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Visualization of Germination Proteins in Putative <i>Bacillus cereus</i> Germinosomes
oleh: Yan Wang, Richard de Boer, Norbert Vischer, Pauline van Haastrecht, Peter Setlow, Stanley Brul
Format: | Article |
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Diterbitkan: | MDPI AG 2020-07-01 |
Deskripsi
<i>Bacillus cereus</i> 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 <i>B. cereus</i> spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of <i>B. cereus</i> 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 <i>B. cereus</i> spores, we have examined the organization of germinant receptors (GRs) in spores′ IM. Previous studies have indicated that in spores of <i>B.</i><i>cereus</i> 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 <i>gerR</i> 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 <i>gerD</i> promoter. We termed all fluorescent foci observed germinosomes, the term used for the IM foci of GRs in <i>Bacillus subtilis</i> spores. Our data are the first evidence for the existence of germinosomes in <i>B. cereus</i> spores.