Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis 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 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.

Maturation of Released Spores Is Necessary for Acquisition of Full Spore Heat Resistance during Bacillus subtilis Sporulation

ABSTRACTThe first ∼10% of spores released from sporangia (early spores) duringBacillus subtilissporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ∼24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca2+but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.

An Unusual Response Regulator Influences Sporulation at Early and Late Stages in Streptomyces coelicolor

ABSTRACT WhiI, a regulator required for efficient sporulation septation in the aerial mycelium of Streptomyces coelicolor, resembles response regulators of bacterial two-component systems but lacks some conserved features of typical phosphorylation pockets. Four amino acids of the abnormal “phosphorylation pocket” were changed by site-directed mutagenesis. Unlike whiI null mutations, these point mutations did not interfere with sporulation septation but had various effects on spore maturation. Transcriptome analysis was used to compare gene expression in the wild-type strain, a D27A mutant (pale gray spores), a D69E mutant (wild-type spores), and a null mutant (white aerial mycelium, no spores) (a new variant of PCR targeting was used to introduce the point mutations into the chromosomal copy of whiI). The results revealed 45 genes that were affected by the deletion of whiI. Many of these showed increased expression in the wild type at the time when aerial growth and development were taking place. About half of them showed reduced expression in the null mutant, and about half showed increased expression. Some, but not all, of these 45 genes were also affected by the D27A mutation, and a few were affected by the D69E mutation. The results were consistent with a model in which WhiI acts differently at sequential stages of development. Consideration of the functions of whiI-influenced genes provides some insights into the physiology of aerial hyphae. Mutation of seven whiI-influenced genes revealed that three of them play roles in spore maturation.

Effect of Temperature on Sporulation of Botryosphaeria dothidea, B. obtusa, and B. rhodina

Three Botryosphaeria spp. were grown on autoclaved apple and peach stems in cotton-plugged tubes with constant moisture at 6, 12, 18, 24, and 30°C to determine the effect of temperature on sporulation. Number of conidia per pycnidium was determined weekly from 4 to 10 weeks after inoculation. The experiment was repeated three times. Maximum sporulation occurred at 24°C with B. dothidea and at 18 and 24°C with B. obtusa. Spore production of both fungi showed a quadratic curvilinear response to temperature. Pycnidia were erumpent, typical of their habit in nature. Maximum sporulation of B. rhodina occurred at 12, 24, and 30°C instead of at a distinctive peak. Of the three fungi, B. rhodina produced the greatest number of conidia per pycnidium at all temperatures. Mycelia and pycnidia of B. rhodina grew on top of the bark, which is atypical of their habit in nature. For spore production by B. dothidea, there was a significant interaction between temperature and time. Maximum sporulation over the 10-week period occurred in week 4 and/or 6 for B. dothidea at 12, 18, and 24°C, with a linear response at 12 and 24°C (P ≤0.05). Conidial maturation of B. obtusa and B. rhodina had a quadratic curvilinear response due to temperature, with a maximum maturation at 12, 18, and 24°C with B. obtusa and at 24°C with B. rhodina. Spore maturation would affect longevity of conidial viability. Maximum spore production over time and percent pigmented spores over time by B. obtusa, and spore maturation over time by B. rhodina occurred in weeks 8, 9, and 10 with a significant linear response (P ≤ 0.05). All three Botryosphaeria spp. produced conidia over the 6 to 30°C range and over the 7-week period (weeks 4 to 10), with maximum sporulation or spore maturation at 18 to 24°C.