scholarly journals Integrative Analysis of Proteome and Transcriptome Dynamics during Bacillus subtilis Spore Revival

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Bhagyashree Swarge ◽  
Wishwas Abhyankar ◽  
Martijs Jonker ◽  
Huub Hoefsloot ◽  
Gertjan Kramer ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Molecular Mechanisms ◽  
Transformation Process ◽  
Comprehensive Overview ◽  
Vegetative Cells ◽  
Content Type ◽  
Protein Levels ◽  
Bacillus Subtilis Spore ◽  
Proteome Changes

ABSTRACT Bacillus subtilis spores can reactivate their metabolism through germination upon contact with germinants and can develop into vegetative cells upon outgrowth. However, the mechanisms at the basis of the molecular machinery that triggers the spore germination and outgrowth processes are still largely unclear. To gain further insights into these processes, the transcriptome and proteome changes occurring during the conversion of spores to vegetative cells were analyzed in the present study. For each time point sampled, the changes in the spore proteome were quantitatively monitored relative to the proteome of metabolically 15N-labeled vegetative cells. Of the quantified proteins, 60% are shared by vegetative cells and spores, indicating that the spores have a minimal protein set, sufficient to resume metabolism upon completion of germination. These shared proteins thus represent the most basic “survival kit” for spore-based life. We observed no significant change in the proteome or the transcriptome until the spore’s completion of germination. Our analysis identified 34 abundant mRNA transcripts in the dormant spores, 31 of which are rapidly degraded after germination. In outgrowing spores, we identified 3,152 differentially expressed genes and have demonstrated the differential expression of 322 proteins with our mass spectrometry analyses. Our data also showed that 173 proteins from dormant spores, including both proteins unique to spores and proteins shared with vegetative cells, were lost after completion of germination. The observed diverse timings of synthesis of different protein sets in spore outgrowth revealed a putative core strategy underlying the revival of ‘life’ from the B. subtilis spore. IMPORTANCE This study demonstrated the progress of macromolecular synthesis during Bacillus subtilis spore germination and outgrowth. The transcriptome analysis has additionally allowed us to trace gene expression during this transformation process. For the first time, the basic survival kit for spore-based life has been identified. In addition, in this analysis based on monitoring of protein levels in germinating and outgrowing spores, the transition from (ribo)nucleotide and amino acid biosynthesis to the restoration of all metabolic pathways can be clearly seen. The integrative multi-omics approach applied in this study thus has helped us to achieve a comprehensive overview of the molecular mechanisms at the basis of spore germination and outgrowth as well as to identify important knowledge gaps in need of further study.

scholarly journals Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

2014 ◽  
Vol 197 (2) ◽  
pp. 252-261 ◽  
Author(s):  
Anthony J. Troiano ◽  
Jingqiao Zhang ◽  
Ann E. Cowan ◽  
Ji Yu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Western Blot ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Interference Microscopy ◽  
Epifluorescence Microscopy ◽  
Focus Formation ◽  
Content Type ◽  
Protein Levels ◽  
Bacillus Subtilis Spore

Germination ofBacillus subtilisspores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the “germinosome,” and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ∼50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.

scholarly journals Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments

2015 ◽  
Vol 81 (19) ◽  
pp. 6725-6735 ◽  
Author(s):  
Katja Nagler ◽  
Peter Setlow ◽  
Kai Reineke ◽  
Adam Driks ◽  
Ralf Moeller
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
High Salinity ◽  
Dipicolinic Acid ◽  
Food Microbiology ◽  
Coat Proteins ◽  
Wild Type ◽  
Protective Function ◽  
Content Type ◽  
Bacillus Subtilis Spore

ABSTRACTThe germination of spore-forming bacteria in high-salinity environments is of applied interest for food microbiology and soil ecology. It has previously been shown that high salt concentrations detrimentally affectBacillus subtilisspore germination, rendering this process slower and less efficient. The mechanistic details of these salt effects, however, remained obscure. Since initiation of nutrient germination first requires germinant passage through the spores' protective integuments, the aim of this study was to elucidate the role of the proteinaceous spore coat in germination in high-salinity environments. Spores lacking major layers of the coat due to chemical decoating or mutation germinated much worse in the presence of NaCl than untreated wild-type spores at comparable salinities. However, the absence of the crust, the absence of some individual nonmorphogenetic proteins, and the absence of either CwlJ or SleB had no or little effect on germination in high-salinity environments. Although the germination of spores lacking GerP (which is assumed to facilitate germinant flow through the coat) was generally less efficient than the germination of wild-type spores, the presence of up to 2.4 M NaCl enhanced the germination of these mutant spores. Interestingly, nutrient-independent germination by high pressure was also inhibited by NaCl. Taken together, these results suggest that (i) the coat has a protective function during germination in high-salinity environments; (ii) germination inhibition by NaCl is probably not exerted at the level of cortex hydrolysis, germinant accessibility, or germinant-receptor binding; and (iii) the most likely germination processes to be inhibited by NaCl are ion, Ca2+-dipicolinic acid, and water fluxes.

