Welcome to the IUMS 2022 Interactive Program

Background and Aims

The progress in synthetic and computational biology has significantly improved our capability to fabricate robust bacterial biosensors. These and other advancements have made possible, for instance, the engineering of E. coli as a programmable living tool for diagnostic and environmental applications. However, the dependency of bacterial biosensors on bioluminiscence, fluorescence, or colorimetric reporters limits their use for those applications requiring the simultaneous detection of multiple targets in the same sample. Surface-enhanced Raman scattering (SERS) spectroscopy is an analytical technique that employs plasmonic nanoparticles as optical enhancers for increasing the inherently weak intensity of the Raman signal. The main features of SERS include its high specificity, sensitivity, and multiplexing capabilities owing to the narrow spectral bandwidths that characterize the Raman spectra. In previous studies we successfully applied SERS for the in situ detection and imaging of secreted bacterial metabolites^{1, 2}. Herein, we report the development of bacterial biosensors based on SERS.

1. Bodelon G., et al. Nature Materials. 2016; 15(11):1203-1211

2. Bodelon G., et al. ACS Nano. 2017; 11(5):4631-4640

Methods

To this aim, we evaluated the inducible expression of heterologous Raman-active metabolites in E. coli and, in combination with multivariate statistics and machine learning tools, we investigated their potential use as SERS reporters.

Results

We show the unambiguous identification of the selected metabolites by SERS, as well as their simultaneous detection in mixtures of biosensor strains.

Conclusions

Our results demonstrate the suitability of the proposed approach, thereby paving the way for a novel class of bacterial biosensors with improved multiplex detection capabilities.

Background and Aims

The type VI secretion system (T6SS) is deployed by numerous Gram-negative bacteria to deliver toxic effectors into neighboring cells. The genome of Pantoea agglomerans pv. betae (Pab) phytopathogenic bacteria contains a gene cluster (T6SS1) predicted to encode a complete T6SS. We examined structural genes of the T6SS1 cluster and associated putative effector and immunity genes. In addition, we experimentally validated a predicted effector and immunity pair and its activity.

Methods

Computational analysis was used to characterize genes in the Pab T6SS1 cluster, while secretion and competition assays were employed to test T6SS1 functionality. These analyses revealed a specialized VgrG antibacterial effector and numerous putative effectors and immunity proteins. We tested functionality of the specialized VgrG using protein expression, toxicity and competition assays.

Results

The Pab genome contains two T6SS clusters: T6SS1 and T6SS2, which encode a complete and a partial T6SS, respectively. T6SS1 can be divided in three rapidly evolving genetic islands. Each island displays arrays of orphan immunity and toxin immunity modules acting as an antibacterial system and possibly conferring Pab the ability to resist aggression by competitors. Furthermore, the VgrG C-terminal domain encodes a peptidoglycan hydrolyzing toxin that targets a periplasmic component of prey cells.

Conclusions

Pab T6SS1 is a functional antibacterial system secreting toxic effectors to eliminate competitors and it is equipped with immunity proteins to avoid self-intoxication. Using bioinformatics, biochemical, and genetic assays, we identified T6SS‐secreted effectors and determined that VgrG is a specialized antibacterial effector.

Background and Aims

Structural colour (SC) is an optical mechanism by which ordered nanostructures reflect light to generate vivid, angle-dependent hues. A familiar example is a feather of the peacock. The optics of SC have been studied for centuries, in birds and insects, since the time of Newton and Hooke. Optical physics has now demonstrated of a rich variety of SCs in many living organisms. However, very little is known about the genes that encode SC in any branch of the tree of life. Some bacterial colonies also display SC, particularly strains from Class Flavobacteriia. Organised and aligned cells, such as those of Flavobacterium IR1, can form a 2D photocrystalline structure, resulting in vivid, angle-dependent colour when illuminated. We aim to use IR1 as a model, genetically amenable system to understand how living matter can organise to create SC.

Methods

Proteomics, transposon mutagenesis and genomics approaches have been used to identify the underlying genes/proteins and pathways involved in SC. A Crispr-Cas12 gene editing system was adapted to IR1 and is being used to make targeted changes to the genome to map the genes involved in SC and create new strains with improved photonic properties.

Results

We have created mutants with altered properties in SC, for example the colony b is 'red shifted' due to a transposon insertion, compared to the WT in panel a. Genomics/proteomics data and Crispr-mediated KOs will also be presented.

Conclusions

We can create strains with improved photonic properties to understand the role of SC in nature and to make sustainable, photonic biomaterials to replace unsustainable pigments.

Background and Aims

Cyanobacteria are a diverse group of photoautotrophic prokaryotes that are found in a variety of environments, and can be grown in the laboratory. With genomic information available for more than 130 cyanobacterial strains, many can be engineered to enhance hydrogen production. Cyanobacteria are ideal cell factories for hydrogen production because they have low nutrient requirements and are capable of using light to generate biomass from water and CO₂.

Cyanobacteria produce hydrogen through two key enzymes, nitrogenase and bidirectional hydrogenase, and oxidize molecular hydrogen by uptake hydrogenase.

Methods

Cyanobacterial uptake hydrogenase has two subunits; the small subunit directs electron transport to the large subunit, which has an active site that binds H₂. These subunits are encoded by hupS and hupL genes, respectively, that can be genetically modified to reduce activity of the uptake hydrogenase and increase hydrogen production.

Results

Genetic deletion of uptake hydrogenase in cyanobacteria affects strains in various ways. In Anabaena sp. PCC 7120 it increased hydrogen production by 4-7 fold while in Anabaena variabilis ATCC 29413 it reached a 5-fold improvement, compared to the wild type strain. Uptake hydrogenase deletion strains can be the starting point for further genetic modifications for the purpose of enhancement of their hydrogen-producing capacity.

Conclusions

Hydrogen production makes cyanobacteria promising for renewable fuel. Accumulation of biomass by cyanobacteria is linked to carbon dioxide sequestering. This reduces the carbon footprint by producing polymers of carbon. The ability to reduce carbon dioxide pollution as well as produce energy dense hydrogen makes cyanobacteria significant in biofuel research.