A Minimal Model of Metabolism Based Chemotaxis

Since the pioneering work by Julius Adler in the 1960's, bacterial chemotaxis has been predominantly studied as metabolism-independent. All available simulation models of bacterial chemotaxis endorse this assumption. Recent studies have shown, however, that many metabolism-dependent chemotactic patterns occur in bacteria. We hereby present the simplest artificial protocell model capable of performing metabolism-based chemotaxis. The model serves as a proof of concept to show how even the simplest metabolism can sustain chemotactic patterns of varying sophistication. It also reproduces a set of phenomena that have recently attracted attention on bacterial chemotaxis and provides insights about alternative mechanisms that could instantiate them. We conclude that relaxing the metabolism-independent assumption provides important theoretical advances, forces us to rethink some established pre-conceptions and may help us better understand unexplored and poorly understood aspects of bacterial chemotaxis

Reconstruction of the Core and Extended Regulons of Global Transcription Factors

The processes underlying the evolution of regulatory networks are unclear. To address this question, we used a comparative genomics approach that takes advantage of the large number of sequenced bacterial genomes to predict conserved and variable members of transcriptional regulatory networks across phylogenetically related organisms. Specifically, we developed a computational method to predict the conserved regulons of transcription factors across α-proteobacteria. We focused on the CRP/FNR super-family of transcription factors because it contains several well-characterized members, such as FNR, FixK, and DNR. While FNR, FixK, and DNR are each proposed to regulate different aspects of anaerobic metabolism, they are predicted to recognize very similar DNA target sequences, and they occur in various combinations among individual α-proteobacterial species. In this study, the composition of the respective FNR, FixK, or DNR conserved regulons across 87 α-proteobacterial species was predicted by comparing the phylogenetic profiles of the regulators with the profiles of putative target genes. The utility of our predictions was evaluated by experimentally characterizing the FnrL regulon (a FNR-type regulator) in the α-proteobacterium Rhodobacter sphaeroides. Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks. Our findings also predict that, at least for the FNR-type regulators, there is a core set of target genes conserved across many species. In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors. The comparative genomics approach we developed is readily applicable to other regulatory networks

The design and realization of synthetic pathways for the fixation of carbon dioxide in vitro

The fixation of inorganic carbon and the conversion to organic molecules is a prerequisite for life and the foundation of the carbon cycle on Earth. Since the industrial revolution, this carbon cycle has become inbalanced and consequently the atmospheric carbon dioxide (CO2) concentration is increasing and is a major cause of global warming. On the contrary, atmospheric CO2 can also be considered as an important carbon feedstock of the future. However, human society has not yet come up with a viable solution to convert this inorganic atmospheric CO2 back into reduced carbon compounds and is still relying on natural CO2 fixation. Nature has evolved multiple solutions to reduce CO2 and incorporate it into organic molecules. The involved pathways differ in their cofactor requirements and are often limited to anoxic conditions. Many attempts have been made to improve natural carbon fixation to a more energy efficient process, but showed little success. The emerging field of synthetic biology offers an alternative approach by designing novel pathways for the fixation of CO2. Although, several such artificial pathways have been designed, none of them have been realized so far. This reveals an existing gap between the design and the realization and implementation of such a synthetic CO2 fixation pathway. In this work we designed several synthetic oxygen-tolerant CO2 fixation pathways in a bottom-up approach, by freely combining enzymes from different biological sources. The pathways were designed around an efficient central carboxylase from the family of enoyl-CoA carboxylases/reductases. Some members of this family belong to the most efficient carboxylases known so far, do not accept oxygen as a substrate and only require the ubiquitous NADPH as co-substrate. The theoretical analysis of thermodynamic and energetic properties of the designed pathways for CO2 fixation also showed that they are comparable or even more energy efficient than naturally occurring oxygen-tolerant CO2-fixing pathways. We were able to realize two of these cycles in vitro and investigated their efficiencies for the fixation of inorganic CO2 into organic molecules. We established the Crotonyl-CoA/EThylmalonyl-CoA/Hydroxybutyryl-CoA (CETCH) and HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle in vitro and their CO2 fixation efficiencies were increased in several rounds of optimization. In this process, we energized the systems by ATP- and NADPH-regeneration modules, applied the principle of metabolic proofreading to recycle undesired side products and engineered several enzymes to efficiently catalyze desired reactions. The CETCH cycle in its current version 5.4 is a reaction network of 17 enzymes originating from nine different organisms of all three domains of life. It converts CO2 into organic molecules at a rate of 5 nmol CO2 per minute and mg enzyme. In comparison, the HOPAC cycle in its current version 4.1 comprises 15 enzymes originating from eight different organisms. A stepwise incorporation of 13CO2 into the intermediates of both synthetic pathway confirmed a continuous operation for multiple rounds of conversion. During the development of the synthetic cycles for CO2 fixation, we solved a novel crystal structure of a key enzyme for both pathways, the methylsuccinyl-CoA dehydrogenase. This is a member of the well described family of flavin dependent acyl-CoA dehydrogenases. We elucidated the substrate specificity of the enzyme for (2S)-methylsuccinyl-CoA, which represents a complex substrate amongst the acyl-CoA dehydrogenase family. In summary, this study laid the foundation for the development of artificial pathways for the fixation of CO2 and narrow the gap between theoretical design of synthetic CO2 fixation pathways and their application in vivo. The CETCH and HOPAC cycle expands the solution space beyond the six naturally evolved CO2 fixation pathways by two man-made alternative that are thermodynamically more efficient than the CBB cycle of plants

