Carotenoid Analysis of Halophilic Archaea by Resonance Raman Spectroscopy

13 Pages·2007·0.32 MB·English

ASTROBIOLOGY
Volume 7, Number 4, 2007
© Mary Ann Liebert, Inc.
DOI: 10.1089/ast.2006.0097
Research Paper
Carotenoid Analysis of Halophilic Archaea
by Resonance Raman Spectroscopy
CRAIG P. MARSHALL,1,2 STEFAN LEUKO,2 CANDACE M. COYLE,3
MALCOLM R. WALTER,2,4 BRENDAN P. BURNS,2,4 and BRETT A. NEILAN2,4
ABSTRACT
Recently, halite and sulfate evaporate rocks have been discovered on Mars by the NASA
rovers, Spirit and Opportunity. It is reasonable to propose that halophilic microorganisms
could have potentially flourished in these settings. If so, biomolecules found in microorgan-
isms adapted to high salinity and basic pH environments on Earth may be reliable biomark-
ers for detecting life on Mars. Therefore, we investigated the potential of Resonance Raman
(RR) spectroscopy to detect biomarkers derived from microorganisms adapted to hypersaline
environments. RR spectra were acquired using 488.0 and 514.5 nm excitation from a variety
of halophilic archaea, including Halobacterium salinarum NRC-1, Halococcus morrhuae, and
Natrinema pallidum. It was clearly demonstrated that RR spectra enhance the chromophore
carotenoid molecules in the cell membrane with respect to the various protein and lipid cel-
lular components. RR spectra acquired from all halophilic archaea investigatedcontained ma-
jor features at approximately 1000, 1152, and 1505 cm(cid:2)1. The bands at 1505 cm(cid:2)1 and 1152
cm(cid:2)1 are due to in-phase C¨C ((cid:2) ) and C–C stretching ((cid:2) ) vibrations of the polyene chain
1 2
in carotenoids. Additionally, in-plane rocking modes of CH groups attached to the polyene
3
chain coupled with C–C bonds occur in the 1000 cm(cid:2)1 region. We also investigated the RR
spectral differences between bacterioruberin and bacteriorhodopsin as another potential bio-
marker for hypersaline environments. By comparison, the RR spectrum acquired from bacte-
riorhodopsin is much more complex and contains modes that can be divided into four groups:
the C¨C stretches (1600–1500 cm(cid:2)1), the CCH in-plane rocks (1400–1250 cm(cid:2)1), the C–C
stretches (1250–1100 cm(cid:2)1), and the hydrogen out-of-plane wags (1000–700 cm(cid:2)1). RR spec-
troscopy was shown to be a useful tool for the analysis and remote in situ detection of
carotenoids from halophilic archaea without the need for large sample sizes and complicated
extractions, which are required by analytical techniques such as high performance liquid chro-
matography and mass spectrometry. Key words: Biomarkers—Halophilic Archaea—Mars—
Raman spectroscopy—Spectroscopic biosignatures. Astrobiology 7, 631–643.
1Vibrational Spectroscopy Facility, School of Chemistry, The University of Sydney, Australia.
2Australian Centre for Astrobiology, Macquarie University, Sydney, Australia.
3Department of Chemistry, The University of Texas at San Antonio, San Antonio, Texas.
4School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia.
631
632 MARSHALL ET AL.
INTRODUCTION photoprotection system (Cockell and Knowland,
1999), but is also important for the adaptation of
RECENTLY, HALITEANDSULFATEevaporate rocks membrane fluidity to changing osmotic condi-
have been discovered on Mars by the NASA tions (D’Souza et al., 1997).
rovers, Spirit and Opportunity. This suggests that Raman spectroscopy is mostly viewed as a spe-
brine pools may have been relatively common on cialist laboratory or research technique. How-
the surface of that planet and, thus, may have pro- ever, in recent years several systems have been
vided regions of high salt concentration. Conse- specifically developed for field-based applica-
quently, it is reasonable to propose that halophilic tions. Coupling recent advances in laser sources,
microorganisms could have potentially flour- optical elements, spectrometers, and detectors
ished under such hypersaline conditions (for ex- has led to the development of robust, compact,
ample, Landis, 2001; Wierzchos et al., 2006). Thus, and miniaturised Raman systems. Consequently,
modern terrestrial salt basin and cultured salt- the potential use of Raman spectroscopy in plan-
tolerant microbes are good analogues for condi- etary exploration as part of a rover or lander in-
tions under which life might have evolved on strumentation package, particularly for the ex-
Mars (for example, Landis, 2001; Wierzchos et al., ploration of Mars, is now being realized. NASA
2006). If so, biomolecules found in microorgan- and ESA currently consider Raman spectroscopy,
isms adapted to high salinity and basic pH envi- either separately or in combination with laser-in-
ronments on Earth may also be reliable biomark- duced breakdown spectroscopy, or fluorescence,
ers for detecting life on Mars. as a fundamental next-generation instrument for
Halophilic archaea are chemo-organotrophs the characterization of mineralogical and organic
that belong to the class Euryarchaeota. These mi- material during the exploration of Mars. Instru-
crobes are often the predominant microorganism mentation for robotic missions is probably the
present in salt lakes, pools of evaporating sea- most important consideration for Mars explora-
water, solar salterns, and other hypersaline envi- tion. It is important to note that Raman applica-
ronments with salt concentrations as high as tions, which add to the knowledge of Mars, cover
halite saturation (Oren, 2002). Halophiles also live other aspects, such as the study of potential ter-
in a variety of warm and cold environments. Ex- restrial martian analogues. Hence, it is crucial to
tremely halophilic archaea have been noted for construct a Raman database of biosignatures of po-
their bright red or purple color. The pigments re- tential martian analogue microbial life found in ex-
sponsible for these colors consist of isoprenoid- treme environments on Earth to facilitate the de-
derived or retinal-protein compounds (Kush- tection of biosignatures on Mars. This work has
waha et al., 1974). The pigment responsible for already commenced; for example, there are a num-
the purple color is a retinal-protein complex, ber of recent pioneering studies on cyanobacterial
bacteriorhodopsin, while the isoprenoid-derived biomolecules using Raman spectroscopy, pre-
carotenoid pigment, bacterioruberin, gives rise to dominantly NIR FT-Raman spectroscopy by
a bright pinkish-red color. Wynn-Williams and Edwards, (2000), Edwards et
Bacteriorhodopsin converts the energy of green al.(2005, 2004), Villar et al.(2005), and, for a more
light (500–650 nm) into an electrochemical proton extensive review, Villar and Edwards (2006).
