Bacterial viruses, also known as phages, are intrinsic components of the Earth’s Critical Zone (CZ). Together with diverse communities of bacteria and fungi, they occupy habitats of the CZ extending from the vegetation canopy, through the soils and into the aquifers. In this thesis, I aimed to study the transport of phages in the upper CZ and their interactions with non-host soil bacteria and fungi to reveal their role in regulating the CZ microbial life. To reflect transport processes of CZ-inhabiting phages in soil tracers are highly useful. Thus, in the first study, I evaluated the transport efficiency and particle intactness of marine tracer phages passing through soil. Marine phages were selected as tracer, because they are non-pathogenic, non-toxic, naturally absent and thus non-multiplying in the terrestrial subsurface. I found that the marine phages PSA-HM1 and PSA-HS2 retained high phage particle intactness in contrast to commonly used Escherichia virus T4. This suggests their potential as particle tracers to mimic the transport of (bio-) colloids of similar traits in soil. Soil in the CZ is often unsaturated and restricts mobility of microorganisms. Fungi bridge unsaturated zones in soil and hence provide network for microbial transport. In the second study, I developed a hyphosphere model system mimicking unsaturated soil environment, and reported on the ability of hyphal-riding bacteria to co-transport lytic phages and to utilize phages as “weapons” for improved colonization of water-unsaturated habitats. As the findings emphasize the importance of hyphal transport of bacteria and associated phages, in the third study, I developed a hyphae-assisted approach and isolated five soil bacteria able to co-transport phages. In analogy to invasion frameworks in macroecology, the hyphosphere model system with bacteria and co-transported phages can be useful models to simulate processes of biological invasion at micro-scale. In the fourth study, I investigated dormant phages (i.e. prophages) that are widespread in the CZ and can be induced under environmental stress. I found that volatile fungal metabolites can act as triggers for prophage induction and may exert long-distance manipulation of prophage activity thereby affecting microbial community and nutrient cycling in soil. Altogether, the findings may help to elucidate transport processes of phages in the CZ and to reveal the role of phages in the CZ microbial ecosystem. Approaches (e.g. phage as tracers) and findings (e.g. phage-bacterial co-transport) may also serve as useful tools for testing hypothesis in other disciplines, such as hydrogeology, invasion ecology and chemical ecology.

The sequence of 1360 base pairs of the P22 PL operon was determined, linking a continuous sequence from PL through abc2. P22 mutants bearing deletions in the sequenced region were constructed and tested for their phenotypes. Plasmids were constructed to express PL operon genes singly and in combinations from PlacUV5. Two previously known genes, 17 and c3, are located within this sequence. In addition, three new genes have been identified: ral, kil and arf. Genes ral and c3 are homologous, as well as functionally analogous, to λ ral and cIII, respectively. P22 kil, like λ kil, kills the host cell when it is expressed. The two kil genes, although analogous in cell killing and map location, have no apparent sequence homology. The functions of the P22 and λ kil genes are unknown; however, P22 kil is essential for lytic growth in the absence of abc. Gene arf (accessory recombination function) is located just upstream of erf; it is essential for P22 growth in the absence of kil or other genes upstream in PL. The growth defect of P22 bearing a deletion that removes arf is complemented by expression of either arf or the λ red genes from plasmids. P22 sequences that include the stop codon for 17 potentially form a small stem-loop structure; these sequences are nearly identical to λ sequences that contain the stop codon for ssb. In λ this potential stem-loop structure occupies a map position near the terminator tL2b. Plasmids that include the potential P22 structure negatively regulate kil gene expression in cis.

Synthetic biology, a field that sits between Biology and Engineering disciplines, has come into its own in the last decade. The decreasing cost of DNA synthesis has lead to the creation of larger and more complex synthetic gene networks, engineered with functional goals rather than simple demonstration. While many methods have been developed to reduce the time required to produce complex networks, none focus upon the considerable tuning needed to turn structurally correct networks into functional gene networks. To this end, we created a Plug-and-Play synthetic gene network assembly that emphasizes character-driven iteration for producing functional synthetic gene networks. This platform enables post-construction modification and easy tuning of networks through its ability to swap individual parts. To demonstrate this system, we constructed a functional bistable genetic toggle and transformed it into two functionally distinct synthetic networks. Once these networks have been created and tuned at the bench, they next must be delivered to bacteria in their target environment. While this is easy for industrial applications, delivering synthetic networks as medical therapeutics has a host of problems, such as competing microbes, the host immune system, and harsh microenvironments. Therefore, we employed bacteriophage technologies to deliver functional synthetic gene networks to specific bacterial strains in various microenvironments. We first sought to deliver functional genetic networks to bacteria present in the gut microbiome. This allows for functionalization of these bacteria to eventually sense disease states and secrete therapeutics. As a proof of concept a simple circuit was created using the Plug-and-Play platform and tested before being moved into the replicative form plasmid of the M13 bacteriophage. Bacteriophage particles carrying this network were used to infect gut bacteria of mice. Infection and functionality of the synthetic network was monitored from screening fecal samples. Next, we employed phagemid technologies to deliver high copy plasmids expressing antibacterial networks to target bacteria. This allows for sustained expression of antibacterial genes that cause non-lytic bacterial death without reliance upon traditional small molecule antibiotics. Phagemid particles carrying our antibacterial networks were then tested against wild type and antibiotic-resistant bacteria in an in vitro and in vivo environment.

