Bruce Applegate

Whole-cell biocomputing

The ability to manipulate systems on the molecular scale naturally leads to speculation about the rational design of molecular-scale machines. Cells might be the ultimate molecular-scale machines and our ability to engineer them is relatively advanced when compared with our ability to control the synthesis and direct the assembly of man-made materials. Indeed, engineered whole cells deployed in biosensors can be considered one of the practical successes of molecular-scale devices. However, these devices explore only a small portion of cellular functionality. Individual cells or self-organized groups of cells perform extremely complex functions that include sensing, communication, navigation, cooperation and even fabrication of synthetic nanoscopic materials. In natural systems, these capabilities are controlled by complex genetic regulatory circuits, which are only partially understood and not readily accessible for use in engineered systems. Here, we focus on efforts to mimic the functionality of man-made information-processing systems within whole cells.

A bioluminescent whole-cell reporter for detection of 2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenol in soil.

ABSTRACT A bioreporter was made containing atfdRPDII-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (R2 = 0.9825) in the range of 2.0 μM to 1.1 × 102 μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 102μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.

Characterization and field trials of a bioluminescent bacterial reporter of iron bioavailability

To better understand Fe cycling in marine and freshwater systems, we have developed a biomolecular tool to track the perceived bioavailability of Fe to heterotrophic bacteria. Bioluminescent reporters, constructed by fusing the fepA–fes promoter of Escherichia coli (an Enterobactin biosynthesis gene regulated by the ferric uptake regulatory [Fur] system) to a luxCDABE cassette, were integrated into the chromosome of a halotolerant Pseudomonas putida, which uses the Fur system to regulate high-affinity Fe uptake. The resultant P. putida bioreporter has been successfully tested both in lab and field studies. Laboratory cultures were maintained at a range of concentrations of total Fe (0–25 nM) or limited by the addition of concentrations of wellcharacterized siderophores (desferrioxamine B [DFB], ferrichrome, 2,2V-dipyridyl [DP] and Rhodotorulic acid [RA], 0–200 nM) and used to establish the dynamic range of this reporter system. Analysis of sample incubations after only 4 h suggest that both of the trihydroxamate-type siderophores DFB and ferrichrome efficiently reduced Fe availability, resulting respectively in a 1.77- and 1.88-fold increase in luminescence relative to Fe-replete conditions. In contrast, additions of the dihydroxamate-type siderophore RA and the synthetic chelator DP resulted in no response from the system, suggesting that cells could access Fe complexed to these compounds without activating high-affinity Fe transport systems. Field studies were performed in the central basin of Lake Erie, which has previously been shown to undergo sporadic Fe limitation during summer stratification. DFB concentrations were titrated across a range of 0–50 nM into unfiltered water to manipulate Fe availabilities. Bioreporters expressed Fe stress (ca. a 2-fold increase in luminescence) at concentrations of DFB equivalent to the total (dissolved+particulate) Fe in the system (c30 nM), indicative of the concentration of bioavailable Fe. In a similar experiment with 0.2-Am pre-filtered water (2.25–5.24 nM Fe), a 6-fold increase in luminescence (relative to controls) was observed at the lowest (15 nM) concentration of chelators. The results of this study demonstrate the validity of bioreporters as a complimentary tool to measurements of total Fe. Moreover, these results suggest that a significant source of

Understanding the role of agricultural practices in the potential colonization and contamination by Escherichia coli in the rhizospheres of fresh produce.

To better protect consumers from exposure to produce contaminated with Escherichia coli, the potential transfer of E. coli from manure or irrigation water to plants must be better understood. We used E. coli strains expressing bioluminescence (E. coli O157:H7 lux) or multiantibiotic resistance (E. coli²(+)) in this study. These marked strains enabled us to visualize in situ rhizosphere colonization and metabolic activity and to track the occurrence and survival of E. coli in soil, rhizosphere, and phyllosphere. When radish and lettuce seeds were treated with E. coli O157:H7 lux and grown in an agar-based growth system, rapid bacterial colonization of the germinating seedlings and high levels of microbial activity were seen. Introduction of E. coli²(+) to soil via manure or via manure in irrigation water showed that E. coli could establish itself in the lettuce rhizosphere. Regardless of introduction method, 15 days subsequent to its establishment in the rhizosphere, E. coli²(+) was detected on the phyllosphere of lettuce at an average number of 2.5 log CFU/g. When E. coli²(+) was introduced 17 and 32 days postseeding to untreated soil (rather than the plant surface) via irrigation, it was detected at low levels (1.4 log CFU/g) on the lettuce phyllosphere 10 days later. While E. coli²(+) persisted in the bulk and rhizosphere soil throughout the study period (day 41), it was not detected on the external portions of the phyllosphere after 27 days. Overall, we find that E. coli is mobile in the plant system and responds to the rhizosphere like other bacteria.

The Use of a Novel NanoLuc -Based Reporter Phage for the Detection of Escherichia coli O157:H7

Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food as they are host-specific and able to differentiate live cells from dead ones. Upon infection, target bacteria become identifiable since reporter genes are expressed from the engineered phage genome. The E. coli O157:H7 bacteriophage ΦV10 was modified to express NanoLuc luciferase (Nluc) derived from the deep-sea shrimp Oplophorus gracilirostris. Once infected by the ΦV10 reporter phage, E. coli O157:H7 produces a strong bioluminescent signal upon addition of commercial luciferin (Nano-Glo®). Enrichment assays using E. coli O157:H7 grown in LB broth with a reporter phage concentration of 1.76 × 102 pfu ml−1 are capable of detecting approximately 5 CFU in 7 hours. Comparable detection was achieved within 9 hours using 9.23 × 103 pfu ml−1 of phage in selective culture enrichments of ground beef as a representative food matrix. Therefore we conclude that this NanoLuc reporter phage assay shows promise for detection of E. coli O157:H7 from food in a simple, fast and sensitive manner.

