| dc.description.abstract | In recent decades, there has been tremendous interest in applying environmental microbiology for waste treatment, bioremediation and sustainable energy production. As one of the most important model environmental microbes, Shewanella species are renowned for their flexible growth, capability of surviving and growing in a wide gradient of various environmental conditions, and excellent respiratory versatility. There is great prospect of using this organism for production of valuable chemicals, treatment of various types of contamination, and for energy generation in Microbial Fuel Cells. However, the real-life application of Shewanella is hindered by the relatively low level of performance. Fundamental knowledge of the physiology, ecology, stress response mechanism, as well as correct engineering, are indispensable for discovering novel properties and to boost the performance of Shewanella-based applications.
In an initial effort of understanding gene functions and regulations, a particularly interesting mutant of Shewanella loihica PV-4 that could accumulate significant amount of red pigmentation was identified via transposon mutant library screening. The mutation was then found to be on gene Shew_2229 which encodes ferrochelatase. Using analytical methods, the red pigment was verified as protoporphyrin IX (PPIX), an important chemical involved in the synthesis of hemoglobin. Quantification of the yield of PPIX in this mutant was estimated to be 11.2mg/g cell dry weight, which was at least hundreds of times higher compared to other reported bacterial strains and similar to the level of a fully engineered strain that require multiple types of antibiotics to be supplied for its fermentation. A patent for using this PV-4 mutant strain for industrial production of PPIX was issued (US patent No. 9273334B2).
Of great interest was that, the genome of PV-4 contains two paralogues of the hemH gene, designated as hemH1 (Shew_2229) and hemH2 (Shew_1140). It was discovered that single disruption of hemH1 resulted in PPIX accumulation, while single disruption of hemH2 had no apparent phenotype. Therefore, it is hypothesized that there is functional redundancy among the two paralogues with hemH1 as the dominant gene and hemH2 playing a supplementary role. To test this hypothesis, the regulation mechanisms of hemH1 and hemH2 were analyzed via comparative genomics. Sequence analysis of the promoter region of the two paralogues indicated that the two genes were regulated differently. The promoter sequence of hemH1 contains the binding box of the constitutively expressed sigma factor RpoD, while the promoter sequence of hemH2 harbors the binding sequence of the extracellular family sigma factor RpoE2, which suggested that the expression of the two paralogues might be different. Consistently, hemH1 was found constitutively expressed while expression of hemH2 was minimal in presence of a non-disrupted hemH1 but increased significantly when hemH1 was disrupted, supporting the supplementary role of hemH2 in PV-4. Besides, the up-regulation of hemH2 was also observed when PV-4 was exposed to the oxidative stress, either by introduction of hydrogen peroxide or due to accumulation of the photosensitive PPIX. We found that expression of hemH2 was significantly higher when the hemH1 mutant was exposed to light, and accumulation of PPIX was no longer observed under such condition. All these findings suggest that hemH2 plays a more important role under oxidative stressed conditions although its expression is minimal under normal conditions. The apparent redundancy of the hemH paralogues may contribute to the survival of this strain under stress conditions.
Salt stress is one of the most commonly observed stresses for microbes in the natural environment. It is often associated with environmental pollution and can be introduced during bioremediation processes, such as neutralizing pH for sites with high acidity or alkalinity. Understanding the mechanism for salt stress response of microbes is a fundamental step toward boosting the performance of environmental microbiology applications. Although there has been ample documentation regarding the salt stress response in various microbes, knowledge about the microbial response to long term salt stress is lacking. The Shewanella putrefaciens CN-32 strain is an excellent candidate for bioremediation or MFCs. Using the strategy of experimental evolution, the response to salt (NaCl) stress over long term lab incubation was investigated. The changes of growth phenotype, metabolite profile, and transcriptome in evolved populations were analyzed. The mutations occurred in evolved populations were captured by sequencing population DNAs to help interpret fitness changes.
Profound differences in phenotypes were observed among the evolved populations. Populations evolved under salt stress (ES) exhibited significant advantage in growth rate and peak biomass compared with populations evolved under control conditions (EC) or ancestor (AN), indicating gain of fitness. However, the evolved populations were inferior in terms of motility compared to the ancestor, likely one of the tradeoffs during the evolutionary course. Analysis of metabolite profile via Detrended Correspondence Analysis showed that culture condition (salt stress vs. control) and the condition of evolution are the key factors shaping the metabolite composition. Interesting patterns of metabolites were identified, such as the significant higher amount of two compatible solutes, proline and ectoine, in the ES populations, indicating that these two metabolites may play critical role for salt tolerance in this organism.
Transcriptome analysis of select populations revealed that significant up-regulation of genes involved in proline uptake and synthesis was shared across the populations, again reflected the importance of proline accumulation for salt stress protection. Whole-genome-resequencing revealed interesting mutations that may contribute to the improved salt tolerance in ES populations.
The potential application of Shewanella species as bioanode for electricity generation in MFCs was investigated. Redox polymer was introduced with the aim of boosting current generation. The effect of various factors was investigated, such as different Shewanella strains, loading of the redox polymer, and methods of bacteria incorporation. The results showed that electrode surface modification by coating of PVP-Os redox polymer led to several folds of increase in current density. Among the four strains studied, namely Shewanella oneidensis MR-1, Shewanella putrefaciens W3-18-1, Shewanella loihica PV-4, and Shewanella putrefaciens CN-32, strain W3-18-1 showed the best current density with or without redox polymer. Using W3-18-1 as the target organism, the effect of polymer loading, and methods of incorporation were further investigated. Increased amount of PVP-OS loading alone did not lead to an increase of current density, as the increased thickness might perturb efficient electron communication between the redox centers. Four different methods of bacteria incorporation with PVP-OS were compared, the suspension method, lay-over method, mix and cast method, and layer-by-layer method using a gold electrode. The layer-by-layer method produced significantly higher current density compared with other methods, but take longer time to reach peak current density. The suspension method showed second highest current density and quickest response, while the LO and MAC methods showed significantly lower current density.
In summary, the mechanisms of PPIX accumulation and oxidative stress response in Shewanella loihica PV-4, the long term salt adaptation in Shewanella putrefaciens CN-32, and current generation in several Shewanella strains, were investigated via systematic analysis of mutagenesis, functional genomics, comparative genomics, and electrochemistry. Results of this study expands our current knowledge of the physiology, genetics, stress response, and evolution of the Shewanella genus, providing important clues for environmental applications such as bioremediation and MFC, as well as using this type of organism for industrial production of valuable chemicals such as PPIX. | en_US |
