1
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Fluorescent fingerprints of endolithic phototrophic cyanobacteria living within 2
halite rocks in the Atacama Desert 3
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Running Title: Fluorescence from the Atacama Desert cyanobacteria 5
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Roldán M.1#, Ascaso C.2, Wierzchos J.2 7
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1Servei de Microscòpia, Universitat Autònoma de Barcelona, Bellaterra 08193 9
Barcelona, Spain 10
2 Museo Nacional de Ciencias Naturales, CSIC, C/ Serrano 115 dpdo. 28006 Madrid 11
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#Corresponding author: 13
Mònica Roldán 14
Servei de Microscòpia 15
Edifici C, Facultat de Ciències 16
Universitat Autònoma de Barcelona 17
Phone: (34) 93 5811516 18
Fax: (34) 93 5812090 19
Email: [email protected] 20
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AEM Accepts, published online ahead of print on 7 March 2014Appl. Environ. Microbiol. doi:10.1128/AEM.03428-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 27
Halite deposits from the hyperarid zone of the Atacama Desert reveal the presence of 28
endolithic microbial colonization dominated by cyanobacteria associated with 29
heterotrophic bacteria and archaea. Using the lambda-scan (λ-scan) confocal laser 30
scanning microscopy (CLSM) option, this study examines the autofluorescence 31
emission spectra produced by single cyanobacterial cells found inside halite rocks and 32
by their photosynthetic pigments. Photosynthetic pigments could be identified 33
according to the shape of the emission spectra and wavelengths of fluorescence peaks. 34
According to their fluorescence fingerprints, three groups of cyanobacterial cells were 35
identified within this natural extreme microhabitat: (i) cells producing a single 36
fluorescence peak corresponding to the emission range of phycobiliproteins and 37
chlorophyll a, (ii) cells producing two fluorescence peaks within the red and green 38
signal ranges, and (iii) cells only emitting low intensity fluorescence within the 39
unspecific green fluorescence signal range. Photosynthetic pigment fingerprints 40
emerged as indicators of the preservation state or viability of the cells. These 41
observations were supported by a cell plasma membrane integrity test based on SYTOX 42
Green DNA staining and by transmission electron microscopy ultrastructural 43
observations of cyanobacterial cells. 44
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46
3
Introduction 47
The Atacama Desert in northern Chile is the driest place on Earth. Its most arid regions 48
lie between the Andes rain shadow and the Coastal Cordillera (1). Until recently, this 49
hyperarid zone, or central depression, of the Atacama Desert, was considered the dry 50
limit for photosynthetic activity (2) and primary production. Indeed, only extremely low 51
concentrations of microorganisms had been detected in the soils of this zone (3). 52
However, despite the major constraint for microbial life in the desert being the scarcity 53
of liquid water, an abundance of photosynthetic life, mainly cyanobacteria associated 54
with heterotrophic bacteria and archaea, has recently been detected in the Atacama’s 55
hyperarid zone (4, 5, 6). This remarkable discovery was made inside the halite deposits 56
that form part of the Neogene salt-encrusted playas of Atacama, also known as salares 57
(7). Chroococcidiopsis-like cells were the only cyanobacteria found inside halite 58
pinnacles, and phylogenetic studies revealed their close genetic affinity to the genus 59
Halothece (5, 6). The presence of this endolithic community indicates that life has 60
found a survival strategy in the hyper-arid zone of Atacama, where all other 61
colonization strategies have failed. These cells would seem to be biologically adapted to 62
conditions of high salinity, and their presence indicates a water source other than the 63
area’s practically non-existent rainfall. Davila et al. (8) showed that water vapour 64
condenses within the halite pinnacles at relative humidity (RH) levels that correspond to 65
the deliquescence point of NaCl (RH=75%). More recently, it was shown that halite 66
endoliths could obtain liquid water through spontaneous capillary condensation at a 67
relative humidity much lower than the deliquescence RH of NaCl (9). 68
All desert microorganisms undergo extended periods in a desiccated, metabolically 69
inactive state, in which individual cells are subjected to a variety of chemical and 70
physiological stresses. These stresses lead to a damage that cannot be repaired until 71
4
metabolism restarts (10, 11). Besides desiccation, microbial communities inside halite 72
crusts have to deal with conditions of extreme salinity (5, 6) and excess light, which has 73
