Investigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

ABSTRACT 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 germinan...

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Autores principales:	Bhagyashree Swarge, Chahida Nafid, Norbert Vischer, Gertjan Kramer, Peter Setlow, Stanley Brul
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	spore maturation B. subtilis MalS protein synthesis spore germination Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/f217045086f4405cb39fedb22ec3b02b
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spelling	oai:doaj.org-article:f217045086f4405cb39fedb22ec3b02b2021-11-15T15:30:50ZInvestigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions10.1128/mSphere.00464-202379-5042https://doaj.org/article/f217045086f4405cb39fedb22ec3b02b2020-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00464-20https://doaj.org/toc/2379-5042ABSTRACT 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.Bhagyashree SwargeChahida NafidNorbert VischerGertjan KramerPeter SetlowStanley BrulAmerican Society for Microbiologyarticlespore maturationB. subtilisMalSprotein synthesisspore germinationMicrobiologyQR1-502ENmSphere, Vol 5, Iss 4 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	spore maturation B. subtilis MalS protein synthesis spore germination Microbiology QR1-502
spellingShingle	spore maturation B. subtilis MalS protein synthesis spore germination Microbiology QR1-502 Bhagyashree Swarge Chahida Nafid Norbert Vischer Gertjan Kramer Peter Setlow Stanley Brul Investigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions
description	ABSTRACT 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.
format	article
author	Bhagyashree Swarge Chahida Nafid Norbert Vischer Gertjan Kramer Peter Setlow Stanley Brul
author_facet	Bhagyashree Swarge Chahida Nafid Norbert Vischer Gertjan Kramer Peter Setlow Stanley Brul
author_sort	Bhagyashree Swarge
title	Investigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions
title_short	Investigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions
title_full	Investigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions
title_fullStr	Investigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions
title_full_unstemmed	Investigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions
title_sort	investigating synthesis of the mals malic enzyme during <named-content content-type="genus-species">bacillus subtilis</named-content> spore germination and outgrowth and the influence of spore maturation and sporulation conditions
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/f217045086f4405cb39fedb22ec3b02b
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Investigating Synthesis of the MalS Malic Enzyme during <named-content content-type="genus-species">Bacillus subtilis</named-content> Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

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