scholarly journals Effects of Sporulation Conditions on the Germination and Germination Protein Levels of Bacillus subtilis Spores

2012 ◽  
Vol 78 (8) ◽  
pp. 2689-2697 ◽  
Author(s):  
Arturo Ramirez-Peralta ◽  
Pengfei Zhang ◽  
Yong-qing Li ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Rich Medium ◽  
Microscope Slide ◽  
Content Type ◽  
Protein Levels ◽  
Germination Characteristics ◽  
The Difference ◽  
Lag Times

ABSTRACTBacillus subtilisspores prepared in rich medium germinated faster with nutrient germinants than poor-medium spores as populations in liquid and multiple individual spores on a microscope slide. Poor-medium spores had longer average lag times between mixing of spores with nutrient germinants and initiation of Ca-dipicolinic acid (CaDPA) release. Rich-medium spores made at 37°C germinated slightly faster with nutrient germinants than 23°C spores in liquid, but not when spores germinated on a slide. The difference in germination characteristics of these spore populations in liquid was paralleled by changes in expression levels of a transcriptionallacZfusion to thegerAoperon, encoding a germinant receptor (GR). Levels of GR subunits were 3- to 8-fold lower in poor-medium spores than rich-medium spores and 1.6- to 2-fold lower in 23°C spores than 37°C spores, and levels of the auxiliary germination protein GerD were 3.5- to 4-fold lower in poor medium and 23°C spores. In contrast, levels of another likely germination protein, SpoVAD, were similar in all these spores. These different spores germinated similarly with CaDPA, and poor-medium and 23°C spores germinated faster than rich-medium and 37°C spores, respectively, with dodecylamine. Since spore germination with CaDPA and dodecylamine does not require GerD or GRs, these results indicate that determinants of rates of nutrient germination of spores prepared differently are primarily the levels of the GRs that bind nutrient germinants and trigger germination and secondarily the levels of GerD.

scholarly journals Ribosome Reconstruction during Recovery from High-Hydrostatic-Pressure-Induced Injury in Bacillus subtilis

2019 ◽  
Vol 86 (1) ◽  
Author(s):  
Huyen Thi Minh Nguyen ◽  
Genki Akanuma ◽  
Tu Thi Minh Hoa ◽  
Yuji Nakai ◽  
Keitarou Kimura ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Growth Arrest ◽  
Recovery Process ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Vegetative Cells ◽  
Content Type ◽  
Growth Recovery

ABSTRACT Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf, whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn2+ or Zn2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn2+ or Zn2+. Moreover, Zn2+, but not Mn2+, promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn2+-dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn2+ and Zn2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells. IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn2+ and Zn2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells.

scholarly journals Both Fidaxomicin and Vancomycin Inhibit Outgrowth of Clostridium difficile Spores

2012 ◽  
Vol 57 (1) ◽  
pp. 664-667 ◽  
Author(s):  
Charlotte A. Allen ◽  
Farah Babakhani ◽  
Pam Sears ◽  
Ly Nguyen ◽  
Joseph A. Sorg
Keyword(s):  
Clostridium Difficile ◽  
Clinical Response ◽  
Recurrence Rate ◽  
Spore Germination ◽  
Vegetative Cells ◽  
Content Type

ABSTRACTFidaxomicin (FDX) is approved to treatClostridium difficile-associated diarrhea and is superior to vancomycin in providing a sustained clinical response (cure without recurrence in the subsequent 25 days). The mechanism(s) behind the low recurrence rate of FDX-treated patients could be multifactorial. Here, we tested effects of FDX, its metabolite OP-1118, and vancomycin on spore germination and determined that none affected the initiation of spore germination but all inhibited outgrowth of vegetative cells from germinated spores.

scholarly journals Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA2mob Operon in Foodborne Strains of Bacillus subtilis

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Antonina O. Krawczyk ◽  
Anne de Jong ◽  
Jimmy Omony ◽  
Siger Holsappel ◽  
Marjon H. J. Wells-Bennik ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Spore Germination ◽  
Food Industry ◽  
Heat Treatments ◽  
Phenotypic Traits ◽  
Content Type ◽  
Heat Resistant ◽  
Heat Activation ◽  
High Level

ABSTRACT Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA 2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA 2mob required higher HA temperatures for efficient germination than spores lacking spoVA 2mob. The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K+ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers. IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores.

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