Engineering Membranes in Escherichia coli: The Magnetosome, LemA Protein Family and Outer Membrane Vesicles

Magnetosomes are membranous organelles found in magnetotactic bacteria (MTB). The organelle consist of ferromagnetic crystals housed within a lipid bilayer chained together by an actin-like filament and allows MTB to orient within magnetic fields. The genetic information required to produce these organelles has been linked to four different operons, encoding for 30 genes. These membranous organelles and the magnetic minerals housed within have various biotechnological applications, therefore enhanced recombinant production of such structures in a model organism holds significant potential. The research described in this thesis is focuses on the production of recombinant magnetosomes in the model organism Escherichia coli. Cloning the genes involved in the generation of the organelle individually or in various combinations resulted in the construction of over 100 different plasmids, compatible with the model organism. SDS-PAGE and electron microscopy analysis was used to characterise E. coli cells harbouring these constructs. The observation of electron dense particles, arranged in a chain structure, show that magnetosome generation in the model organism is possible, but is highly dependent on the growth conditions used. The need for specific growth conditions is later backed up by the analysis of the maturation of the cytochrome c proteins involved in magnetosome biomineralisation, which can only be correctly processed under certain conditions. Individual production of two different magnetosome proteins, MamQ or MamY, allowed the generation of various membranous structures in E. coli observed in 48.9% and 56.2% of the whole population of cells respectively. Combinations of these with MamI, MamL or MamB in a variety of combinations led to a variation in the phenotype observed. Bioinformatics analysis of MamQ led to the discovery of a novel membrane restructuring protein family, the LemA protein family, present in a broad range of bacteria. Four different LemA proteins from Bacillus megaterium, Clostridium kluyveri, Brucella melitensis or Pseudomonas aeruginosa were then produced in E. coli and the analysis of the resulting strains revealed the presence of novel intracellular membranous structures which vary in size, form and localisation. Furthermore, when attempts were made to target these proteins for the modification of the outer membrane, a mechanism for increased outer membrane vesicle generation was serendipitously discovered and different effects of these proteins were once again observed. Together, the results described shows good evidence for recombinant magnetosome production in E. coli and opens a new avenue of membrane engineering in this commonly used organism. Such membranous structures have various biotechnological applications, such as enhanced metabolic engineering potential or specialised lipid vesicle production

Effect of metabolosome encapsulation peptides on enzyme activity, co-aggregation, incorporation and bacterial microcompartment formation

Metabolosomes, catabolic bacterial microcompartments, are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multi-component protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation prior to the formation of the outer shell. We investigated the effect of three distinct encapsulation peptides on foreign cargo in a recombinant BMC system. Our data demonstrate that these peptides cause variation with respect to enzyme activity and protein aggregation. We observed that the level of protein aggregation generally correlates with the size of metabolosomes, while in the absence of cargo BMCs self-assemble into smaller compartments. The results agree with a flexible model for BMC formation based around the ability of the BMC shell to associate with an aggregate formed due to the interaction of encapsulation peptides

Differential temporal release and lipoprotein loading in B. thetaiotaomicron bacterial extracellular vesicles

Bacterial extracellular vesicles (BEVs) contribute to stress responses, quorum sensing, biofilm formation and interspecies and interkingdom communication. However, the factors that regulate their release and heterogeneity are not well understood. We set out to investigate these factors in the common gut commensal Bacteroides thetaiotaomicron by studying BEV release throughout their growth cycle. Utilising a range of methods, we demonstrate that vesicles released at different stages of growth have significantly different composition, with early vesicles enriched in specifically released outer membrane vesicles (OMVs) containing a larger proportion of lipoproteins, while late phase BEVs primarily contain lytic vesicles with enrichment of cytoplasmic proteins. Furthermore, we demonstrate that lipoproteins containing a negatively charged signal peptide are preferentially incorporated in OMVs. We use this observation to predict all Bacteroides thetaiotaomicron OMV enriched lipoproteins and analyse their function. Overall, our findings highlight the need to understand media composition and BEV release dynamics prior to functional characterisation and define the theoretical functional capacity of Bacteroides thetaiotaomicron OMVs

Extracellular vesicles produced by the human gut commensal bacterium Bacteroides thetaiotaomicron elicit anti-inflammatory responses from innate immune cells