a direct effect on their physiology. In response to varying light conditions, 74
photosynthetic microorganisms undergo structural, behavioural, physiological and 75
chemical modifications (12, 13) such as changing the quality and concentration of their 76
light-harvesting pigments (12, 14, 15), and pigment degradation (11, 16, 17). This 77
suggests that the state of photosynthetic pigments can be an indicator of cell viability. 78
The physiology of photosynthetic microorganisms can be investigated by examining 79
their photosynthetic capacity. Because photosynthesis is such a rapid process and the 80
fact that it can only be indirectly inferred from measurements of related variables (e.g. 81
oxygen and/or carbon dioxide), microscopy techniques used in multidisciplinary studies 82
have emerged as powerful tools for the in vivo quantification of photosynthesis. 83
Although these techniques are all non-invasive, each method has technical, practical and 84
physiological advantages and limitations to be considered (18, 19). Thus, some 85
microscopy procedures can be used to detect physiological and biochemical changes in 86
photosynthetic microorganisms allowing for the detection of fluorescence properties of 87
photosynthetic pigments (17). Significant progress is constantly being made in detectors 88
and computer technology, image analysis, visualization, laser sources and optical 89
technologies. These developments have allowed the non-invasive detection of 90
photosynthetic pigment changes occurring in microorganisms in their natural 91
environment (18, 20). 92
The present study was designed to detect autofluorescence emission spectra emitted by 93
both single cells and the photosynthetic pigments of cyanobacteria living within halite 94
rocks in the Atacama Desert. The fluorescent fingerprints obtained were used to identify 95
5
cyanobacterial photosynthetic pigments and as a measure of cyanobacterial cell 96
viability. 97
98
Materials and Methods 99
Samples and study area 100
This study compares the microbial colonization of halites from two different areas of 101
the Atacama Desert (N Chile): Yungay (24º 05’ 53’’S; 069º 55’ 59’’W) and Salar 102
Grande (21º 08’54’’S; 070º 01’04’’W). Although both sampling areas occur in the 103
hyperarid area of the Atacama Desert, their environmental conditions vary slightly (see 104
Table 1). The Yungay area shows a mean annual precipitation below 2 mm yr-1 and is 105
considered the driest place on Earth (21). The area is 60 km from the coast and lies at an 106
altitude of 962 m between the Coastal Cordillera to the west (1000 - 3000 m high) and 107
the Domeyko Mountains to the east (about 4000 m high). The other sampling site (Salar 108
Grande) lies 300 km north of Yungay. However, its location close to the Pacific coast (8 109
km) and its lower altitude results in this region often experiencing the arrival of moist 110
air and fog locally known as “camanchaca” (6, 22, 23). 111
In both areas, the halite crust occurs as pinnacles that show characteristic irregular 112
shapes formed by wind action and partial long-term dissolution and reprecipitation of 113
evaporite deposits. At both sampling sites, air temperature (T) and relative humidity 114
(RH) were collected over a four-year period (May 2008-2012) using data loggers 115
(Onset, HOBO Pro v2) as described in Wierzchos et al. (9). These RH/T sensors were 116
placed close to the halite pinnacles 20 cm above the soil surface in the shade. Thus, the 117
temperature recorded is a function of the air temperature and heat radiation from the 118
nearby halite crust and soil. 119
6
According to detailed analyses of RH/T, photosynthetic active radiation and electrical 120
conductivity sensor readings, as well as personal information obtained by two 121
permanent workers at a remote water pump station 2 km from Yungay area, there was 122
no rainfall at the sampling sites from 2007 until May 2012. The RH/T data for the 123
sampling sites are provided in Table 1. 124
The halite samples used for the present study were collected in January 2010 and May 125
2011 in expeditions to the sites mentioned above. Samples were stored dry in the dark at 126
room temperature for no longer than one month until their preparation for the different 127
microscopy techniques. The day before preparation they were left in a chamber in 128
day/night light conditions at 75% RH to allow deliquescence and the adsorption of 129
water by microbial cells. 130
131
Transmission electron microscopy (TEM) 132
Representative colonized halite layers were dissolved in 20% aqueous NaCl solution 133
and made up to a NaCl concentration of 5M. After a short period (5 min) of 134
precipitation of scarce mineral particles, the supernatant was centrifuged at 12,000 g for 135
10 min. The precipitated microbial cells were fixed according to the protocol described 136
