Browsing by Subject "Bacteriophage"

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

Many vital biological tasks, such as protein degradation, DNA strand separation, and viral DNA packaging are performed by ring NTPase assemblies. These assemblies harvest energy from NTP binding and hydrolysis in order to translocate their biopolymer substrate through their central pores. Single-molecule characterization demonstrated that these assemblies are highly coordinated and produce forces an order of magnitude larger than most molecular motors. Recently, many structures of these assemblies have been experimentally solved and resulting globular translocation models have been proposed. While these static structures have provided great insights into how molecular motors assemble, the specific molecular mechanisms that promote, regulate, and coordinate the dynamic translocation processes remain poorly understood. In this dissertation, I use computational tools to model ring ATPase molecular motors in order to elucidate such mechanisms. Initially, I focus on viral packaging ATPases and then generalize my findings to a broader class of motors by studying FtsK-like and AAA+ motors. For all systems, atomistic molecular dynamics simulations were used to calculate free-energy landscapes that predict conformational changes, predict mutual-information-based signaling pathways that couple enzymatic and mechanical activities, predict principal components of motion that describe the enzyme’s native function, and predict the effects of mutagenesis in silico. For viral packaging ATPases, I first predicted that a strictly conserved Walker A arginine residue functions analogously to a sensor II motif arginine found in AAA+ systems, and that it is used to couple ATP binding to lid subdomain rotation. Second, I predicted how mutations in the Walker A and Walker B motifs could abrogate enzyme function. All these predictions were corroborated by collaborators’ extensive experimental characterization. Third, I helped build the first structure of an actively packaging viral ATPase motor into the cryo-EM reconstruction and led the biological interpretation of the resulting structure. Fourth, I used molecular dynamics simulations of pentameric ATPase assemblies to predict how the assemblies respond to nucleotide-occupancy and presence of double-stranded DNA substrate. Based on the structure and simulations, I proposed the helical-to-planar model of viral DNA packaging, which is the first atomistic model that can predict the salient features of viral DNA packaging. Further, this model lays the groundwork of future work by predicting specific conformational changes and interactions that were otherwise obscure from experimental studies. Fifth, I tested a key proposal in my helical-to-planar model by using molecular dynamics simulations to investigate how nucleotide binding is coupled to substrate gripping. The resulting glutamate switch signaling pathway was corroborated by structural data and functional mutagenesis assays. Lastly, I investigated FtsK-like and AAA+ enzymes to probe for molecular mechanisms common to a broad class of translocating ring ATPases. From these studies, I identified a core set of principles that can be modularly added together to describe a number of different translocation models. In summary, the results presented in this dissertation describe fundamental mechanisms of translocating ring ATPase motors. When possible, my computational predictions were corroborated by experimental characterization. When experimental characterization was not yet possible, my predictions and derived models serve as a guide for future studies. The models I derived provide the first comprehensive description of the coordinated conformational changes that drive viral DNA packaging. Further, they have the potential to inform rational design of synthetic molecular motors and anti-viral therapeutics that target the genome packaging step.

Outer membrane vesicles are an important constitutive product of all Gram-negative bacteria. Bacteria have evolved many responses to alleviate all different types of stress. The primary objective of this dissertation is to investigate the role of outer membrane vesicles (OMVs) as a method by which Gram-negative bacteria can quickly act to protect themselves against particular threats. Generally, we find that stressors whose primary effect is on the outer membrane can be protected against by OMVs. Throughout this study, a variety of different microbiological and biochemical methods are used to answer key questions in the innate ability of OMVs to protect against particular antimicrobials. Using Escherichia coli as well as Pseudomonas aeruginosa as model organisms we tested the ability of purified vesicles from each species to protect themselves and other hosts. Using bacteriophage T4, we investigated the ability of OMVs purified from E. coli to adsorb phage as well as how this interaction affected the efficiency of infection. We found that OMVs are protective against antimicrobial peptides, as well as bacteriophage. In the course of understanding this protection we also observed and characterized the cross species effects of both OMV protection as well as phage infection. Where typically a phage infects a specific species, we found that T4 associated OMVs treating a non-native host P. aeruginosa resulted in the production of a novel prophage. Upon further examination, we determined that this induction was occurring via a novel pathway that we attempted to further characterize by performing a genetic screen to identify genes important to this induction. The work within this dissertation fully supports the hypothesis of a regulated response to outer membrane acting stimuli, resulting in the induction of vesiculation and the adsorption of stressor in the extra-cellular milieu. This model of protection agrees with the idea of a bacterial innate defense system, which acts in the short term before the adaptive response can fully occur, resulting in a bridge between the untreated to the treated and resistant culture.