CHAPTER 7: METHOD DEVELOPMENT FOR ASTROBIOLOGIAL EXPLORATION EXPLORATION
7.2 Sporulation of psychrophilic endospores
Studying of psychrophilic bacterial spores in cyrospheres such as ice cores and permafrost has shed light on the understanding of how microorganisms can survive at subfreezing temperatures on Earth and possibly on Mars and other cold planetary bodies.
This chapter describes the isolation of psychrophilic Bacillus species from soils in Jet Propulsion Laboratory and Lake Vida in Antarctica, as well as a comparison between environmental psychrophilic strains with laboratory cultured psychrophilic endospores in terms of endospore properties, heat resistance and germination kinetics. Novel sporulation techniques have been developed to produce synchronized and homogeneous psychrophilic Bacillus endospores. Psychrophilic endospores are more demanding than
their mesophilic counterparts in terms of sporulation requirement. Aeration and germination inhibitor, such as D-alanine, were added to the nutrient exhaustion sporulation medium in order to obtain large, pure and homogeneous spore batches for germination and resistance studies.
7.2.1 Introduction
Endospores under psychrophilic conditions are of great significance because 3-quarter of the earth’s surface are covered with cold oceans and large land masses are maintained at low temperatures, which finds relevancy with lots of different icy celestial bodies. Study of endospores as the most resistant and long-lived survivor in various extreme cold biospheres, such as ice cores, polar lakes and alpine permafrost, hints to the exobiological search for life. When encountering nutritional deficiency, rod-shaped Gram-positive bacteria of the genera Bacillus and Clostridium differentiate to produce heat- and solvent- resistant endospores. Endospores have also been detected from these cold environments1-
4. In the past several decades, mesophilic endospores such as Bacillus subtilis, B.
megaterium and B. anthracis have been thoroughly studied due to their easy handling and fast growth rate. Yet endospore psychrophily has received little attention since Larkin and Stokes have reported on spore formation and germination at 0°C by aerobic spore- formers from soil, mud and water in 19665,6. Cold-adapted microbes can broadly be categorized into two groupings, psychrophiles and psychrotolerants. Psychrophiles are defined to be microorganisms that have a growth temperature range from 22°C to 0°C or lower, with an optimum growth at about 15°C. On the other hand, psychrotolerants generally do not grow at zero but do at 3-5°C, and have optimum and maximum growth
temperatures above 20°C7,8. For simplicity, in what follows, the term psychrophile is used to cover both groups, unless there are specific and relevant differences. In this study, we revisit and study sporulation, germination, and resistance properties of psychrophilic endospores using an array of new microbiological techniques.
Work with Earth ice has raised the importance to develop proper protocols and method at low temperatures. Nevertheless, most of our understanding on sporulation, germination and resistance of endospores rest on the most-studied B. subtilis and its close mesophilic relatives. In order to study cold biosphere properly, we need to understand more about cold-tolerant endospore-forming bacteria. Towards this goal, the first step is to produce clean psychrophilic endospores in a synchronized and homogeneous way.
Psychrophilic pores are routinely produced on fortified agar or in rich liquid media in previous studies, which results in heterogeneous batches. It has also been well established that bacterial spore properties are affected by the conditions during sporulation. Metal and lipid contents in particular are affected greatly by the preparatory method. Therefore, a novel sporulation protocol for psychrophilic spores have been developed that is of great importance for obtaining reproducible and homogeneous endospore batches for a thorough characterization of germination and other properties.
Considerable differences have been shown to exist between laboratory strain and environmental mesophilic endospores, including B. subtilis and B. atrophaeus, which are typically used as model strains in all types of spore work. Prolonged cultivation of bacteria in laboratory conditions can cause changes in gene expression patterns and loss of key properties for survival in the natural environment9-11. It is of prime importance to know the variability of key spore properties such as germination and heat resistance in
the natural psychrophilic population. Knowing the boundary conditions for heat tolerance in the environment can help determine an optimal heat shock temperature for screening endospores from vegetative cells. Gould et al. have investigated the germination behavior of crude spores isolated directly from soil and reported that a significant proportion of the spores did not respond to any germinative compound12. Response to various germinants is crucial to the application of endospore viability assays.
7.2.2 Methods
7.2.2.1 Bacterial strains and growth conditions
Bacillus longisporus (DSM 477) and Bacillus psychrodurans (DSM 11713) were obtained from the German Collection of Microorganisms and Cell Cultures. All solutions and agar media were pre-chilled to 4°C and held at this temperature on a cold tray during the whole inoculation procedure. Both of them were growth in nutrient broth incubated at 20°C in a shaking bath at 120 rev min-1.
Environmental strains were isolated from JPL soils and Lake Vida from Antarctica using the single cell technique13. JPL subsurface soil samples were collected aseptically in sterile jars and Lake Vida brine sample was received as a gift. Soil and mud samples were diluted 1:10 in sterile 0.1% peptone and heated at 80°C for 15 min to inactivate vegetative cells. Lake Vida samples were heated without prior dilution. A 100- µl amount of the soil extract was inoculated onto tryptic soy agar (TSA) (Difco, Sparks, MD). 5-mL of the brine sample was inoculated into 25 ml of a medium consisting of 12.5 g of NaCl, 2 g of K2HPO4, 1.25 g of KH2PO4, 0.5 g of MgSO4 · 7H2O, 0.125 g of CaCl2 · 2H2O, 0.05 g of MnSO4 · H2O, 2.5 g KNO3 and 2.5 g of yeast extract in 1 liter of water.
0.2-µm filter sterilized Wolfe’s mineral (0.5%) and vitamin (0.5%) solutions (ATCC) were added aseptically to the sterile medium at 60°C. The basal medium was made of the 1/5 of the autoclaved lake sample.
The samples were incubated for 2 to 4 weeks at 7°C. The cells harvested on agar plates and the broth were diluted accordingly and streaked on respective solid medium and incubated at 7°C for 2 weeks. Isolated colonies were restreaked several times to insure purity and were transferred to slants of agar for long-term storage. The strain identities were confirmed by analyzing the first 500 base pairs of the 16S ribosomal DNA gene.
7.2.2.2 Sporulation and endospore purification
Mesophilic endospores were prepared by using 2×SG sporulation agar because it is reported to give high spore densities14. All ingredients were obtained from Sigma unless otherwise noted. The medium consisted of: 16.0 g nutrient broth (Difco), 2.0 g KCl, 0.5 g MgSO4 and 17.0 g agar in 1 litre of water. The medium pH was adjusted to 7, then autoclaved and cooled. The following components were added after cooling: 1.0 mL 1 M Ca(NO3)2, 1.0 mL 0.1 M MnCl2 · 4H2O, 1.0 mL 1 mM FeSO4 and 2.0 ml 0.5% glucose (w/v). 200 µL of fresh Bacillus vegetative cells in tryptic soy broth in exponential phase were added and spread over the agar surface. The plates were incubated at 30°C for 1 week. Spores were harvested using cotton applicators and suspended in sterile water. This mixture of spores and cells was centrifuged at 1300 g for 20 min, washed with sterile water 3 times. Lysozyme (50 µg mL-1) was added in the presence of buffer (¼ volume TrisCl, 0.05 M, pH 7.2), and incubated with constant stirring at 37°C overnight.
Lysozyme was removed by centrifuging 8 times (at 1,300 g) and washing with sterile deionized water. The resultant spore suspension was stored at 4°C.
B. longisporus and B. psychrodurans and spore-forming isolates from JPL soil and Lake Vida were all sporulated using the same way except that the sporulation media for Lake Vida was supplemented with sea water broth15 to mimic the natural environments. The sporulation media was composed of nutrient broth (Difco) with 0.5%
yeast extract, 0.1% glucose, 0.7 mM CaCl2 · 2H2O, 1mM MgCl2 · 6H2O and 50 mM MnCl2 · 4H2O, supplemented with 1 mM methyl anthranilate and 1 mM D-alanine16, at final pH of 7.0. Media for Lake Vida was further fortified with 1.5 g Bacto Peptone, 0.01 g FePO4 · H2O, 750 ml sea water and 250 ml distilled water, adjusted to pH 7.0. Actively growing culture, consisting of vegetative cells and endospores, was heat treated at 65°C for 15 minutes and transferred to fresh medium several times17 and finally inoculated into sporulation media augmented with an airlift system. The sporulation media was added to a 250-mL vacuum filtration flask fitted with a fritted glass gas silicone dispersion tube, two outlets, and a magnetic stir bar. The tube was connected to a fish tank pump through a 0.2-µm membrane filter. Sterile air was bubbled through the medium at a constant rate with an aspirator. The degree of sporulation was determined by direct microscopic counts.