Advances in the Biology of Phototrophic Bacteria

The application of genomic, transcriptomic, and proteomic analyses brings new dimensions to our understanding of the biology of phototrophic bacteria. Comparing gene sequences of photosynthetic reaction center proteins and a key enzyme of bacteriochlorophyll biosynthesis from more than 150 genomes demonstrates the ancient roots of phototrophic bacteria. The presence and phylogeny of biosynthetic pathways of the compatible solutes ectoine and glycine betaine define groups of marine and halophilic phototrophic bacteria. The wide range of ecological niches conquered during evolution is demonstrated by the adaptation of cyanobacterial genera Scytonema, Tolypothrix, and Nostoc to different temperature ranges and the adaptation of Heliorestis species to alkaline habitats. Differences between phototrophic purple bacteria from marine and freshwater habitats are reflected in the preference for sulfidic and non-sulfidic niches. Also, a high proportion of siderophore producers was found among isolates from freshwater sources opposed to those from salty habitats . The primary colonization of carbonate rocks by a group of novel endolithic cyanobacteria and the following successions were studied over 9 months. The genomic characterization of the aerobic Dinoroseobacter strain AAP5, the strictly anaerobic and syntrophic Prosthecochloris ethylica, and the strictly anaerobic Heliorestis convoluta is reported. Significant differences in relation to oxygen are reflected in oxygen production by some species, oxygen tolerance over a wide range of concentrations, and the use of oxygen for energy generation or a strictly anaerobic lifestyle. Relations to oxygen are highlighted in papers on photooxidative stress, regulation of iron–sulfur cluster formation, and interactions of redox regulators. In situ metatranscriptomic and proteomic studies demonstrate the high metabolic flexibility of Chloroflexus aggregans in a hot spring microbial mat and show its adaptation to the changing conditions over day and night periods by a well-coordinated regulation of key metabolic processes for both phototrophic and chemotrophic growth

Transcriptional Analysis of Shewanella oneidensis MR-1 with an Electrode Compared to Fe(III)Citrate or Oxygen as Terminal Electron Acceptor

Shewanella oneidensis is a target of extensive research in the fields of bioelectrochemical systems and bioremediation because of its versatile metabolic capabilities, especially with regard to respiration with extracellular electron acceptors. The physiological activity of S. oneidensis to respire at electrodes is of great interest, but the growth conditions in thin-layer biofilms make physiological analyses experimentally challenging. Here, we took a global approach to evaluate physiological activity with an electrode as terminal electron acceptor for the generation of electric current. We performed expression analysis with DNA microarrays to compare the overall gene expression with an electrode to that with soluble iron(III) or oxygen as the electron acceptor and applied new hierarchical model-based statistics for the differential expression analysis. We confirmed the differential expression of many genes that have previously been reported to be involved in electrode respiration, such as the entire mtr operon. We also formulate hypotheses on other possible gene involvements in electrode respiration, for example, a role of ScyA in inter-protein electron transfer and a regulatory role of the cbb3-type cytochrome c oxidase under anaerobic conditions. Further, we hypothesize that electrode respiration imposes a significant stress on S. oneidensis, resulting in higher energetic costs for electrode respiration than for soluble iron(III) respiration, which fosters a higher metabolic turnover to cover energy needs. Our hypotheses now require experimental verification, but this expression analysis provides a fundamental platform for further studies into the molecular mechanisms of S. oneidensis electron transfer and the physiologically special situation of growth on a poised-potential surface

