Towards the minimal proteome of life: Proteome profiles of Bacillus subtilis vegetative cells and spores

Vischer

et al. 2019

Preprint

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Spore forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and food-borne diseases. The spores remain in a dormant state for extended periods due to their highly resistant features. When environmental conditions become favourable, they can germinate as the germinant receptors located on the spore's inner membrane get activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth. The present study focuses on the synthesis of protein MalS during B. subtilis spore germination by investigating the dynamics of the fluorescence level of a MalS-GFP fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase within the first 15 minutes of germination, followed by a drop and stabilization of the fluorescence throughout the spore ripening period. Western blot analyses, however, indicate no increase in the levels of the MalS-GFP fusion protein during the first 15 minutes after the addition of the germinants. Thus the instantaneous increase in fluorescence of MalS-GFP may likely due to a change in the physical environment as the spore germination is triggered. Our findings also show that the different sporulation conditions and the maturation time of spores affect the expression of MalS-GFP and the germination behaviour of the spores.

Section: Introductionsupporting

confidence: 65%

Investigating the influence of spore maturation and sporulation conditions on MalS expression during Bacillus subtilis spore germination

Vischer

et al. 2019

Preprint

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“…The B. subtilis strains PY79 and PB2 were used in this study. Growth conditions for sporulation are described elsewhere 5 . Mature spores (96 hrs) were purified as described previously 13 .…”

Section: Methodsmentioning

confidence: 99%

“…Mature spores (96 hrs) were purified as described previously 13 . For 15 N metabolic labelling, B. subtilis PY79 cells were grown as described previously 5 . Briefly, NH 4 Cl supplied in MOPS medium was replaced with 15 NH 4 Cl.…”

Section: Methodsmentioning

confidence: 99%

“…In Approach I, samples from were subjected to one pot isolation method and peptides were fractionated using ZIC-HILIC 5,13 . For approach II and III fractionation was omitted.…”

Section: Methodsmentioning

confidence: 99%

“…Through the germinant sensing and calcium dipicolinic acid transport signal transduction system, B. subtilis channels information related to external stimuli to an internal response, enabling the above mentioned morphogenetic transformation. The dormant spores harbour the transcriptional and translational machinery that might facilitates the early stages of outgrowth 5 . The protein cargo that transfers from progenitor cell to the spore, the ‘tool-kit for life’, is key to the spore’s memory 6 which in turn modulates its germination response 7 .…”

Section: Introductionmentioning

confidence: 99%

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Integrative analysis of proteome and transcriptome dynamics duringBacillus subtilisspore revival

Jonker

Abhyankar

et al. 2019

Preprint

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Bacillus subtilis forms highly resistant, metabolically inactive dormant spores upon nutrient limitation. These endospores pose challenges to the food and medical sectors. Spores reactivate their metabolism upon contact with germinants and develop into vegetative cells.The activation of the molecular machinery that triggers the progress of germination and spore outgrowth is still unsettled. To gain further insight in spore germination and outgrowth processes, the transcriptome and proteome changeover during spore germination and outgrowth to vegetative cells, was analysed. B. subtilis transcriptome analysis allow us to trace the different functional groups of genes expressed. For each time-point sample, the change in the spore proteome was quantitatively monitored relative to the reference proteome of 15 N metabolically labelled vegetative cells. We observed until the phase transition, i.e. completion of germination, no significant change in the proteome. We have identified 36 transcripts present abundantly in the dormant spores. This number is in close agreement with the previous findings. These transcripts mainly belong to the genes encoding small acid soluble proteins (sspE, sspO, sspI, sspK, sspF) and proteins with uncharacterized functions.We observed in total 3152 differentially expressed genes, but 'only' 323 differentially expressed proteins (total 451 proteins identified and quantified). Our data shows that 173 proteins from dormant spores, both spore unique proteins and protein shared with vegetative cells, are lost during the phase transitioning period. This loss is in addition to the active protein degradation, undertaken by the spore proteases such as Gpr, as germination and outgrowth proceeds. Further analysis is required to functionally interpret the observed protein loss. The observed diverse timing of the synthesis of different protein sets reveals a putative core-strategy of the revival of 'life' starting from the B. subtilis spore.

Artificial Sporulation Induction (ASI) by kinA Overexpression Affects the Proteomes and Properties of Bacillus subtilis Spores

Abhyankar

et al. 2020

IJMS

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To facilitate more accurate spore proteomic analysis, the current study focuses on inducing homogeneous sporulation by overexpressing kinA and assesses the effect of synchronized sporulation initiation on spore resistance, structures, the germination behavior at single-spore level and the proteome. The results indicate that, in our set up, the sporulation by overexpressing kinA can generate a spore yield of 70% within 8 h. The procedure increases spore wet heat resistance and thickness of the spore coat and cortex layers, whilst delaying the time to spore phase-darkening and burst after addition of germinant. The proteome analysis reveals that the upregulated proteins in the kinA induced spores, compared to spores without kinA induction, as well as the ‘wildtype’ spores, are mostly involved in spore formation. The downregulated proteins mostly belong to the categories of coping with stress, carbon and nitrogen metabolism, as well as the regulation of sporulation. Thus, while kinA overexpression enhances synchronicity in sporulation initiation, it also has profound effects on the central equilibrium of spore formation and spore germination, through modulation of the spore molecular composition and stress resistance physiology.