Bacterial extracellular vesicles (BEVs) produced by gut commensal bacteria have been proposed to play an important role in maintaining host homeostasis via interactions with the immune system. Details of the mediators and pathways of BEV-immune cell interactions are however incomplete. In this study, we provide evidence for the anti-inflammatory and immunomodulatory properties of extracellular vesicles produced by the prominent human gut commensal bacterium Bacteroides thetaiotaomicron (Bt BEVs) and identify the molecular mechanisms underlying their interaction with innate immune cells. Administration of Bt BEVs to mice treated with colitis-inducing dextran sodium sulfate (DSS) ameliorates the symptoms of intestinal inflammation, improving survival rate and reducing weight loss and disease activity index scores, in association with upregulation of IL-10 production in colonic tissue and in splenocytes. Pre-treatment (conditioning) of murine bone marrow derived monocytes (BMDM) with Bt BEVs resulted in higher ratio of IL-10/TNFα production after an LPS challenge when compared to LPS pre-conditioned or non-conditioned BMDM. Using the THP-1 monocytic cell line the interactions between Bt BEVs and monocytes/macrophages were shown to be mediated primarily by TLR2. Histone (H3K4me1) methylation analysis showed that Bt BEVs induced epigenetic reprogramming which persisted after infectious challenge, as revealed by increased levels of H3K4me1 in Bt BEV-conditioned LPS-challenged BMDM. Collectively, our findings highlight the important role of Bt BEVs in maintaining host immune homeostasis and raise the promising possibility of considering their use in immune therapies

Effect of metabolosome encapsulation peptides on enzyme activity, co-aggregation, incorporation and bacterial microcompartment formation

Metabolosomes, catabolic bacterial microcompartments, are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multi-component protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation prior to the formation of the outer shell. We investigated the effect of three distinct encapsulation peptides on foreign cargo in a recombinant BMC system. Our data demonstrate that these peptides cause variation with respect to enzyme activity and protein aggregation. We observed that the level of protein aggregation generally correlates with the size of metabolosomes, while in the absence of cargo BMCs self-assemble into smaller compartments. The results agree with a flexible model for BMC formation based around the ability of the BMC shell to associate with an aggregate formed due to the interaction of encapsulation peptides

Nutrient smuggling: Commensal gut bacteria‐derived extracellular vesicles scavenge vitamin B12 and related cobamides for microbe and host acquisition

Abstract The processes by which bacteria proactively scavenge essential nutrients in crowded environments such as the gastrointestinal tract are not fully understood. In this context, we observed that bacterial extracellular vesicles (BEVs) produced by the human commensal gut microbe Bacteroides thetaiotaomicron contain multiple high‐affinity vitamin B12 binding proteins suggesting that the vesicles play a role in micronutrient scavenging. Vitamin B12 belongs to the cobamide family of cofactors that regulate microbial communities through their limited bioavailability. We show that B. thetaiotaomicron derived BEVs bind a variety of cobamides and not only deliver them back to the parental bacterium but also sequester the micronutrient from competing bacteria. Additionally, Caco‐2 cells, representing a model intestinal epithelial barrier, acquire cobamide‐bound vesicles and traffic them to lysosomes, thereby mimicking the physiological cobalamin‐specific intrinsic factor‐mediated uptake process. Our findings identify a novel cobamide binding activity associated with BEVs with far‐reaching implications for microbiota and host health

Employing bacterial microcompartment technology to engineer a shell-free enzyme-aggregate for enhanced 1,2-propanediol production in Escherichia coli.

Bacterial microcompartments (BMCs) enhance the breakdown of metabolites such as 1,2-propanediol (1,2-PD) to propionic acid. The encapsulation of proteins within the BMC is mediated by the presence of targeting sequences. In an attempt to redesign the Pdu BMC into a 1,2-PD synthesising factory using glycerol as the starting material we added N-terminal targeting peptides to glycerol dehydrogenase, dihydroxyacetone kinase, methylglyoxal synthase and 1,2-propanediol oxidoreductase to allow their inclusion into an empty BMC. 1,2-PD producing strains containing the fused enzymes exhibit a 245% increase in product formation in comparison to un-tagged enzymes, irrespective of the presence of BMCs. Tagging of enzymes with targeting peptides results in the formation of dense protein aggregates within the cell that are shown by immuno-labelling to contain the vast majority of tagged proteins. It can therefore be concluded that these protein inclusions are metabolically active and facilitate the significant increase in product formation