Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA.

Genetic exchange mediated by viruses of bacteria (bacteriophages) is the primary driver of rapid bacterial evolution. The priority of viruses is usually to propagate themselves. Most bacteriophages use the small terminase protein to identify their own genome and direct its inclusion into phage capsids. Gene transfer agents (GTAs) are descended from bacteriophages, but they instead package fragments of the entire bacterial genome without preference for their own genes. GTAs do not selectively target specific DNA, and no GTA small terminases are known. Here, we identified the small terminase from the model Rhodobacter capsulatus GTA, which then allowed prediction of analogues in other species. We examined the role of the small terminase in GTA production and propose a structural basis for random DNA packaging.IMPORTANCE Random transfer of any and all genes between bacteria could be influential in the spread of virulence or antimicrobial resistance genes. Discovery of the true prevalence of GTAs in sequenced genomes is hampered by their apparent similarity to bacteriophages. Our data allowed the prediction of small terminases in diverse GTA producer species, and defining the characteristics of a "GTA-type" terminase could be an important step toward novel GTA identification. Importantly, the GTA small terminase shares many features with its phage counterpart. We propose that the GTA terminase complex could become a streamlined model system to answer fundamental questions about double-stranded DNA (dsDNA) packaging by viruses that have not been forthcoming to date.

Phosphate concentration and the putative sensor kinase protein CckA modulate cell lysis and release of the Rhodobacter capsulatus gene transfer agent.

The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles.

Characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting Shiga toxin encoding bacteriophage.

Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. Here, increasing levels of Stx phage Φ24(B) integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The Φ24(B) int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that Φ24(B) must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the Φ24(B) prophage is proposed.

Thermoluminescence dosimetry for skin dose assessment during intraoperative radiotherapy for early breast cancer.

Dosimetry for intraoperative radiotherapy (IORT) after wide local excision for breast cancer using a 50 kV X-ray needle (Intrabeam) was performed in vivo using thermoluminescence dosimetry. Eight LiF:Mg,Ti chips were placed on the skin around the incision site after wide local excision while the tumour bed was irradiated to a prescribed dose of 5 Gy 10 mm from the applicator surface. The maximum and mean measured skin dose for 57 patients ranged from 0.64 to 7.1 Gy and 0.56 to 4.78 Gy, respectively, reflecting different tissue thicknesses overlying the applicator. The average maximum dose of 2.93+/-1.46 Gy was below the threshold for severe radiation skin toxicity.

Medium-term performance and maintenance of SUDS: a case-study of Hopwood Park Motorway Service Area, UK.

One of the main barriers to implementing SUDS is concern about performance and maintenance costs since there are few well-documented case-studies. This paper summarizes studies conducted between 2000 and 2008 of the performance and maintenance of four SUDS management trains constructed in 1999 at the Hopwood Park Motorway Service Area, central England. Assessments were made of the wildlife value and sedimentation in the SUDS ponds, the hydraulic performance of the coach park management train, water quality in all management trains, and soil/sediment composition in the grass filter strip, interceptor and ponds. Maintenance procedures and costs were also reviewed. Results demonstrate the benefits of a management train approach over individual SUDS units for flow attenuation, water treatment, spillage containment and maintenance. Peak flows, pond sediment depth and contaminant concentrations in sediment and water decreased through the coach park management train. Of the 2007 annual landscape budget of pounds 15,000 for the whole site, the maintenance costs for SUDS only accounted for pounds 2,500 compared to pounds 4,000 for conventional drainage structures. Furthermore, since sediment has been attenuated in the management trains, the cost of sediment removal after the recommended period of three years was only pounds 554 and, if the design is not compromised, less frequent removal will be required in future.

Evidence of traffic-related pollutant control in soil-based sustainable urban drainage systems (SUDS).

SUDS are being increasingly employed to control highway runoff and have the potential to protect groundwater and surface water quality by minimising the risks of both point and diffuse sources of pollution. While these systems are effective at retaining polluted solids by filtration and sedimentation processes, less is known of the detail of pollutant behaviour within SUDS structures. This paper reports on investigations carried out as part of a co-ordinated programme of controlled studies and field measurements at soft-engineered SUDS undertaken in the UK, observing the accumulation and behaviour of traffic-related heavy metals, oil and PAHs. The field data presented were collected from two extended detention basins serving the M74 motorway in the south-west of Scotland. Additional data were supplied from an experimental lysimeter soil core leaching study. Results show that basin design influences pollutant accumulation and behaviour in the basins. Management and/or control strategies are discussed for reducing the impact of traffic-related pollutants on the aqueous environment.

The appearance and effects of metallic implants in CT images.

The computed tomography (CT) imaging artefacts that metallic medical implants produce in surrounding tissues are usually contoured and over-ridden during radiotherapy treatment planning. In cases where radiotherapy treatment beams unavoidably pass though implants, it is especially important to understand the imaging artefacts that may occur within the implants themselves. This study examines CT images of a set of simple metallic objects, immersed in water, in order to evaluate reliability and variability of CT numbers (Hounsfield units, HUs) within medical implants. Model implants with a range of sizes (heights from 2.2 to 49.6 mm), electron densities (from 2.3 to 7.7 times the electron density of water) and effective atomic numbers (from 3.9 to 9.0 times the effective atomic number of water in a CT X-ray beam) were created by stacking metal coins from several currencies. These 'implants' were CT scanned within a large (31.0 cm across) and a small (12.8 cm across) water phantom. Resulting HU values are as much as 50 % lower than the result of extrapolating standard electron density calibration data (obtained for tissue and bone densities) up to the metal densities and there is a 6 % difference between the results obtained by scanning with 120 and 140 kVp tube potentials. Profiles through the implants show localised cupping artefacts, within the implants, as well as a gradual decline in HU outside the implants that can cause the implants' sizes to be over estimated by 1.3-9.0 mm. These effects are exacerbated when the implants are scanned in the small phantom or at the side of the large phantom, due to reduced pre-hardening of the X-ray beam in these configurations. These results demonstrate the necessity of over-riding the densities of metallic implants, as well as their artefacts in tissue, in order to obtain accurate radiotherapy dose calculations.

Investigations into the causes of residue variability on carrots in the UK.

A pilot study was established to determine whether the preferential flow of water and thus insecticide in carrot beds might be responsible for the high residues of organophosphorus insecticides detected in individual carrot roots grown in the UK. A field site typical of UK carrot growing conditions was selected on a sandy soil with a low organic carbon content. Brilliant blue dye was applied in water to a small number of field plots located in the carrot beds to trace water movement through the bed and not to simulate insecticide application or irrigation. The plots were excavated following sufficient time for infiltration and drainage. Horizontal and vertical cross-sections through the soil profile were cut and descriptions of the dye presence in relation to soil features and the position of the carrot roots were made. Dye tracing and soil analyses showed there was a clear mechanical cultivation effect which generated a preferential movement of dye and water within the bed. The subsequent growth of carrots also created additional pathways of preferential movement due to stem-flow or canopy drip. A second study which increased replication of samples and allowed analysis of triazophos and chlorfenvinphos residues in the carrots could not identify any single factor which was conclusively responsible for initiating high residues in individual roots.