Influence of fullerene (C60) on soil bacterial communities: aqueous aggregate size and solvent co-introduction effects.

Fullerene C60 nanoparticles are being used in broad range of applications. It is important to assess their potential impacts in the environment. We evaluated the effects of C60 introduced as aqueous suspensions of nC60 aggregates of different particle size or via organic solvents on soils with different organic matter contents in this study. Impacts of the application were evaluated by measuring total microbial biomass, metabolic activity and bacterial community structure. Results show that nC60 aggregates, introduced as an aqueous suspension, had size-dependent effects on soil bacterial community composition in the low organic matter system, but induced minimal change in the microbial biomass and metabolic activity in soils with both high and low organic matter contents. Fullerene C60, co-introduced via an organic solvent, did not influence the response of soil microbes to the organic solvents. Our results suggest that nC60 aggregates of smaller size may have negative impact on soil biota and soil organic matter may play a key role in modulating the environmental effect of nanomaterials.

A model system for pathogen detection using a two-component bacteriophage/bioluminescent signal amplification assay

Microbial contamination has become a mounting concern the last decade due to an increased emphasis of minimally processed food products specifically produce, and the recognition of foodborne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes. This research investigates a detection approach utilizing bacteriophage pathogen specificity coupled with a bacterial bioluminescent bioreporter utilizing the quorum sensing molecule from Vibrio fischeri, N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL). The 3-oxo-C6-HSL molecules diffuse out of the target cell after infection and induce bioluminescence from a population of 3-oxo-C6-HSL bioreporters (ROLux). E. coli phage M13, a well-characterized bacteriophage, offers a model system testing the use of bacteriophage for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains. These assays showed detection limits of 102cfu after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105cfu target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time.

Ultraviolet A and B wavelength-dependent inactivation of viruses and bacteria in the water

UVA and UVB can be applied to solar disinfection of water. In this study, the inactivation and photoreactivation of viruses and bacteria in the UVA-B range were analyzed. MS2 and T4 bacteriophages, and Escherichia coli were used as surrogates to quantify dose-response behaviors. Inactivation in UVC was used to validate the methodology and to expand the inactivation action spectra. The results showed log-linear inactivation for MS2 and T4 in the 254-320 nm wavelength range. T4 inactivation was consistently faster than MS2 (except at 320 nm), and for both phages, inactivation decreased with increasing wavelength. The dose-response of bacteria exhibited a lag at low doses, possibly because the photons must strike a discrete number of critical targets before growth stops. A tail was present at high doses for some wavelengths, perhaps due to clumping or the presence of subgroups with higher resistance. The inactivation action spectra for bacteria exhibited a reduction in inactivation as wavelength increased. No bacterial inactivation was observed beyond 320 nm at doses applied. After inactivation at 297 nm (UVA), bacteria regained viability through photoreactivation, and repair increased with increase in photoreactivating light exposure time. This implies additional doses above inactivation thresholds are required to cause irreversible damage. These results are useful for designing solar disinfection systems.

High voltage atmospheric cold plasma treatment of fresh cantaloupe to improve safety and quality

Raw agricultural products naturally contain a diverse surface population of microbial organisms. Cantaloupes, like other agricultural products, rarely undergo antimicrobial treatment before reaching the consumer, and are most often consumed raw. Many consumers fail to wash this fruit prior to consumption, contributing to the recent outbreaks of foodborne illness (1). This, along with poor handling practices makes it a desirable model for decontamination treatments. The goal of this research was to evaluate High Voltage Atmospheric Cold Plasma (HVACP) treatment for the reduction of surface microbial load on fresh cantaloupe. HVACP is a novel in-package atmospheric cold plasma process developed at Purdue University by Keener et al. Previous research (2) has shown HVACP is effective at eliminating spores (>6 log10), reducing microbial load (3-5 log10 of E. coli O157:H7 on lettuce; Salmonella spp. on tomatoes and spinach (3)) while extending shelf life. It indirectly exposes packaged, sliced cantaloupe to a high voltage / low power (90 Kv / 200W) HVACP field for 180 seconds, followed by 24 hours of room temperature (21 C) storage. During the 24 hour storage the reactive gas species (RGS) generated during HVACP treatment slowly convert back to their more forms (O2 and N2). Measurements include microbial load analysis, oxygen, nitrous oxides, and ozone concentrations. Consequent analysis of the cantaloupe exocarp by BARDOT (BActerial Rapid Detection using Optical light scattering Technology), showed a 2-3 log10 reduction in microbial load across treated samples and 74% reduction in microbial diversity. Results indicate that this HVACP process effectively reduces microbial populations on fresh, raw cantaloupe surfaces.

Microbial Bioluminescent Sensing of Bioavailable Contaminants in Environmental Matrices

and potential risks of various remedial approaches including natural attenuation, capping and dredging. Expertise in a broad range of technical disciplines was required to conduct this evaluation. The effectiveness of a remedy was judged to be directly related to the speed at which surficial sediment average concentrations could be reduced. Most of the remedial action objectives were expected to be directly related to the surficial sediment average concentration. Capping was found to be an effective means of rapidly reducing exposure and risk, although there remains a residual risk of cap failure and contaminant release far into the future. Conservative approaches to cap design were investigated to ensure long term stability of the cap and containment of the contaminants. Capping also provided opportunities for habitat improvement, efforts that would be consistent with the long term goals of the river, although not required to meet remedial goals.