by de los Ríos and Ascaso (25) with some modifications. In brief, these precipitates 137
containing microbial cells were fixed in 3% glutaraldehyde in 5M NaCl at room 138
temperature for 3 hours and then in 1 % osmium tetroxide, dehydrated in a graded series 139
of ethanol, and embedded in Spurr's resin. Poststained ultrathin sections were observed 140
on a Zeiss EM910 transmission electron microscope equipped with a Gatan CCD 141
camera. 142
143
Light, fluorescence and confocal microscopy 144
7
Halite samples taken from 3-5 mm below the crust surface containing pigmented 145
microbial communities were scraped and dissolved in a 20% NaCl aqueous solution and 146
made up to a NaCl concentration of 5M. Following a short period (5 min) of 147
precipitation of scarce mineral particles, the supernatant was centrifuged at 12,000 g for 148
10 min. Pellets of microorganisms were resuspensed in 20 μL of 5M NaCl and cells 149
were visualized by bright field DIC light microscopy using an AxioImager D1 Zeiss 150
instrument equipped with a CCD colour camera (AxioCam MRc Zeiss) and Plan-Apo 151
60x/1.4 Zeiss oil immersion objective. These same cell preparations were observed by 152
fluorescence microscopy (FM) using specific filters for eGFP (Zeiss Filter Set 38; 153
Ex/Em: 450-490/500-550 nm) to visualize both the weak green autofluorescence of 154
unidentified substances, and specific intensive fluorescence of the SYTOX Green (S-155
7020, Molecular Probes) dye. Also the rhodamine filter set (Zeiss Filter Set 20; Ex/Em: 156
540-552/567-647 nm) was used to visualize the red autofluorescence of photosynthetic 157
pigments. 158
To detect cyanobacterial cells with damaged membranes, the samples were stained 159
using a specific fluorescence SYTOX Green dye. Some extent of cell membrane 160
damage increases SYTOX Green influx (10). This nucleic acid stain was used according 161
to Wierzchos et al. (24). The original solution containing 5 mM SYTOX Green in 162
anhydrous DMSO was diluted at 1:100 in water and added to the suspension of halite-163
extracted microorganisms. The cells were stained during 10 min at room temperature 164
and after that examined by FM using a specific filter for eGFP (SYTOX Green signal) 165
using Plan-Apo 60x/1.4 and 100x/1.4 Zeiss oil immersion objectives. 166
Autofluorescence (green and red signal) was also visualized using a Leica TCS-SP5 167
confocal laser scanning microscope (Leica Microsystems Heidelberg GmbH, 168
Mannheim, Germany), and a x63 (1.4 NA) Plan Apochromat oil immersion objective. 169
8
Red autofluorescence was viewed in the red channel (640-785 nm emission) using a 561 170
nm laser diode and green autofluorescence was observed in the green channel (495-560 171
nm emission) using a 488 nm line from an Ar laser respectively. The samples were 172
mounted on Mat-Teck culture dishes (Mat Teck Corp., Ashland, Massachusetts, United 173
States). Optical sections were acquired in x-y planes every 0.3 m along the optical axis 174
with 1 Airy confocal pinhole. Different projections were generated by the Leica LAS 175
AF software and the Imaris software package, version 2.7 (Bitplane AG Zürich, 176
Switzerland) for 3D reconstructions of cell aggregates. 177
Autofluorescence intensity was determined as an indicator of the integrity of the 178
photosynthetic apparatus as described by Billi et al. (10). 179
The emission spectra of cyanobacterial pigments were obtained using a wavelength λ-180
scan function of the confocal laser scanning microscopy (CLSM) based on a 181
fluorescence method that determines the complete spectral distribution of the 182
fluorescence signals emitted (20). Images were acquired with the same CLSM and 183
objective. Series of images (xy ) were taken to determine the emission spectra of the 184
samples and to establish peaks. Photosynthetic pigments and other unknown 185
autofluorescent molecules were excited with a 488 mm line of an argon laser. 186
Fluorescence emission was captured in 10 nm bandwidth increments (lambda step size 187
= 5 nm) in the range 495 nm to 780 nm. A Region of Interest (ROI) in the thylakoid 188
area was defined to determine mean fluorescence intensity (MFI) in relation to the 189
emission wavelength. A set of 20 ROIs of 1 m2 was used to analyse the mean 190
fluorescence intensity and peak emission range of the samples. Fluorescence 191
measurements were expressed in arbitrary units (a.u.). 192
193
194
9
Results 195
Cyanobacterial cell ultrastructure 196
Microbial communities inhabiting halite rocks in the Atacama Desert are mainly 197
comprised of one cyanobacterial morphotype accompanied by heterotrophic bacteria 198
and archaea (5, 6). The TEM images in Figure 1 show the different types of aggregate 199
containing cyanobacterial cells observed. Some of the round-shaped multicellular 200
aggregates were composed of several (Fig. 1a and b) cyanobacterial cells. Other 201