Seven strains of Streptococcus cremoris and Streptococcus lactis were tested for temperature sensitivity on a temperature gradient bar. A temperature of 37 C was the optimum for differentiating between temperature-sensitive and insensitive strains. Temperature-insensitive strains produced cell masses with absorbance values of 0.1 or higher and activity levels of 1.0 (expressed as a decrease in pH) or above at 37 C. Temperature-sensitive strains failed to produce these cell masses and activity levels, Strain selection and resistant- mutant isolation made it possible to identify ten strains of Streptococci sufficiently free of phage sensitivity for use in a cheese plant rotation program. These resistant mutants failed after being successfully used in mixed cultures for a short period of time, They became either slow acid producers or were again attacked by new bacteriophages. More work is needed to successfully isolate phage-resistant mutants suitable for cheese starters.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18693号 / 工博第3971号 / 新制||工||1611(附属図書館) / 31626 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 北川 進, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Microorganisms interact frequently with each other and with higher organisms. This contact and communication takes place at the molecular level. Microbial interactions with eukaryotes can be pathogenic or mutualistic. One of the best-studied symbioses is the complex interaction between nitrogen-fixing soil bacteria, termed rhizobia, and legumes. This symbiosis culminates in the elaboration of a new organ, the root nodule. Many of the molecular signals exchanged between the host plant and the invading rhizobia have been deduced, but there is still much that remains to be discovered. The molecular determinant of host range at the genus level of the plant host has been determined to be lipochitooligomers called Nod factors. The molecular determinants of host range at the species and cultivar level are less well-defined. Part of my work has been to identify and characterize accessory plasmids that disrupt the normal progression of symbiosis between legumes of the genus Medicago and their rhizobial symbiont, Sinorhizobium meliloti. A cre--loxP-based system capable of making large, defined deletions was developed for the analysis of these plasmids. This system is also being employed to cure the laboratory strain, S. meliloti Rm1021 of its two megaplasmids-a loss of nearly half of its genome. I have also done work to determine whether locally-collected sinorhizobia are native, invasive, or native with symbiosis genes acquired horizontally from invasive sinorhizobia. Finally, I have studied Sinorhizobium meliloti as a host by identifying an outer membrane porin that several bacteriophages use to adsorb to the S. meliloti cell surface.

Control Method for Invasive Aquatic Species introduced via Ballast Water: Effects of Carbon Dioxide Supersaturation on Survivorship of Digesia tigrina (Planaria: Maculata) and Lirceus brachyurus (Isopoda: Crustacea) Survivorship of an aquatic species of planaria (Digesia tigrina) and isopods (Lirceus barchyurus) to elevated levels of carbon dioxide (CO₂) was determined. Both planaria and isopods were exposed to levels of freshwater supersaturated with carbon dioxide, and percent mortality was calculated for various exposure durations, and at various pressure levels. The data collected were graphically analyzed to determine the time necessary to produce mortality in 50% (LT50) of any given sample of specimens tested at a certain pressure level. At 38.6 kPa, 103.4 kPa and 172.4 kPa, the LT50 for planaria was calculated to be 150.3 ± 10.1, 58.6 ± 11.1, and 27.8 ± 6.2 minutes, respectively. At 38.6 kPa, 103.4 kPa and 172.4 kPa , the LT50 for isopods was calculated to be 181.1 ± 52.5, 79.7 ± 21.9, and 40.5 ± 17.0 minutes, respectively. These results suggest that CO₂ supersaturation may be an easily applied, efficient method that would end the unwanted introduction of nonnative aquatic species to habitats via ballast water released from shipping vessels. Effect of High Hydrostatic Pressure Processing on Freely Suspended and Shellfish Associated T7 Bacteriophage The effectiveness of hydrostatic pressure processing (HPP) for inactivating viruses has only been evaluated in a limited number of studies and most of the work has been performed with freely suspended viruses. In this work, the inactivation of freely suspended, as well as shellfish associated bacteriophage T7, by HPP was studied. T7 was selected in hopes that it could potentially serve as a model for animal virus behavior. Both clams (Mercenaria mercenaria) and oysters (Crassostrea virginica) were homogeneously blended separately and inoculated with bacteriophage T7. The inoculated shellfish meat, as well as freely suspended virus samples, were subjected to HPP under the following conditions: 2, 4 and 6 min durations; 241.3, 275.8 and 344.7 Megapascals (MPa) pressure levels; and temperature ranges of 29.4 – 35, 37.8 – 43.3 and 46.1 – 51.7Ë C. Plaque forming unit (PFU) reductions of 7.8 log10 (100% inactivation) were achieved for freely suspended T7 at 344.7 MPa, 2 min and 37.8 – 43.3Ë C. At 46.1 – 51.7Ë C, T7 associated with either clams or oysters was inactivated at nearly 100% (> 4 log10) at all pressure levels and durations tested. The results indicated that T7 is readily inactivated by HPP under the proper conditions, may be protected or made more susceptible by shellfish meat, and may serve as a viable model for the response of several animal viruses to HPP. / Master of Science