Genome-scale reconstruction and system level investigation of the metabolic network of Methylobacterium extorquens AM1

Background: Methylotrophic microorganisms are playing a key role in biogeochemical processes - especially the global carbon cycle - and have gained interest for biotechnological purposes. Significant progress was made in the recent years in the biochemistry, genetics, genomics, and physiology of methylotrophic bacteria, showing that methylotrophy is much more widespread and versatile than initially assumed. Despite such progress, system-level description of the methylotrophic metabolism is currently lacking, and much remains to understand regarding the network-scale organization and properties of methylotrophy, and how the methylotrophic capacity emerges from this organization, especially in facultative organisms. Results: In this work, we report on the integrated, system-level investigation of the metabolic network of the facultative methylotroph Methylobacterium extorquens AM1, a valuable model of methylotrophic bacteria. The genome-scale metabolic network of the bacterium was reconstructed and contains 1139 reactions and 977 metabolites. The sub-network operating upon methylotrophic growth was identified from both in silico and experimental investigations, and 13C-fluxomics was applied to measure the distribution of metabolic fluxes under such conditions. The core metabolism has a highly unusual topology, in which the unique enzymes that catalyse the key steps of C1 assimilation are tightly connected by several, large metabolic cycles (serine cycle, ethylmalonyl- CoA pathway, TCA cycle, anaplerotic processes). The entire set of reactions must operate as a unique process to achieve C1 assimilation, but was shown to be structurally fragile based on network analysis. This observation suggests that in nature a strong pressure of selection must exist to maintain the methylotrophic capability. Nevertheless, substantial substrate cycling could be measured within C2/C3/C4 inter-conversions, indicating that the metabolic network is highly versatile around a flexible backbone of central reactions that allows rapid switching to multi-carbon sources. Conclusions: This work emphasizes that the metabolism of M. extorquens AM1 is adapted to its lifestyle not only in terms of enzymatic equipment, but also in terms of network-level structure and regulation. It suggests that the metabolism of the bacterium has evolved both structurally and functionally to an efficient but transitory utilization of methanol. Besides, this work provides a basis for metabolic engineering to convert methanol into value-added products

Identification of Small RNAs and Differential Gene Expression in Rhodobacter Sphaeroides under Gold Chloride Stress

Small, regulatory RNAs (sRNA) play an important role in mediating transcriptional and translational processes within bacterial organisms. Understanding how these sRNAs play a role in heavy metal stress is of importance for bacteria involved in bioremediation. The following study aims to (i) identify novel sRNA sequences within Rhodobacter sphaeroides using RNAspace, a bioinformatic approach, (ii) validate a set of sRNAs expressed when the bacterium is grown under an aerobic and/or gold chloride stress condition, and (iii) analyze the gene expression profiles to identify specific target genes involved in the gold chloride stress condition. A total of 712 sRNAs were predicted within the genome of R. sphaeroides using the bioinformatic approach. R. sphaeroides growth characteristics were observed under different concentrations of gold chloride and were found to withstand up to a 1.0 µM concentration. Total RNA isolated from the untreated control group and the 1.0 µM AuCl3 treated group were selected for small RNA and total RNA sequencing. A total of three differentially expressed sRNA sequences were detected in the 1.0µM AuCl3 group, thus implying the role of these sRNAs in gold chloride stress. Additionally, targets were predicted for each sRNA utilizing the CopraRNA prediction program. A transcriptomic analysis was performed to identify differentially expressed genes between the control and 1.0 µM AuCl3 groups at lag/early-log and late-log/stationary growth phases. A total of 121 genes representing a wide variety of gene functions exhibited up- or down- gene regulation at the lag/early-log phase, while 604 genes were up-/down-regulated at the late-log/stationary phase. A majority of commonly differentially expressed genes were observed to be involved in membrane alteration, chemotactic response, energy production, and intracellular/extracellular transport across the membrane. Small RNAs that were detected by sRNA sequencing were predicted to additionally target differentially expressed genes observed within this comparison. A compiled list of identified sRNAs and their corresponding target genes were used to further elucidate the regulatory roles of these sRNAs under gold chloride stress

