Dataset Information

The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

ABSTRACT: Bacterial spores spontaneously interact and tightly bind heterologous proteins. A variety of antigens and enzymes have been efficiently displayed on spores of Bacillus subtilis, the model system for spore formers. Adsorption on B. subtilis spores has then been proposed as a non-recombinant approach for the development of mucosal vaccine/drug delivery vehicles, biocatalysts, bioremediation, and diagnostic tools. We used spores of B. megaterium QM B1551 to evaluate their efficiency as an adsorption platform. Spores of B. megaterium are significantly larger than those of B. subtilis and of other Bacillus species and are surrounded by the exosporium, an outermost surface layer present only in some Bacillus species and lacking in B. subtilis. Strain QM B1551 of B. megaterium and a derivative strain totally lacking the exosporium were used to localize the adsorbed monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp., used as a model heterologous protein. Our results indicate that spores of B. megaterium adsorb mRFP more efficiently than B. subtilis spores, that the exosporium is essential for mRFP adsorption, and that most of the adsorbed mRFP molecules are not exposed on the spore surface but rather localized in the space between the outer coat and the exosporium.

SUBMITTER: Lanzilli M

PROVIDER: S-EPMC5095127 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

Lanzilli Mariamichela M Donadio Giuliana G Addevico Roberta R Saggese Anella A Cangiano Giuseppina G Baccigalupi Loredana L Christie Graham G Ricca Ezio E Isticato Rachele R

Frontiers in microbiology 20161104

Bacterial spores spontaneously interact and tightly bind heterologous proteins. A variety of antigens and enzymes have been efficiently displayed on spores of Bacillus subtilis, the model system for spore formers. Adsorption on B. subtilis spores has then been proposed as a non-recombinant approach for the development of mucosal vaccine/drug delivery vehicles, biocatalysts, bioremediation, and diagnostic tools. We used spores of B. megaterium QM B1551 to evaluate their effic ...[more]

PMID: 27867376

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Project description:The exosporium of Bacillus megaterium QM B1551 spores is morphologically distinct from exosporia observed for the spores of many other species. Previous work has demonstrated that unidentified genes carried on one of the large indigenous plasmids are required for the assembly of the Bacillus megaterium exosporium. Here, we provide evidence that pBM600-encoded orthologues of the Bacillus subtilis CotW and CotX proteins, which form the crust layer in spores of that species, are structural components of the Bacillus megaterium QM B1551 spore exosporium. The introduction of plasmid-borne cotW and orthologous cotX genes to the PV361 strain, which lacks all indigenous plasmids and produces spores that are devoid of an exosporium, results in the development of spores with a rudimentary exosporium-type structure. Additionally, purified recombinant CotW protein is shown to assemble at the air-water interface to form thin sheets of material, which is consistent with the idea that this protein may form a basal layer in the Bacillus megaterium QM B1551 exosporium.IMPORTANCE When starved of nutrients, some bacterial species develop metabolically dormant spores that can persist in a viable state in the environment for several years. The outermost layers of spores are of particular interest since (i) these represent the primary site for interaction with the environment and (ii) the protein constituents may have biotechnological applications. The outermost layer, or exosporium, in Bacillus megaterium QM B1551 spores is of interest, as it is morphologically distinct from the exosporia of spores of the pathogenic Bacillus cereus family. In this work, we provide evidence that structurally important protein constituents of the Bacillus megaterium exosporium are different from those in the Bacillus cereus family. We also show that one of these proteins, when purified, can assemble to form sheets of exosporium-like material. This is significant, as it indicates that spore-forming bacteria employ different proteins and mechanisms of assembly to construct their external layers.

Project description:Bacillus megaterium is a bacterium of great importance as a plant-beneficial bacterium in agricultural applications and in industrial bioproduction of proteins. Understanding intracellular processing of carbohydrates in this species is crucial to predicting natural carbon utilization as well as informing strategies in metabolic engineering. Here, we applied stable isotope-assisted metabolomics profiling and metabolic flux analysis to elucidate, at high resolution, the connections of the different catabolic routes for carbohydrate metabolism immediately following substrate uptake in B. megaterium QM B1551. We performed multiple 13C tracer experiments to obtain both kinetic and long-term 13C profiling of intracellular metabolites. In addition to the direct phosphorylation of glucose to glucose-6-phosphate (G6P) prior to oxidation to 6-phosphogluconate (6P-gluconate), the labeling data also captured glucose catabolism through the gluconate pathway involving glucose oxidation to gluconate followed by phosphorylation to 6P-gluconate. Our data further confirmed the absence of the Entner-Doudoroff pathway in B. megaterium and showed that subsequent catabolism of 6P-gluconate was instead through the oxidative pentose-phosphate (PP) pathway. Quantitative flux analysis of glucose-grown cells showed equal partition of consumed glucose from G6P to the Embden-Meyerhof-Parnas (EMP) pathway and from G6P to the PP pathway through 6P-gluconate. Growth on fructose alone or xylose alone was consistent with the ability of B. megaterium to use each substrate as a sole source of carbon. However, a detailed 13C mapping during simultaneous feeding of B. megaterium on glucose, fructose, and xylose indicated non-uniform intracellular investment of the different carbohydrate substrates. Flux of glucose-derived carbons dominated the gluconate pathway and the PP pathway, whereas carbon flux from both glucose and fructose populated the EMP pathway; there was no assimilatory flux of xylose-derived carbons. Collectively, our findings provide new quantitative insights on the contribution of the different catabolic routes involved in initiating carbohydrate catabolism in B. megaterium and related Bacillus species.

Dataset Information

The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

Publications

The Exosporium of <i>Bacillus megaterium</i> QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral <i>Discosoma</i> sp.

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