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Plasmid-encoded genes influence exosporium assembly and morphology in Bacillus megaterium QM B1551 spores.

ABSTRACT: Spores of Bacillus megaterium QM B1551 are encased in a morphologically distinctive exosporium. We demonstrate here that genes encoded on the indigenous pBM500 and pBM600 plasmids are required for exosporium assembly and or stability in spores of this strain. Bioinformatic analyses identified genes encoding orthologues of the B. cereus-family exosporium nap and basal layer proteins within the B. megaterium genome. Transcriptional analyses, supported by electron and fluorescent microscopy, indicate that the pole-localized nap, identified here for the first time in B. megaterium QM B1551 spores, is comprised of the BclA1 protein. The role of the BxpB protein, which forms the basal layer of the exosporium in B. cereus spores, is less clear since spores of a null mutant strain display an apparently normal morphology. Retention of the localized nap in bxpB null spores suggests that B. megaterium employs an alternative mechanism to that used by B. cereus spores in anchoring the nap to the spore surface.

SUBMITTER: Manetsberger J

PROVIDER: S-EPMC4674009 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Plasmid-encoded genes influence exosporium assembly and morphology in Bacillus megaterium QM B1551 spores.

Manetsberger Julia J Hall Elizabeth A H EA Christie Graham G

FEMS microbiology letters 20150826 18

Spores of Bacillus megaterium QM B1551 are encased in a morphologically distinctive exosporium. We demonstrate here that genes encoded on the indigenous pBM500 and pBM600 plasmids are required for exosporium assembly and or stability in spores of this strain. Bioinformatic analyses identified genes encoding orthologues of the B. cereus-family exosporium nap and basal layer proteins within the B. megaterium genome. Transcriptional analyses, supported by electron and fluorescent microscopy, indica ...[more]

PMID: 26316548

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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

Plasmid-encoded genes influence exosporium assembly and morphology in Bacillus megaterium QM B1551 spores.

Publications

Plasmid-encoded genes influence exosporium assembly and morphology in Bacillus megaterium QM B1551 spores.

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