After incubation of 2 weeks at room temperature, 70% of the biological particles present were free “phase-bright” endospores.
After two washing steps and following resuspension in PBS, samples were heat shocked at 65°C and sonicated for 5 min to damage vegetative cells. The suspension was then incubated in lysozyme (50 mg mL-1) at 37°C overnight to lyse the vegetative cells.
The lysozome-treated suspension was put on top of an sodium bromide density gradient
and centrifuged at 5000 g (4°C) for 45 min. This gradient allowed the formation of successive stages of NaBr density with the addition of density beads: 1.5, 1.4, 1.3, 1.2, 1.1 and 1.0 g mL-1.18 The endospores formed a solid pellet with a light crème color at 1.33 g mL-1 density level, where debris and vegetative cells were found at the 1.1 and 1.2 g mL-1 levels, respectively, as thin, loosely packed, dark brownish upper layers. The isolated endospores were washed 10 times in 1 mL sterile water to get rid of any remaining lysozyme. More than 99% of the samples were composed of free ‘phase-bright’
endospores. The suspension was either kept at 4°C with the addition of 50 mM D-alanine to avoid germination or freeze-dried and stored as lyophilized powder at -20°C under the dark. The usual mesophilic endospore storage condition in deionized water at 4°C was not sufficient because psychrophilic endospores manage to germinate at a considerable rate at this temperature.
7.2.2.3 Growth characteristics of strains
Optimal and maximal growth temperatures of the endospores were determined at 4, 7, 15, 20, 30 and 37°C in TSB inoculated in 96-well microtiter plates. Growth was measured by monitoring optical density at 660 nm with a spectrophotometer (NanoDrop) after 2, 7 and 14 days of incubation and turbidity increases of >0.1 OD unit were scores as positive.
7.2.2.4 Characterization procedure
Concentration of bacterial endospores was determined by turbidity measurement and phase-contrast microscopy under a 60× oil immersion objective (Nikon Eclipse 80i, AG Heinze Co., Lake Forest, CA). Size and shape of endospores were determined from
malachite green staining. The Schaeffer-Fulton modification of Wirtz’s spore stain was used19. DPA was measured with a fluorescent assay modified from Shafaat and Ponce20. Endospore suspension were autoclaved (134°C for 45 min), a treatment known to release all DPA21, and pelleted at maximum speed for 10 minutes. The supernatant was assayed for DPA in a final volume of 800 µL containing 100 µL of supernatant and 700µL sodium acetate buffer (20 mM at pH 5.6) with freshly added TbCl3 at a final concentration of 100 µM. Excitation spectra (λex = 250 to 360 nm; λem = 544 nm) and emission spectra (λex = 270 nm; λem = 515 to 580 nm) were measured with a Fluorolog-3 fluorimeter (Jobin Yvon, NJ) consisting of a 450-W xenon lamp for excitation, two double-monochromators set with 3 nm bandpass filter, and a Pelletier-cooled photomultiplier tube. A 500 nm cutoff filter (Omega Filters, Brattleboro, VT) was placed at the entrance of the emission monochromator. Standard curves of 0-100 µM DPA demonstrated straight lines (R2> 0.99) with this method. Endospores were induced to germinate by a combination of L-alanine, inosine and sodium bicarbonate quantified by the described fluorimetric analysis and OD660 measurement.
