Dataset Information

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes.

ABSTRACT: Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge of B. cereus spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of B. cereus spores are a core with the genetic material encased by multiple protective layers, an important one being the spores' inner membrane (IM), the location of many important germination proteins. To study mechanisms involved in germination of B. cereus spores, we have examined the organization of germinant receptors (GRs) in spores' IM. Previous studies have indicated that in spores of B. cereus ATCC 14579 the L-alanine responsive GR, GerR, plays a major role in the germination process. In our study, the location of the GerR GR subunit, GerRB, in spores was examined as a C-terminal SGFP2 fusion protein expressed under the control of the gerR operon's promoter. Our results showed that: i) the fluorescence maxima and integrated intensity in spores with plasmid-borne expression of GerRB-SGFP2 were significantly higher than in wild-type spores; ii) western blot analysis confirmed the expression of the GerRB-SGFP2 fusion protein in spores; and iii) fluorescence microscopy visualized GerRB-SGFP2 specific bright foci in ~30% of individual dormant spores if only GerRB-SGFP2 was expressed, but, noticeably, in ~85% of spores upon co-expression with GerRA and GerRC. Our data corroborates the notion that co-expression of GR subunits improves their stability. Finally, all spores displayed bright fluorescent foci upon expression of GerD-mScarlet-I under the control of the gerD promoter. We termed all fluorescent foci observed germinosomes, the term used for the IM foci of GRs in Bacillus subtilis spores. Our data are the first evidence for the existence of germinosomes in B. cereus spores.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7432890 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes.

Wang Yan Y de Boer Richard R Vischer Norbert N van Haastrecht Pauline P Setlow Peter P Brul Stanley S

International journal of molecular sciences 20200722 15

Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge of B. cereus spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of B. cereus spores are a core with the genetic material encased by multiple protective layers, an important one being the spores' inner membr ...[more]

PMID: 32707970

Similar Datasets

Project description:The Bacillus cereus group of bacteria includes seven closely related species, three of which, B. anthracis, B. cereus and B. thuringiensis, are pathogens of humans, animals and/or insects. Preliminary investigations into the transport capabilities of different bacterial lineages suggested that genes encoding putative efflux systems were unusually abundant in the B. cereus group compared to other bacteria. To explore the drug efflux potential of the B. cereus group all putative efflux systems were identified in the genomes of prototypical strains of B. cereus, B. anthracis and B. thuringiensis using our Transporter Automated Annotation Pipeline. More than 90 putative drug efflux systems were found within each of these strains, accounting for up to 2.7% of their protein coding potential. Comparative analyses demonstrated that the efflux systems are highly conserved between these species; 70-80% of the putative efflux pumps were shared between all three strains studied. Furthermore, 82% of the putative efflux system proteins encoded by the prototypical B. cereus strain ATCC 14579 (type strain) were found to be conserved in at least 80% of 169 B. cereus group strains that have high quality genome sequences available. However, only a handful of these efflux pumps have been functionally characterized. Deletion of individual efflux pump genes from B. cereus typically had little impact to drug resistance phenotypes or the general fitness of the strains, possibly because of the large numbers of alternative efflux systems that may have overlapping substrate specificities. Therefore, to gain insight into the possible transport functions of efflux systems in B. cereus, we undertook large-scale qRT-PCR analyses of efflux pump gene expression following drug shocks and other stress treatments. Clustering of gene expression changes identified several groups of similarly regulated systems that may have overlapping drug resistance functions. In this article we review current knowledge of the small molecule efflux pumps encoded by the B. cereus group and suggest the likely functions of numerous uncharacterised pumps.

Project description:Bacillus cereus spores, like most Bacillus spores, can survive for years and germinate when their surroundings become suitable, and germination proteins play an important role in the initiation of germination. Because germinated spores lose the extreme resistance of dormant spores, information on the function of germination proteins could be useful in developing new strategies to control B. cereus spores. Prior work has shown that (i) the channel protein SpoVAEa exhibits high-frequency movement in the outer leaflet of the inner membrane (IM) in dormant B. subtilis spores and (ii) the formation of the foci termed germinosomes between two germination proteins, the germinant receptor GerR and the scaffold protein GerD, in developing B. cereus spores is slower than foci formation by GerR and GerD individually. However, the movement dynamics of SpoVAEa in B. cereus spores, and the behavior of the germinosome upon B. cereus spore germination, are not known. In this study, we found that SpoVAEa fluorescent foci in dormant B. cereus spores move on the IM, but slower than in B. subtilis spores, and they likely co-localize transiently with GerD-mScarlet-I in the germinosome. Our results further indicate that (i) the expression of GerR-SGFP2 and SpoVAEa-SGFP2 with GerD-mScarlet-I from a plasmid leads to more heterogeneity and lower efficiency of spore germination in B. cereus, and (ii) germinosome foci observed by Fluorescence resonance energy transfer (FRET) between GerR-SGFP2 and GerD-mScarlet-I can be lost soon after the spore-phase transition. However, this is not always the case, as some GerR-SGFP2 and GerD-mScarlet-I foci continued to exist, co-localize, and even show a weak FRET signal. These data highlight the heterogeneous behavior of spore germination protein complexes and indicate that some complexes may persist beyond the initiation of germination. IMPORTANCE Bacillus cereus is commonly present in soil and infects humans via contaminated food. In this study, we used B. cereus spores to investigate the movement of the spore-specific inner membrane (IM) channel protein SpoVAEa, the interaction between SpoVAEa and the germinosome scaffold protein GerD, and the dynamics of germinosomes with GerR and GerD in spore germination. Our results expand upon observations of interactions between specific B. cereus spore germination proteins, in particular the GerR germinant receptor A, B, and C subunits and GerD, as well as those between SpoVAEa and GerD. The approaches used in this work could also be used to examine the interactions between GerD and SpoVAEa and other germination proteins in spores of other Bacillus species.

Dataset Information

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes.

Publications

Visualization of Germination Proteins in Putative <i>Bacillus cereus</i> Germinosomes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets