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

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.


Introduction
Sporulation and germination of genetically identical bacterial cells or spores have been extensively studied and shown to occur heterogeneously. This observation has challenged the food industries and the medical sector to extensively investigate both these processes to minimise microbiological risks by developing novel targeted strategies to eliminate spores.
However, empirical studies on spores, fail to reveal the mechanistic basis of the spore's resistance to the generally deployed bacterial elimination methods [1][2][3] . Therefore, gaining a detailed knowledge of sporulation and spore germination is of utmost importance. As discussed throughout this thesis, the resistance properties of spores are mainly caused by their layered structure and the chemical composition of those various layers 2,4 and the germination receptor proteins (GRs), located in the inner membrane (IM), facilitate spore germination 5,6 .
In B. subtilis three GRs are well known -GerA, GerB and GerK. These proteins are each composed of three subunits and can be triggered 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 B-subunit of these receptors, thereby causing their activation 4,8 . Once the GRs are activated, monovalent and divalent cations are released from the spores along with the dipicolinic acid (DPA), and the degradation of the peptidoglycan cortex is ensued 7 . The latter is a prerequisite for the subsequent spore outgrowth. The occurrence of all these phases leads to complete re-hydration of the spore and restores its macromolecular synthesis and endogenous enzyme activity 6 .
During the outgrowth of spores, complex sugars, carbohydrates and organic acids can be used as carbon sources. In case of B. subtilis, glucose and malate are generally the preferred carbon sources 9 . In fact, any available malate is suggested to enter the TCA cycle where it can be oxidized by malic enzymes (MalS, MaeA, MleA or YtsJ) to pyruvate through oxidative decarboxylation 9 . Recently, Sinai et al. 10 investigated the synthesis of a MalS-GFP reporter fusion protein over time, and predicted that MalS is one of the earliest proteins produced in the germinating B. subtilis spores. They observed a striking increase in the GFP intensity within the first 5 minutes of germination in spores. Remarkably, these spores remained phase-bright while the fluorescence intensity increased leading to their conclusion that protein synthesis is necessary during germination. On the contrary, a following study proved that protein synthesis is not essential for germination 11 . Parallel to these observations, in our proteomics studies 12,13 the relative quantity of MalS in the dormant spores is considerably high and during germination, there is no apparent change in its levels. The above-mentioned studies however, differ with respect to the age of the spores used in each study and the medium used for sporulation. Previously, it has been reported that the sporulation conditions and maturation time affect the spore characteristics and resistance properties 14 . Spores formed on solid rich media are generally more heat resistant and are characterised by a higher degree of coat proteins involved in cross-linking 14 . It has also been shown that higher spore coat protein cross-linking correlates with slight delayed germination times 11 . Therefore, with respect to MalS synthesis and MalS-GFP expression during germination the following research questions require attention: 1) Do the different sporulation conditions and spore maturation times affect MalS-GFP expression in B. subtilis spores? 2) Is MalS synthesized in phase-bright spores?
To answer these questions, we have compared the spores of B. subtilis strain PY79 and AR71 (MalS-GFP) prepared in liquid minimal medium and on solid agar medium. The germination of young (day 2) and old or mature (day 4) spores is triggered with a mixture of Lasparagine, D-glucose, D-fructose and KCl (AGFK), and with L-alanine. The time when germination starts and the actual germination time, which is the time required for the phase transition from phase-bright to phase-dark, have been analysed using time-lapse phasecontrast microscopy. The expression of MalS-GFP in young and matured spores, obtained from the liquid and solid media, is also monitored in a time dependent manner using fluorescence microscopy. In addition, the synthesis of MalS-GFP fusion protein, during the initial stages of germination, is followed by western blot analysis.