The heat resistance of microbes was determined by subjecting a 100-µL mixture of 107 endospores and 107 vegetative cells in PCR tubes to controlled thermal cycles using a PCR machine. The bacterial suspension was kept at 4°C before and after the heat treatment. The temperature was ramped up quickly with 1 minute to the following temperatures: 50, 60, 70, 75, 80, 85, 90, and 95°C. A replicate of 8 tubes were used for each heat inactivation temperature. Heat treated bacterial suspension were pipetted into 96-well microtiter plates and incubated at the approximate temperature with mild agitation until visible turbidity was observed.
Spore surface hydrophobicity was measured according to the method described by Rosenberg et al.22 Spores were suspended in water, and after measurement of the absorbance at 660 nm using a Nanodrop (Abefore, values at 0.4-0.5), 20 µL of n- hexadecane (Sigma Aldrich) was added to 100 µL of spore suspension in a centrifuge tube. This mixture was vortexed for 1 min, after which the phases were allowed to separate for 15 min. Then, the absorbance of the aqueous phase was determined again (Aafter), and the % transfer to the n-hexadecane was deduced by calculating 100 – (Aafter/Abefore) × 100.
The wet density of the whole endospore was measured with a Percoll gradient according to the method described by Tisa et al.23 Spores were concentrated in 0.15 M NaCl, added to 90% (v/v) Percoll (Amersham Pharmacia Biotech) solution with 0.15 M NaCl, and subsequently centrifuged for 40 min at 10,000 g. While spore wet density was derived by comparison of the positions of the spores with those of density marker beads (Amersham Pharmacia Biotech) in the self-established gradient.
The spore core density was determined with Nycodenz density gradients24 according to Lindsay et al.25 as follows: spores were permeabilized with a decoating solution containing 0.1 M DTT, 0.1 M NaCl and 0.5% SDS according to Vary (1973)26. Permeabilized spores were equilibrated in 30% Nycodenz (1.159 g mL-1). Semi- continuous gradients were made by letting discontinuous gradients diffuse overnight at 5°C. After centrifugation for 45 min at 3700 rpm, the spores formed a band at a certain position. The refractive index, which was linearly correlated with the density, from samples taken just above and below the band was measured with a spectrophotometer, and the average yielded the spore core density. Core water content was calculated
according to Lindsay et al.25.
7.2.3 Results
Two psychrophilic isolates of Bacillus were successfully isolated and sporulated from JPL soil; 4 were isolated and produced as clean endospore suspensions from Lake Vida lake brine water. They grew well at 4°C, optimally at 20°C, and failed to grow at temperatures higher than 30°C. Their minimal and maximal growth temperatures were lower than those for mesophilic species of Bacillus by 10°C. Both sporulation and germination were observed to occur at lower temperatures than their mesophilic counterparts. The Lake Vida grew optimally in media supplemented with sea water broth.
One of the Lake Vida isolates contained two endospores in a single mother cell.
Sporulation was observed via the typical seven steps27. DPA can be detected and the degree of heat and UV resistance were comparable to other psychrophilic endospores.
Micro-cycle sporulation and germination events were observed in the sporulation medium, resulting in a heterogeneous mixture of vegetative, sporulating and sporulated cells. Endospores were observed to transition from phase bright to phase dark constantly in the sporulation medium. To remedy this situation, 1 mM methyl anthranilate and 1 mM D-alanine were added into the sporulation medium to inhibit the action of L-alanine- α-ketoglutarate aminotransferase28, thereby preventing endospores from going through the germination pathway. A more synchronous and higher percentage of sporulation rate (~70%) could be obtained afterwards. Aerated sporulation broth fortified with yeast extract, glucose, and D-alanine was found to be the best sporulation medium for psychrophilic endospore-forming bacteria. With further cleaning and purification, final
pure psychrotolerant endospore suspensions of >95% were produced. Table 7.1 summarizes the morphological and physiological characterization of the spore samples.
Figure 7.1 shows the phase contrast micrograph of some of the isolates.