Improvements in Fermentative Hydrogen Production through Physiological Manipulation and Metabolic Engineering

La production biologique d'hydrogène (H2) représente une technologie possible pour la production à grande échelle durable de H2 nécessaire pour l'économie future de l'hydrogène. Cependant, l'obstacle majeur à l'élaboration d'un processus pratique a été la faiblesse des rendements qui sont obtenus, généralement autour de 25%, bien en sous des rendements pouvant être atteints pour la production de biocarburants à partir d'autres processus. L'objectif de cette thèse était de tenter d'améliorer la production d'H2 par la manipulation physiologique et le génie métabolique. Une hypothèse qui a été étudiée était que la production d'H2 pourrait être améliorée et rendue plus économique en utilisant un procédé de fermentation microaérobie sombre car cela pourrait fournir la puissance supplémentaire nécessaire pour une conversion plus complète du substrat et donc une production plus grande d'H2 sans l'aide de l'énergie lumineuse. Les concentrations optimales d’O2 pour la production de H2 microaérobie ont été examinées ainsi que l'impact des sources de carbone et d'azote sur le processus. La recherche présentée ici a démontré la capacité de Rhodobacter capsulatus JP91 hup- (un mutant déficient d’absorption-hydrogénase) de produire de l'H2 sous condition microaérobie sombre avec une limitation dans des quantités d’O2 et d'azote fixé. D'autres travaux devraient être entrepris pour augmenter les rendements d'H2 en utilisant cette technologie. De plus, un processus de photofermentation a été créé pour améliorer le rendement d’H2 à partir du glucose à l'aide de R. capsulatus JP91 hup- soit en mode non renouvelé (batch) et / ou en conditions de culture en continu. Certains défis techniques ont été surmontés en mettant en place des conditions adéquates de fonctionnement pour un rendement accru d'H2. Un rendement maximal de 3,3 mols de H2/ mol de glucose a été trouvé pour les cultures en batch tandis que pour les cultures en continu, il était de 10,3 mols H2/ mol de glucose, beaucoup plus élevé que celui rapporté antérieurement et proche de la valeur maximale théorique de 12 mols H2/ mol de glucose. Dans les cultures en batch l'efficacité maximale de conversion d’énergie lumineuse était de 0,7% alors qu'elle était de 1,34% dans les cultures en continu avec un rendement de conversion maximum de la valeur de chauffage du glucose de 91,14%. Diverses autres approches pour l'augmentation des rendements des processus de photofermentation sont proposées. Les résultats globaux indiquent qu'un processus photofermentatif efficace de production d'H2 à partir du glucose en une seule étape avec des cultures en continu dans des photobioréacteurs pourrait être développé ce qui serait un processus beaucoup plus prometteur que les processus en deux étapes ou avec les co-cultures étudiés antérieurément. En outre, l'expression hétérologue d’hydrogenase a été utilisée comme une stratégie d'ingénierie métabolique afin d'améliorer la production d'H2 par fermentation. La capacité d'exprimer une hydrogénase d'une espèce avec des gènes de maturation d'une autre espèce a été examinée. Une stratégie a démontré que la protéine HydA orpheline de R. rubrum est fonctionnelle et active lorsque co-exprimée chez Escherichia coli avec HydE, HydF et HydG provenant d'organisme différent. La co-expression des gènes [FeFe]-hydrogénase structurels et de maturation dans des micro-organismes qui n'ont pas une [FeFe]-hydrogénase indigène peut entraîner le succès dans l'assemblage et la biosynthèse d'hydrogénase active. Toutefois, d'autres facteurs peuvent être nécessaires pour obtenir des rendements considérablement augmentés en protéines ainsi que l'activité spécifique des hydrogénases recombinantes. Une autre stratégie a consisté à surexprimer une [FeFe]-hydrogénase très active dans une souche hôte de E. coli. L'expression d'une hydrogénase qui peut interagir directement avec le NADPH est souhaitable car cela, plutôt que de la ferrédoxine réduite, est naturellement produit par le métabolisme. Toutefois, la maturation de ce type d'hydrogénase chez E. coli n'a pas été rapportée auparavant. L'opéron hnd (hndA, B, C, D) de Desulfovibrio fructosovorans code pour une [FeFe]-hydrogénase NADP-dépendante, a été exprimé dans différentes souches d’E. coli avec les gènes de maturation hydE, hydF et hydG de Clostridium acetobutylicum. L'activité de l'hydrogénase a été détectée in vitro, donc une