Development Of An Engineered Bioluminescent Reporter Phage For Detection Of Bacterial Blight Of Crucifers

Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant >light>-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wildtype phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.National Science Foundation Small Business Innovative Research 1012059U.S. Department of EducationU.S. Department of AgricultureCellular and Molecular Biolog

Experimental evolution of UV resistance in a phage

The dsDNA bacteriophage T7 was subjected to 30 cycles of lethal ultraviolet light (UV) exposure to select increased resistance to UV. The exposure effected a 0.9999 kill of the ancestral population, and survival of the ending population was nearly 50-fold improved. At the end point, a 2.1 kb deletion of early genes and three substitutions in structural-genes were the only changes observed at high frequency throughout the 40 kb genome; no changes were observed in genes affecting DNA metabolism. The deletion accounted for only a two-fold improvement in survival. One possible explanation of its benefit is that it represents an error catastrophe, whereby the genome experiences a reduced mutation rate. The mechanism of benefit provided by the three structural-gene mutations remains unknown. The results offer some hope of artificially evolving greater protection against sunlight damage in applications of phage therapy to plants, but the response of T7 is weak compared to that observed in bacteria selected to resist ionizing radiation. Because of the weak response, mathematical analysis of the selection process was performed to determine how the protocol might have been modified to achieve a greater response, but the greatest protection may well come from evolving phages to bind materials that block the UV

Role of osmotic and hydrostatic pressures in bacteriophage genome ejection

A critical step in the bacteriophage life cycle is genome ejection into host bacteria. The ejection process for double-stranded DNA phages has been studied thoroughly \textit{in vitro}, where after triggering with the cellular receptor the genome ejects into a buffer. The experimental data have been interpreted in terms of the decrease in free energy of the densely packed DNA associated with genome ejection. Here we detail a simple model of genome ejection in terms of the hydrostatic and osmotic pressures inside the phage, a bacterium, and a buffer solution/culture medium. We argue that the hydrodynamic flow associated with the water movement from the buffer solution into the phage capsid and further drainage into the bacterial cytoplasm, driven by the osmotic gradient between the bacterial cytoplasm and culture medium, provides an alternative mechanism for phage genome ejection \textit{in vivo}; the mechanism is perfectly consistent with phage genome ejection \textit{in vitro}.Comment: 5 pages, 2 figures, references update

Synergy as a rationale for phage therapy using phage cocktails

Where phages are used to treat bacterial contaminations and infections, multiple phages are typically applied at once as a cocktail. When two or more phages in the cocktail attack the same bacterium, the combination may produce better killing than any single phage (synergy) or the combination may be worse than the best single phage (interference). Synergy is of obvious utility, especially if it can be predicted a priori, but it remains poorly documented with few examples known. This study addresses synergy in which one phage improves adsorption by a second phage. It first presents evidence of synergy from an experimental system of two phages and a mucoid E. coli host. The synergy likely stems from a tailspike enzyme produced by one of the phages. We then offer mathematical models and simulations to understand the dynamics of synergy and the enhanced magnitude of bacterial control possible. The models and observations complement each other and suggest that synergy may be of widespread utility and may be predictable from easily observed phenotypes

Therapeutic Application of Phage Capsule Depolymerases against K1, K5, and K30 Capsulated E. coli in Mice

Capsule depolymerase enzymes offer a promising class of new antibiotics. In vivo studies are encouraging but it is unclear how well this type of phage product will generalize in therapeutics, or whether different depolymerases against the same capsule function similarly. Here, in vivo efficacy was tested using cloned bacteriophage depolymerases against Escherichia coli strains with three different capsule types: K1, K5, and K30. When treating infections with the cognate capsule type in a mouse thigh model, the previously studied K1E depolymerase rescued poorly, whereas K1F, K1H, K5, and K30 depolymerases rescued well. K30 gp41 was identified as the catalytically active protein. In contrast to the in vivo studies, K1E enzyme actively degraded K1 capsule polysaccharide in vitro and sensitized K1 bacteria to serum killing. The only in vitro correlate of poor K1E performance in vivo was that the purified enzyme did not form the expected trimer. K1E appeared as an 18-mer which might limit its in vivo distribution. Overall, depolymerases were easily identified, cloned from phage genomes, and as purified proteins they proved generally effective

Diagnostic Bioluminescent Phage for Detection of Yersinia pestis▿

Yersinia pestis is the etiological agent of the plague. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak, whether it is natural or deliberate, be detected and diagnosed quickly. The objective of this research was to generate a recombinant luxAB (“light”)-tagged reporter phage that can detect Y. pestis by rapidly and specifically conferring a bioluminescent signal response to these cells. The bacterial luxAB reporter genes were integrated into a noncoding region of the CDC plague-diagnostic phage φA1122 by homologous recombination. The identity and fitness of the recombinant phage were assessed through PCR analysis and lysis assays and functionally verified by the ability to transduce a bioluminescent signal to recipient cells. The reporter phage conferred a bioluminescent phenotype to Y. pestis within 12 min of infection at 28°C. The signal response time and signal strength were dependent on the number of cells present. A positive signal was obtained from 102 cells within 60 min. A signal response was not detectable with Escherichia coli, although a weak signal (100-fold lower than that with Y. pestis) was obtained with 1 (of 10) Yersinia enterocolitica strains and 2 (of 10) Yersinia pseudotuberculosis strains at the restrictive temperature. Importantly, serum did not prevent the ability of the reporter phage to infect Y. pestis, nor did it significantly quench the resulting bioluminescent signal. Collectively, the results indicate that the reporter phage displays promise for the rapid and specific diagnostic detection of cultivated Y. pestis isolates or infected clinical specimens