aggregates showed a linear organization of phototrophic cells (Fig. 1c). The in situ 202
visualization of this cryptoendolithic microbial ecosystem using LT-SEM, as well as 203
FM and CLSM, suggests that the given aggregate type often depends on the shape of 204
the pore spaces among the halite crystals occupied by microorganisms. Cells within the 205
aggregates divide by binary fission to give rise to polygon-shaped structures consisting 206
of cyanobacterial cells embedded within an extracellular polymeric substance (EPS). In 207
the TEM images, this can be seen as a nano-porous network surrounding the 208
cyanobacterial cytoplasm (asterisks in Fig. 1). Cyanobacterial aggregates appear 209
enveloped by an electron-dense fibrous outer layer and, in some cases (Fig. 1c), the 210
cytoplasm of single cyanobacterial cells is also enveloped by this electron-dense fibrous 211
layer (open arrows in Fig. 1). In many of the cells observed by TEM, the well-212
developed thylakoid system showed parallel membranes in interthylakoid spaces (e.g. 213
white arrows in Fig. 1b). However, some cells revealed signs of senescence at the 214
ultrastructural level (black arrows in Fig. 1). In these cyanobacterial cells, the thylakoid 215
structure becomes disorganized and/or deteriorated clearly indicating the degradation of 216
cell structure, which will likely disrupt photosynthetic activity and metabolism. Albeit 217
indirectly, senescence is by far the leading cause of cell viability loss. Note that these 218
senescent cells can be found within the same aggregates where cells with undisturbed 219
10
ultrastructural elements, or potentially viable cells, are also present. The space around 220
the cyanobacterial aggregates was filled with remains of microbial cells (Fig. 1b). The 221
cells of heterotrophic bacteria and archaea were frequently observed as attached to the 222
outer sheath of cyanobacterial aggregates (Fig. 1a) or, in some cases, isolated bacterial 223
cells were found just beneath the outer layer of the cyanobacterial aggregates (black 224
arrowhead in Fig. 1c). 225
226
Red and green autofluorescence from cyanobacterial cells vs. membrane integrity 227
When examined by FM, the cyanobacterial cells forming the multicellular aggregates 228
showed two distinct ranges of autofluorescence emission signal. Some of the cells 229
emitted autofluorescence in the red signal range (detected using the rhodamine filter 230
set), which corresponds to photosynthetic pigment autofluorescence (PAF). Other cells 231
emitted less intense unspecific broad-spectrum autofluorescence in the green signal 232
range (GAF), detected using the eGFP filter set. Both kinds of emission signal were 233
observed even among cyanobacterial cells within the same aggregate, as shown in 234
Figure 2a and 2b. The GAF signal observed suggests that some of the cyanobacterial 235
cells contained degraded photosynthetic pigments. In GAF signal-emitting cells, 236
autofluorescence was generally distributed evenly across the cell cytoplasm. However, 237
the PAF signal showed a heterogeneous pattern in the cell cytoplasm, supposedly 238
reflecting the position of cyanobacterial thylakoids (data not shown). 239
The SYTOX Green assay revealed the different viability states of cyanobacteria from 240
the Yungay halites. This method identified cyanobacterial cells within an aggregate with 241
damaged plasma membranes. These cells showed a SYTOX Green (green signal) 242
labelled DNA structure, indicating a loose membrane architecture (white arrows in Fig. 243
2 e, f, i and j). This signal was detected using the eGFP filter set and was much more 244
11
intense compared to the weak GAF autofluorescence. Low intensity or null red 245
autofluorescence was also observed within the cells positive for SYTOX Green. In 246
contrast, cyanobacteria cells emitting a high intensity PAF signal showed no SYTOX 247
Green labelling (black arrows in Fig. 2 d, f, h and j). 248
The same observations were made when SYTOX Green was used on the endolithic 249
microbial community from the Salar Grande halite. Figure 3 shows cyanobacterial 250
aggregates and associated heterotrophic bacteria and archaea in DIC images after 251
SYTOX Green staining. This time, we were able to distinguish three types of aggregate 252
containing: cyanobacterial cells showing an intense PAF signal and a negative SYTOX 253
Green signal (aggregates with a blue dotted outline in Fig. 3b), cyanobacterial cells 254
showing distinct PAF and SYTOX Green signals (aggregates with a yellow dotted 255
outline in Fig. 3b), or cyanobacterial cells showing a weak GAF signal yet distinct 256
SYTOX Green signal (aggregates with a white dotted outline in Fig. 3b). We were also 257
able to observe SYTOX Green signals from dead bacteria and/or archaea outside the 258
aggregates (white arrows in Fig. 3 b). 259
260
