Structural and functional analyses of the N-terminal domain of the A subunit of a<i>Bacillus megaterium</i>spore germinant receptor

Li, Yunfeng; Jin, Kai; Pérez-Valdespino, Abigail; Federkiewicz, Kyle; Davis, Andrew John; Maciejewski, Mark W.; Setlow, Peter; Hao, Bing

doi:10.1073/pnas.1903675116

Cited by 16 publications

(18 citation statements)

References 68 publications

(85 reference statements)

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“…Generally, subunit A is comprised of five or six predicted transmembrane (TM) domains and hydrophilic domains at the N-and C-termini, but the B subunit is comprised of only 10-12 TM domains. The A and B GR subunits are the ones most likely to be involved in recognition of amino acid germinants [12]. The GR C-subunit includes a predicted pre-lipoprotein signal sequence, suggesting that the C subunit is anchored to the outer surface of the membrane via an N-terminally attached lipid moiety [13].…”

Section: Introductionmentioning

confidence: 99%

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes

Wang

Boer

Vischer

et al. 2020

IJMS

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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.

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Section: Introductionmentioning

confidence: 99%

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes

Wang

Boer

Vischer

et al. 2020

IJMS

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“…Each of these proteins is composed of three subunits and can be activated by several types of germinants, including specific amino acids ( 4 , 7 ). It is inferred that amino acids, such as alanine or valine, initially bind to the A and or B subunits of these receptors, thereby causing their activation ( 4 , 8 , 9 ). Once the GRs are activated, monovalent and divalent cations are released from the spores along with the spore core’s huge deposit (∼20% of core dry weight) of dipicolinic acid (DPA).…”

Section: Introductionmentioning

confidence: 99%

Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

Swarge

Vischer

et al. 2020

mSphere

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Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore’s inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695–707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination. IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

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“…Ger receptors form membrane-spanning complexes in the IsM and are often implicated in recognition of free amino acids ( Ross and Abel-Santos, 2010 ). Preliminary investigations into the structure of Ger receptors reveal that they are similar to inner membrane-associated small molecule transporters and signal transducers, possibly forming clusters ( Li et al, 2014 , 2019 ). Small PG fragments produced during spore germination stimulate germination of B. subtilis spores by binding to PrkC, a Ser/Thr kinase containing a peptidoglycan binding PASTA domain ( Shah et al, 2008 ).…”

Section: Germinationmentioning

confidence: 99%

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

et al. 2021

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Sporulation is a specialized developmental program employed by a diverse set of bacteria which culminates in the formation of dormant cells displaying increased resilience to stressors. This represents a major survival strategy for bacteria facing harsh environmental conditions, including nutrient limitation, heat, desiccation, and exposure to antimicrobial compounds. Through dispersal to new environments via biotic or abiotic factors, sporulation provides a means for disseminating genetic material and promotes encounters with preferable environments thus promoting environmental selection. Several types of bacterial sporulation have been characterized, each involving numerous morphological changes regulated and performed by non-homologous pathways. Despite their likely independent evolutionary origins, all known modes of sporulation are typically triggered by limited nutrients and require extensive membrane and peptidoglycan remodeling. While distinct modes of sporulation have been observed in diverse species, two major types are at the forefront of understanding the role of sporulation in human health, and microbial population dynamics and survival. Here, we outline endospore and exospore formation by members of the phyla Firmicutes and Actinobacteria, respectively. Using recent advances in molecular and structural biology, we point to the regulatory, genetic, and morphological differences unique to endo- and exospore formation, discuss shared characteristics that contribute to the enhanced environmental survival of spores and, finally, cover the evolutionary aspects of sporulation that contribute to bacterial species diversification.

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Structural and functional analyses of the N-terminal domain of the A subunit of aBacillus megateriumspore germinant receptor

Cited by 16 publications

References 68 publications

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes

Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

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