Bacterial strains and sporulation conditions
The B. subtilis wild-type strain PY79 and the mutant strain AR71 (MalS-GFP, Spc + ; obtained from Sinai et al. 10 ) were used in this study. Similarly, to MalS, PdhD (pyruvate dehydrogenase subunit D) was reported to be newly synthesized at an early stage during spore outgrowth 2 . Therefore, as an independent control for the fluorescence measurements, we used a B. subtilis strain (Erm + ) harbouring a PdhD-IpHluorin (PC) fusion protein prepared in our laboratory. The two different sporulation media used in this study were: defined liquid medium buffered with 80mM 3-(N-morpholino) propanesulfonic acid (MOPS) and 2x Schaeffer's medium with glucose (2x SG) agar solid medium as described previously 11,15 .
Primarily, bacterial cells were cultured on a Tryptic Soy agar (TSA) plate and incubated overnight at 37°C. A single colony was inoculated in Tryptic Soy Broth (TSB) medium and incubated at 37°C at 200 rpm until the exponential growth phase was reached (OD 600 ~ 0.3 to 0.4). A serial dilution was subsequently performed of the culture in MOPS liquid medium or 2x SG liquid medium and incubated overnight at 37°C while shaking at 200 rpm. A single dilution containing exponentially growing cells was selected and the bacterial culture was further enriched in 20 ml MOPS liquid medium or 2xSG liquid medium. For sporulation, cells cultured in MOPS liquid medium were inoculated into 300 ml MOPS liquid medium and cultured at 37°C while shaking at 200 rpm. In contrast, cells cultured in 2x SG liquid medium were spread on 10 plates containing 2x SG agar and incubated at 37°C.

Spore harvesting
Spores cultured in MOPS liquid medium and on 2x SG solid medium were harvested after 48 and 96 hrs 14 . Spore samples were extensively washed with pre-chilled milliQ-water at 4°C.
Subsequently, Tween-20 (0.01% v/v) was added to the spores in milliQ-water to kill vegetative cells and to improve purification. The harvested spores were examined under the microscope using a haemocytometer and the spore yield was estimated. Remaining vegetative cells and/or germinated cells (phase dark) were removed using the Histodenz gradient centrifugation method describe by Abhyankar et al. 14 . The purified harvested spores contained more than 99% phase bright spores. Spores were stored at -80°C until further use.

Germination and Fluorescence measurements
For time-lapse germination experiments, spores were heat activated at 70°C for 30 minutes at 100 rpm and placed on ice for 10 min. Immediately after incubation on ice, the spores were transferred to an agarose pad containing 2% w/v agarose mixed with a double concentration of MOPS medium and germinants (AGFK and L-alanine) in a 1:1 ratio. The set-up of the microscope slide for time-lapse microscopy imaging has been described by Pandey et al. 16 .
Time-lapse microscopy imaging was performed at 37°C in a time dependant manner for 2 hours using a temperature-controlled incubation system. A Nikon ECLIPSE Ti-E inverted

Western Blot analysis
Spores

Determining the effect of maturation and sporulation conditions on the germination behaviour of spores of B. subtilis strain AR71 (MalS-GFP) and the wild type strain PY79.
All the young (day 2) spores of stain AR71 prepared in MOPS liquid medium differ significantly from their matured (day 4) partners with respect to the time required to start germination. At the population level, the relatively young spores are less prone to initiate germination in comparison to the older spores (45% young spores vs. 61% matured spores in 10 min; Table 1(A)). However, these two spore populations do not differ significantly in their germination time (Figure 1, Table 1(A)). The time required to start germination and the germination time differ significantly in case of the young and mature spores of the wild type strain PY79 (Table 1 (B), Figure 1). Furthermore, the young wild-type spores initiate germination later than the matured spores (35% vs. 44% in 10 min, Table 1

(B)).
In case of the sporulation on 2x SG solid medium, the younger AR71 spores are more prone to start germination than their matured counterparts (82% vs 71% in 10 min,  1 (B)). In general, with the exception of AR71 old spores, the wild type PY79 and mutant strain AR71 spores prepared on the solid medium start germination earlier than those prepared in a liquid medium. (Figure 2, Table 1).