The optimal germinant for mesophilic endospores was 50 mM L-alanine, whereas the optimal germinant for psychrophilic endospores was 50 mM L-alanine and 1 mM inosine. The optimal combination of germinants was studied on the psychrophilic endospores. The germination rate of environmental psychrophilic endospores was very low. Upon heat activation at 50°C and pre-incubation at 50 mM calcium dipicolinate, a ten-told increase in germination rate was observed among environmental psychrophilic endospores at 22°C. Figure 7.2 shows a comparison between the optimal germination temperature between mesophilic and psychrophilic endospores when induced by 50 mM L-alanine and 50 mM L-alanine plus 1 mM inosine. Mesophilic endospores germinated fastest and optimally at 37°C and psychrophilic endospores generally germinated best between 15°C and 22°C. Germination still proceeded at a slow but measurable rate at 4°C.
However, the lowest possible germination for mesophilic endospores was 12°C.
The inhibitory effect of sodium bicarbonate on endospore germination was concentration dependent. The higher concentration of sodium bicarbonate, the slower the rate of germination. For instance, 1 mM sodium bicarbonate reduced L-alanine germination rate of B. atrophaeus and B. longisporus endospores by 42% and 46%, respectively. The effect of sodium bicarbonate on mesophilic and psychrophilic endospores was statistically insignificant (p < 0.001). The effect of the cationic surfactant, dodecylamine, in inducing endospore germination was also studied. 1 mM dodecylamine was used as the germinant without any buffer29,30. The highest rate of germination rate of
mesophilic endospores was 65°C and of psychrophilic endospores was 45°C.
Psychrophilic endospores were found to have a higher concentration of DPA per unit volume of endospores than mesophilic endospores. The endospore size of psychrophilic also tended to be smaller than that of mesophilic endospores. Vegetative cells and endospores were subjected to heat shock treatment at various temperatures.
Optimal heat shock temperature was chosen at the temperature at which most of the vegetative cells were inactivated while most of the spore population remained. 60°C – 70°C was the range of optimal heat shock temperature for psychrophilic spores (Figures 7.3, 7.4). The UV resistance of psychrophilic endospores is statistically higher than that of mesophilic endospores as manifested by their high pigmented colonies.
Slope of the middle section of the inactivation curves, which yielded a reproducible D-value of 1 minute at 85°C, in line with previous reports. Spore surface hydrophobicity is caused by the presence of an exosporium31 and plays a key role in adherence of the spores to surfaces32, for example of food or food processing equipment.
The obtained endospores were highly hydrophobic since over 95% of the endospores transferred from water to the n-hexadecane phase in the BATH assay22. In contrast, of B.
cereus vegetative cells and spores from B. subtilis, which lack an exosporium, over 98%
remained in the aqueous phase.
When determining the DPA content using spectroEVA, the spectrum tended to overshadow the characteristic dual peak of Tb-DPA because of the very background fluorescence and autofluorescence upon deep UV excitation. A phosphorimeter was used to performing time gating using the fluorimeter. Promising initial study has revealed a much better resolved spectrum.
7.2.4 Discussion
In this study, we have isolated a combination of psychrophilic and psychrotolerant endospore-forming bacteria from permanently low temperature environments, such as the Antarctic Lake Vida, from cold desert, such as the Atacama Desert in Chile, and in soil and fresh water in California. Although psychrophilic endospores are more likely to be isolated from the cold biosphere, they can often be found everywhere because of their ubiquity33. Endospores are exemplified by their extreme resistance, and the ability of psychrophilic endospore-forming bacteria to thrive at low temperatures harbors a wider range of survival characteristics (e.g., ability to withstand cold shock, desiccation, pH, salinity, cold shock.) Study of psychrophilic endospores is often hampered by the difficulty in obtaining pure spore suspension because of the extra challenge encountered during the sporulation process. In Bacillus the degree of synchrony during sporulation is usually relatively high among mesophilic species. Sporulation can occur in suitable media, 6 to 8 hours after development has started at the end of exponential growth34,35. The entire sporulation process takes 3–5 days to complete. Their psychrophilic counterparts, however, take approximately 2 weeks. Some sporulate faster and some sporulate slower. During this extended period, some of the endospores undergo germination and subsequently microcycle sporogenesis when germinating spores commit and reenter the sporulation cycle in the presence of utilizable nutrients36. Germination and microcycle sporulation are prevalent during sporulation of psychrophilic endospores as indicated by recurrent phase bright and phase dark transition due to the prolonged