyphi B strain. The modified cocktail, SalmoLyse™, was able to significantly (p < 0.05) reduce surface contamination of the Paratyphi B strain by > 99% (2.1-4.1 log). The data show that both phage cocktails were effective in significantly reducing the levels of Salmonella on hard surfaces, provided the contaminating strains were susceptible in the spot-test (i.e., spot-test susceptibility was indicative of efficacy in subsequent surface decontamination studies). The data also support the concept that phage preparations can be customized to meet the desired antibacterial application.

Intrinsic translocation barrier as an initial step in pausing by RNA polymerase II.

Pausing of RNA polymerase II (RNAP II) by backtracking on DNA is a major regulatory mechanism in control of eukaryotic transcription. Backtracking occurs by extrusion of the 3' end of the RNA from the active center after bond formation and before translocation of RNAP II on DNA. In several documented cases, backtracking requires a special signal such as A/T-rich sequences forming an unstable RNA-DNA hybrid in the elongation complex. However, other sequence-dependent backtracking signals and conformations of RNAP II leading to backtracking remain unknown. Here, we demonstrate with S. cerevisiae RNAP II that a cleavage-deficient elongation factor TFIIS (TFIIS(AA)) enhances backtracked pauses during regular transcription. This is due to increased efficiency of formation of an intermediate that leads to backtracking. This intermediate may involve misalignment at the 3' end of the nascent RNA in the active center of the yeast RNAP II, and TFIIS(AA) promotes formation of this intermediate at the DNA sequences, presenting a high-energy barrier to translocation. We proposed a three-step mechanism for RNAP II pausing in which a prolonged dwell time in the pre-translocated state increases the likelihood of the 3' RNA end misalignment facilitating a backtrack pausing. These results demonstrate an important role of the intrinsic blocks to forward translocation in pausing by RNAP II.

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.

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.

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.