Fluorescence emission spectra of phototrophic cyanobacteria cells 261
The lambda-scan confocal microscopy option records a series of individual images 262
obtained using a defined emission fluorescence wavelength range. This procedure has 263
been successfully used to assess the physiological state of photosynthetic 264
microorganisms at the single-cell level (11, 17). We used the CLSM lambda-scan 265
feature to characterize the fluorescence emitted by cyanobacterial photosynthetic 266
pigments. When excited at a wavelength of 488 nm, cyanobacterial cells isolated from 267
the halite samples showed three types of emission: some emitted weak fluorescence 268
12
within the green range, some within the red range, and others in both the red and green 269
range (Figs. 4-6). 270
Some emission spectra produced by cyanobacterial cells isolated from halites from both 271
Yungay and Salar Grande featured a wide curve with a low maximum intensity at ca. 272
560 nm (Table 2 and Figs. 4a and 5a). These spectra were recorded in cells showing a 273
GAF type emission that was confirmed by simultaneous (CLSM and FM) visualization. 274
In addition, many of the cyanobacterial cells from both locations exhibited a distinct 275
emission peak between 657.6 - 662.7 nm (Table 2 and Figs. 4b and 5b). We consider 276
that this high intensity emission peak is produced by overlapping of the spectra of 277
phycobiliprotein photosynthetic pigments whose characteristic emission peaks 278
correspond to phycocyanin (PC) and allophycocyanin (APC) (20). Moreover, our 279
spectra show an asymmetric slope with a small shoulder at ca. 680 nm, which could be 280
the outcome of overlapping of the characteristic emission spectra of phycobiliproteins 281
and chlorophyll a (Chl a) (20). These spectra were recorded in cells showing a PAF- 282
type emission, as confirmed by simultaneous CLSM and FM visualization. 283
Some cyanobacterial cells isolated from the Yungay halite showed an emission 284
spectrum with two broad low intensity peaks (Table 2 and Fig. 6). These peaks were 285
recorded in cells showing both GAF- and PAF- type emission as confirmed by 286
simultaneous CLSM and FM visualization. 287
Mean fluorescence intensity (MFI) and the half-band width of the spectra differed for 288
the GAF, PAF and GAF+PAF emission spectra patterns. Generally speaking, MFI 289
values were higher for the PAF- than GAF- type spectra though this difference was 290
more evident for cyanobacteria cells isolated from the Salar Grande halite. The 291
bandwidths of the two types of emission spectra also differed, being wider for the green 292
emission than the red. 293
13
Collectively, our TEM, FM and CLSM, SYTOX Green assay and lambda-scan data 294
indicate that: 295
i. Some cyanobacterial cells emitted red fluorescence (PAF) derived only from 296
photosynthetic pigments and were negative for the SYTOX Green stain. These cells 297
showed well-organized parallel thylakoids and preserved their ultrastructural integrity 298
as observed by TEM (Fig. 1a). The emission spectra likely corresponding to these cells 299
showed a peak in the range characteristic for phycobiliproteins and chlorophyll a (Figs. 300
2 f, j - black arrows; Fig. 3 – blue outline; Figs. 4b and 5b). These cells may be classed 301
as intact and healthy. 302
ii. Other cyanobacterial cells were stained with SYTOX Green, but still showed weak 303
photosynthetic pigment fluorescence (PAF) (Figs. 2 f and j - white arrows; Fig. 3b -304
yellow outline). These cells probably gave rise to the emission spectra showing two low 305
intensity peaks (Fig 6). Although these cells emit fluorescence attributable to 306
photosynthetic pigments, their cell integrity has been lost (SYTOX Green penetrates the 307
cell) such that they are not vital. 308
iii. Finally, yet other cyanobacterial cells were stained with SYTOX Green and only 309
emitted an unspecific green autofluorescence signal (GAF). These cells generated 310
emission spectra with a peak in the green region (Fig. 3 - white outline; Figs. 4a and 311
5a). These cells could correspond to the cells showing extensive thylakoid 312
disorganization as observed by TEM. We interpret these cells as non viable. 313
314
Discussion 315
In extreme environments, photosynthetic microorganisms develop different strategies to 316
survive the more hostile time intervals (26). For endoliths found within halite pinnacles 317
14
in the Atacama Desert, intensive sun light radiation, salinity and a lack of water (9) are 318
the most important factors threatening their survival. 319
320
Cyanobacterial cell viability 321
As the dominant group of photosynthetic halite colonizers, cyanobacteria appeared in 322
different physiological/viability states within a single aggregate. Knowing the viability 323
of photosynthetic organisms in their natural environment is essential to understand the 324