NADP-dépendante [FeFe]-hydrogénase multimérique active a été exprimée avec succès chez E. coli pour la première fois. Les recherches futures pourraient conduire à l'expression de cette enzyme chez les souches de E. coli qui produisent plus de NADPH, ouvrant la voie à une augmentation des rendements d'hydrogène via la voie des pentoses phosphates.Biological hydrogen (H2) production represents a possible technology for the large scale sustainable production of H2 needed for a future hydrogen economy. However, the major obstacle to developing a practical process has been the low yields that are obtained, typically around 25%, well below those achievable for the production of other biofuels from the same feedstock. The goal of this thesis was to improve H2 production through physiological manipulation and metabolic engineering. One investigated hypothesis was that H2 production could be improved and made more economical by using a microaerobic dark fermentation process since this could provide the extra reducing power required for driving substrate conversion to completion and hence more H2 production might be obtained without using light energy. The optimal O2 concentrations for microaerobic H2 production were examined as well as the impact of carbon and nitrogen sources on the process. The research reported here proved the capability of Rhodobacter capsulatus JP91 hup- (an uptake-hydrogenase deficient mutant) to produce H2 under microaerobic dark conditions with limiting amounts of O2 and fixed nitrogen. Further work should be undertaken to increase H2 yields using this technology. In addition, a photofermentation process was established to improve H2 yield from glucose using R. capsulatus JP91 hup- strain either in batch and/or continuous culture conditions. Some technical challenges in establishing the proper operational conditions for increased H2 yield were overcome. A maximum yield of 3.3 mols of H2/ mol of glucose was found for batch cultures whereas in continous cultures it was 10.3 mols H2/ mol glucose, much higher than previously reported and close to the theoretical maximum value of 12 mols H2/ mol glucose. In batch cultures the maximum light conversion efficiency was 0.7% whereas it was 1.34% in continuous cultures with a maximum conversion efficiency of the heating value of glucose of 91.14%. Various approaches to further increasing yields in photofermentation processes are proposed. The overall results suggest that an efficient single stage photofermentative H2 production process from glucose using continuous cultures in photobioreactors could be developed which would be a much more promising alternative process to the previously studied two stage photofermentation or co-culture approaches. Furthermore, the heterologous expression of hydrogenases was used as a metabolic engineering strategy to improve fermentative H2 production. The capability of expressing a hydrogenase from one species with the maturation genes from another was examined. One strategy demonstrated that the orphan hydA of R. rubrum is functional and active when co-expressed in E. coli with hydE, hydF and hydG from different organisms. Co-expression of the [FeFe]-hydrogenase structural and maturation genes in microorganisms that lack a native [FeFe]-hydrogenase can successfully result in the assembly and biosynthesis of active hydrogenases. However, other factors may be required for significantly increased protein yields and hence the specific activity of the recombinant hydrogenases. Another strategy was to overexpress one of the highly active [FeFe]-hydrogenases in a suitable E. coli host strain. Expression of a hydrogenase that can directly interact with NADPH is desirable as this, rather than reduced ferredoxin, is naturally produced by its metabolism. However, the successful maturation of this type of hydrogenase in E. coli had not been previously reported. The Desulfovibrio fructosovorans hnd operon (hndA, B, C, and D genes), encoding a NADP-dependent [FeFe]-hydrogenase, was expressed in various E. coli strains with the maturation genes hydE, hydF and hydG of Clostridium acetobutylicum. Hydrogenase activities were detected in vitro, thus a multi-subunit NADP-dependent [FeFe]-active hydrogenase was successfully expressed and matured in E. coli for the first time. Future research could lead to the expression of this hydrogenase in E. coli host strains that overproduce NADPH, setting the stage for increased hydrogen yields via the pentose phosphate pathway