GFP fluorescence
The    changing localization and distribution of the GFP fluorescence over time. The young phasebright MalS-GFP spores, sporulated in both liquid and on solid medium, show a clustered GFP fluorescence. Upon completion of germination, the clustered GFP diffuses throughout the spore. However, in some of the control (PdhD-IpHluorin) spores (day 2, MOPS) the GFP clusters are still present even after germination is completed (Supplementary figure 2).

Effect of sporulation conditions and maturation time on the levels of MalS-GFP fusion protein
The western blot analyses show that in young and mature AR71 spores, prepared in liquid and on solid media, the levels of the MalS-GFP fusion protein do not increase significantly within 15 minutes after the addition of the germinants. The microscopy images show that by this point all spores turn phase dark i.e. their germination is completed. and CaDPA from the core. This triggers cortex hydrolysis, further CaDPA release and full core hydration. Completion of germination results in a phase-dark spore which is ready to start macromolecular synthesis. The germination process itself is very heterogeneous and germination kinetics appears to be affected by different sporulation conditions 18 and spore maturation 11 . Also, the recent evidence shows that the spore-coat characteristics differ in the spores prepared in the liquid and on solid media. Nevertheless, the precise effect of spore maturation and sporulation conditions on germination remains unclear. Evidences over decades of work on spore germination indicate that there is no significant metabolic activity and no protein synthesis in a spore until completion of germination [4][5][6]19 . Albeit, a recent report has shown protein synthesis to be necessary during germination, and dormant spores to be metabolically active 10 . The discrepancy between the earlier studies and the work of Sinai and colleagues may stem from the differences in the sporulation conditions and age of spores used in the respective studies. Therefore, in the present work we attempt to observe the effect of sporulation conditions and spore maturation on behaviour of spore and protein synthesis during germination.
With regards to the germination behaviour, we focus on two different aspects: the effect on (a) the time required start germination and (b) the actual germination time (total duration to achieve phase darkening). In general, the spores prepared on solid medium start germination earlier as compared to spores sporulated in the liquid medium. This is in accordance with a previous report 18 . Moreover, the young AR71 (MalS-GFP) spores, prepared in both liquid and solid media, complete germination faster than the matured spores. Interestingly, the young wild type spores prepared in liquid medium behave similarly by having a shorter germination time than that of the matured spores, but the spores prepared on the solid medium differ in this aspect. Thus, for the wild type spores prepared on solid medium the age or the maturation time of spores appears to affect the germination time as well. This could be due to the difference in (a) thickness of the spore-coat layers and the protein-crosslinking therein 14 or (b) the number of germination related proteins in the spores, as suggested previously 8 .
Next, we analyse the influence of maturation times and sporulation conditions on MalS-GFP expression over time. Interestingly, for the spores prepared on solid medium the MalS-GFP fluorescence is always higher than that for spores prepared in liquid medium. This distinction might be a variation in GFP maturation behaviour caused by the differing growth conditions, as suggested previously 20 . Furthermore, MalS-GFP in the spores appears to be clustered until germination is completed. Previously, it has been shown that a pH of 6.5 promotes aggregation of EGFP 21 . Since the pH in the spore core is between 6.0 and 6.4 22,23 , the observed clustering of MalS-GFP could represent GFP aggregation. The observation that these clusters diffuse throughout the spores during the ripening stage can be attributed to the phase transition in the state of core water as a result of initial heat activation 24 . The decrease in MalS-GFP fluorescence after 30 min may also result from the changed state of core water, the water intake by a germinating spore or due to utilization of MalS by the reviving spore.
However, our proteomics and transcriptomic data 13  In conclusion, our study shows that the sporulation conditions and maturation period of spores affect their germination behaviour and the fluorescence intensities whereas expression of MalS-GFP and thereby protein synthesis in germinating spore remain unaffected.