ecology of extreme-environment microbial ecosystems (27, 28, 29, 30). Knowledge in 325
this area is still far from complete. 326
The viability of photosynthetic organisms has been so far examined in different ways. 327
In the microbial endolithic ecosystem examined here, prior CLSM/TEM studies 328
confirmed the presence of viable cells (4) and characterized the ultrastructure and the 329
integrity of cyanobacterial and bacterial cells (5). More recently, quantum yield 330
fluorescence measurements (31) and carbon cycling rates, as indicated by the isotope 331
contents (13C and 14C) of phospholipid fatty acids (PLFA) and glycolipid fatty acids 332
(GLFA) (32), have been reported. Although a universally applicable viability method 333
could be a utopian ideal, there is a clear need to determine how broadly current viability 334
methods can be applied. Some studies have reported different results using the same 335
viability method (33). Culture-independent viability indicators such as those proposed 336
here might not be conclusive, especially in complex extreme micro-environments. Their 337
use to investigate complex populations in natural conditions, however, has been 338
encouraged (34). The SYTOX Green method has been described as broadly applicable 339
to photosynthetic microorganisms (10, 11). Multiparameter techniques have also 340
clarified some questions regarding single cell viability (35). In this study, as a measure 341
15
of cell health we assessed both cell membrane integrity and autofluorescence patterns 342
produced by different emission wavelength ranges. 343
344
PAF and GAF fluorescence 345
Emission spectra in both the red and green emission ranges were observed for the 346
cyanobacteria isolated from halite pinnacles. The bandwidth of these emissions was 347
wider in the green than the red range. This is because red fluorescence is produced by 348
photosynthetic pigments (mostly Chl a and phycobiliproteins) with defined emission 349
peaks (11, 15, 17, 20). In contrast, GAF may be attributable to fluorescence emitted by 350
different molecules with different widely overlapping emission spectra. In 351
photosynthetic microorganisms, photosynthetic pigment autofluorescence is considered 352
an indicator of cell viability (10, 35, 37, 38). A loss of pigment fluorescence 353
(“chlorosis”, 39) has been correlated with decreased enzyme activity and increased 354
membrane permeability (38) and may therefore be a useful indicator of senescence for 355
any species of alga (38). 356
When red autofluorescence fades, a green unspecific fluorescence observable at the 357
same excitation wavelength may appear. Different species of microalgae and higher 358
plants in different physiological states exhibit GAF of varying intensity (40). For algae 359
or/and cyanobacteria, the use of this indicator has only been scarcely explored and the 360
molecules responsible for GAF have not yet been clearly identified (40). Green 361
autofluorescence can be induced by a variety of different molecules such as flavonoids, 362
flavins (e.g. FADH) (41), cinnamic acids, betaxanthine, luciferin compounds (40) and 363
pyridine nucleotides (e.g. NADH) (36, 37). NADH has been often associated with 364
viability under UV excitation (350-360 nm) and may be found within most 365
metabolically active prokaryotic and eukaryotic cells (36). The molecule FADH is a 366
16
likely viability indicator candidate because it fluoresces at the same wavelength as these 367
cyanobacteria (42). Thus, the bright-green autofluorescence of FADH at 530 nm 368
(excited by a 488 nm laser) provides information on the oxidation state and metabolism 369
of both bacteria and eukaryotes (43, 44). 370
A number of different factors affecting the efficiency of energy transfer from PC to Chl 371
a will cause a drop in the fluorescence intensity of the photosynthetic pigments in each 372
cell (45). Environmental factors, such as low light stress (17), high light stress and 373
nutrient stress, or death of part of the population due to ageing (10, 11, 37) have so far 374
been identified. In general, increased concentrations of chlorophyll oxidation products 375
have been observed in nutrient-depleted cells, but it is likely that specific chlorophyll 376
transformation pathways vary between species (38). In the case of endolithic 377
cyanobacteria living inside halite pinnacles, excessive solar radiation with a high UV 378
fraction is known to produce significant stress. Effectively, in Yungay, these 379
cyanobacteria produce considerable amounts of scytonemin – a UV protective pigment 380
(46, 47). Nitrogen starvation also leads to low levels of photosynthesis during nitrogen 381
limitation (48, 49). Nitrogen stress affects energy transfer from PC and Chla, and 382
therefore their spectral properties under both in vivo and in vitro conditions (45). 383
Another important factor causing photosynthetic pigments damage could be the 384
constant high salinity, long periods of dryness (9) and low RH values inside the halite 385
pinnacles. Recently Davila et al. (31) observed that the PSII of phototrophic 386
cyanobacteria inhabiting halite pinnacles in the Yungay area was inactive below a RH 387
of 60%. However, when the RH increased above 70%, fluorescence appeared within 388
minutes and stabilized at relatively low, but significantly positive values. It is 389
significant that activation of PSII in the halite cyanobacteria did not occur until liquid 390
water was produced through deliquescence (8) and/or water vapour condensed within 391
17
the nano-pores of halite (9). Hence it seems that photosynthesis in cyanobacteria can 392
only be activated in the presence of liquid water (50). All these factors can cause 393
different viability states of the cells. Automortality is closely associated with non-394
viability (51, 52). Non-viable cells are defined as cells that still have an intact cell shape 395
but can no longer grow or divide. The loss of membrane integrity occurs in the later 396
stages of automortality, resulting in the total disintegration of the cell (53, 54). But 397
before this final damage, cells can suffer different forms of injury to the cytoplasm’s 398
ultrastructural elements, as shown here by TEM (Fig. 1). Once this process begins, 399
degradation of the photosynthetic pigments, in particular chlorophyll (55), and finally 400
fragmentation of the genome lead to the final stage of autolysis. Billi et al. (10) were 401
able to correlate DNA fragmentation and loss of red fluorescence in photosynthetic 402
organisms. Our observations do not exclude the possibility that the unspecific 403
autofluorescence in the green region (GAF) observed in some cells is related to the 404
degradation products of chlorophyll, as reported by Zhong Tang & Dobbs (40). GAF 405
could also be the consequence of increased levels of denatured proteins, for example 406
following the rapid degradation of photosynthetic pigments (56). Thus, cells showing 407
the degradation of their photosynthetic pigments will have completely lost their 408
photosynthetic capacity (53, 57). This means the non-viable cells will no longer show 409
photosynthetic pigment fluorescence allowing them to be clearly identified in situ and at 410
the single cell level by means of CLSM λ-scanning, even in communities composed of 411
numerous cells. However, the persistence of phycobiliprotein autofluorescence serves as 412
a survival marker and has been related to genome stability, undamaged plasma 413
membranes and dehydrogenase activity upon rewetting (10). It therefore seems that 414
viable and non-viable phototrophic endolithic cyanobacterial cells can be distinguished 415
according to culture-independent viability indicators and fluorescent fingerprints 416
18
supported by cell plasma membrane integrity testing and cell ultrastructural 417
observations. 418
419
Conclusions 420
The findings of this study indicate that the -scan option of CLSM microscopy can be 421
used to determine the viability of cyanobacteria cells according to the autofluorescence 422
of their photosynthetic pigments (PAF) and to an unspecific green autofluorescence 423
(GAF). Testing is performed in situ without disrupting the spatial integrity of structured 424
microbial communities or denaturing their biomolecules. We propose the use of this 425
method as an efficient tool for in vivo studies designed to address the cell physiology of 426
unculturable endolithic phototrophic microorganisms inhabiting extreme environments. 427
428
Acknowledgments 429
This study was funded by grant CGL2010-16004 from MINECO and grant 430
NNX12AD61G awarded to J.W. by NASA. 431
The authors thank A. Burton for editorial assistance and Mariona Hernández-Mariné 432
(Universitat de Barcelona) for useful comments and suggestions. 433
434
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601
26
Figure Legends: 602
Figure A1. TEM images of cryptoendolithic microorganisms found in halites. (a) 603
Round-shaped multicellular aggregate composed of cyanobacteria and heterotrophic 604
bacteria (and/or archaea) (black arrowheads) adhered to the outer electron-dense multi-605
layered structure (open arrows, also in b and c) enveloping the aggregate. Note the 606
extracellular polymeric substances surrounding cyanobacterial cells (asterisks in Figs. a-607
c). (b) Aggregate with four cyanobacterial cells at different stages of senescence. White 608
arrows point to a cell with well-organized parallel thylakoids; black arrows indicate 609
cells with a high level of thylakoid disorganization; open arrowheads point to the 610
remains of dead microorganisms. (c) Aggregate with linearly organized phototrophic 611
cells, one of which (black arrow) shows a high level of thylakoid disorganization. Scale 612
bar = 2 m. 613
614
Figure A2. Green and red autofluorescence patterns observed for cyanobacterial cell 615
within aggregates and plasma membrane-damaged cells in halite samples from Yungay. 616
(a-b). Photosynthetic pigments emitting in the red (PAF) signal range and unspecific 617
green (GAF) signal range, respectively. (c and g) DIC images of cyanobacterial 618
aggregates. (d-h) Photosynthetic pigment autofluorescence (black arrows indicate an 619
intense PAF signal). (e and i) SYTOX Green signal (white arrows) emitted by cells 620
with damaged plasma membranes; cells with intact membranes are not labelled (black 621
arrows in d, f, h and j). (f and j), merged colour images of d + e, and h + i, respectively. 622
Scale bar = 10 m for all images. 623
624
Figure A3. An endolithic microbial community found in the Salar Grande halites. (a) 625
DIC image (b) Fluorescence microscopy image. Aggregates containing cyanobacteria 626
27
showing an intense photosynthetic pigment autofluorescence (PAF) signal (blue dotted 627
outline); aggregates containing cyanobacteria showing both a PAF and a SYTOX Green 628
signal (yellow-dotted outline); aggregates containing cyanobacteria showing a weak 629
GAF signal, but strong SYTOX Green signal (white-dotted outline). White arrows point 630
to SYTOX Green-stained dead bacteria and/or archaea in inter-aggregate spaces. 631
632
Figure A4. Autofluorescence CLSM -λ-scan images and the corresponding emission 633
spectra recorded in cyanobacteria isolated from halite (Yungay). (a) CLSM - λ-scan 634
image and spectral profile corresponding to unspecific cell autofluorescence in the 635
green region (GAF) in response to 488 nm laser excitation. Plot of mean fluorescence 636
intensity (MFI) versus emission wavelengths of the cells ( -scan emission max = 556.12 637
nm). (b) CLSM -λ-scan image and spectral profile corresponding to the emission peaks 638
of photosynthetic pigments (PAF) excited with the 488 nm laser. Plot of MFI versus 639
emission wavelengths of the cells. Note the peak at 662.75 nm for phycocyanin (PC) 640
and allophycocyanin (APC), and the shoulder at 685.2 nm for chlorophyll a. The data 641
from both spectra represent MFI (n = 15) ± S.E., standard error; a.u., arbitrary units. 642
643
Figure A5. Autofluorescence CLSM -λ-scan images and the corresponding emission 644
spectra recorded in cyanobacteria isolated from halite (Salar Grande). (a) CLSM - λ-645
scan image and spectral profile corresponding to unspecific cell autofluorescence in the 646
green region (GAF) in response to 488 nm laser excitation. Plot of mean fluorescence 647
intensity (MFI) versus emission wavelengths of the cells ( -scan emission max = 567.35 648
nm). (b) CLSM -λ-scan image and spectral profile corresponding to the emission peaks 649
of photosynthetic pigments (PAF) excited with the 488 nm laser. Plot of MFI versus 650
emission wavelengths of the cells. Note the peak at 657.59 nm for phycocyanin (PC) 651
28
and allophycocyanin (APC) and the shoulder at 679.6 nm for chlorophyll a. The data 652
from both spectra represent MFI (n = 17) ± S.E., standard error; a.u., arbitrary units. 653
654
Figure A6. Autofluorescence emission spectrum recorded for cyanobacteria isolated 655
from halite (Yungay). The spectral profile reveals weak unspecific autofluorescence in 656
the green region and weak emission in the range of photosynthetic pigments. Cells were 657
excited using the 488 nm laser. Plotted are MFI values (n = 15 ± S.E., standard error) 658
against the emission wavelength of the cells. 659
660
Table A1. Microclimate data recorded at the sampling sites from May 2008 to May 661
2011; S.D., standard deviation. 662
663
Table A2. Numerical values of maximum (Max [nm]) and mean fluorescence intensity 664
(MFI in arbitrary units [a.u.]) of the λ-scan spectra emitted by different 665
autofluorescence sources within the cyanobacterial cells; n.o., not observed. 666
667
Table A1. Microclimate data recorded at the sampling sites from May 2008 to May
2011; S.D., standard deviation.
Site Temperature ºC / year
Mean Max. Min. S.D.
Relative humidity % / year
Mean Max. Min. S.D.
Yungay 17.93 46.01 -8.20 11.30 34.52 76.55 2.40 21.38
Salar Grande 20.24 42.33 3.77 8.82 51.45 93.22 3.37 22.45
Table A2. Numerical values of maximum (Max [nm]) and mean fluorescence intensity
(MFI in arbitrary units [a.u.]) of the λ-scan spectra emitted by different
autofluorescence sources within the cyanobacterial cells; n.o., not observed.
Yungay Salar Grande
Source of autofluorescence Max [nm] MFI [a.u.] Max [nm] MFI [a.u.]
Pigments from degraded cells /
GAF emission
556.12 43.33 ± 2.91 567.35 144.16 ± 8.65
Non-degraded photosynthetic
pigments /PAF emission
662.75 151.40 ± 3.98
657.59 225.13 ± 19.06
Pigments from transition phase
cells / GAF+PAF emission
550.51
662.75
27.65 ± 2.57
36.2 ± 6.35
n.o. n.o.