gradient, which in turn is used for ATP produc- Motivated by the growing importance of being
tion by ATP-synthases. For example, the plasmic able to detect biomolecules during planetary ex-
membrane of Halobacterium salinarumNRC-1 con- ploration, this study aimed to investigate the po-
tains membrane patches known as the purple tential of resonance Raman (RR) spectroscopy of
membrane with a protein:lipid ratio of 75:25. The carotenoids biosynthesized by halophilic archaea
only protein in the purple membrane is bacteri- as a potential new biomarker for hypersaline en-
orhodopsin, which forms a hexagonal 2-dimen- vironments on Mars.
sional crystal that consists of bacteriorhodopsin
trimers (Haupts et al., 1999). Bacterioruberin is a
ubiquitous and abundant pinkish-red pigment in RESONANCE RAMAN SPECTROSCOPY
moderately (Rønnekleiv and Liaaen-Jensen, 1995) OF CAROTENOIDS
to extremely halophilic archaea (Liaaen-Jensen,
1979). This red pigment, located in the membrane Raman spectroscopy is a form of vibrational
of halophilic archaea, not only plays a role in the spectroscopy that has long been routinely used
RAMAN SPECTROSCOPY OF ARCHAEA CAROTENOIDS 633
to identify and quantify chemical compounds. we observe Raman active modes shown as Ra-
A Raman spectrum is a spectrum of the light man bands in the spectrum.
scattered from a sample, which is irradiated If a biological molecule absorbs light in the vis-
with monochromatic radiation in the visible or ible portion of the electromagnetic spectrum, then
near-infrared region. The light may be scattered new possibilities are opened up for Raman spec-
either elastically (Rayleigh scattering) or inelas- troscopy for these compounds of interest, via the
tically (Raman scattering) as shown schemati- resonance Raman effect (Spiro, 1987). Judicious
cally by the possible consequences of a photon- selection of tuning the excitation wavelength to
molecule interaction (Carey, 1982) in Fig. 1. In the electronic absorption spectrum can produce
the case of Raman scattering, the emergent light selective enhancement of certain Raman bands
is shifted from its original frequency by a quan- (Carey, 1982; Spiro, 1987) (Fig. 1). These Raman
tum of energy that corresponds to a molecular bands correspond to vibrational modes that in-
transition of the sample. The transition may be volve motions of the atoms in the chromophore,
translational, rotational, vibrational, or elec- which is that portion of the molecule where the
tronic in nature. For chemical and biological electronic transition is localized. There are two
purposes, the vibrational Raman effect is the types of resonance-enhanced vibrational modes,
most important. For a vibrational mode to be which are termed type A (A-term) and B (B-term)
Raman active, a change in polarizability is (Carey, 1982; Spiro, 1987). Type A modes connect
needed as the molecule vibrates. Molecules con- the ground state to the resonant excited state
sist of a nuclear structure surrounded by a com- through the Franck-Condon overlap, while type
plex field or cloud of electrons. Application of B modes couple the resonant excited state to an-
a potential field causes the electrons to ebb and other excited state at higher frequency through
flow so that they are slightly concentrated to- vibronic mixing. Type A modes are totally sym-
ward the (cid:2)and away from the (cid:3)of the applied metric, since the ground state wave function has
field. The ease at which electrons respond to a the full symmetry of the molecule. Type B modes,
given field is described as polarizability. If the however, may possess any symmetry that is a re-
polarizability changes as the molecule vibrates, sult of the direct product of the two electronic
FIG. 1. Some of the possible consequences of a photon-molecule interaction.The lengths of the upward arrows
are proportional to the frequencies of the incoming light while the lengths of the downward arrows are proportional
to the frequencies of the scattered light. The vibrational quantum numbers in the upper and lower electronic states
are (cid:2)(cid:4)and (cid:2)(cid:5)respectively (modified from Carey, 1982).
634 MARSHALL ET AL.
transition representations. Therefore, RR spec- this (cid:3)-(cid:3)* absorption band indicates an increase
troscopy provides a means whereby vibrations of in the conjugation length, which is reflected in its
biological chromophores can be distinguished color, progressing from yellow to orange to red.
from many of the vibrational modes associated For example, (cid:4)-carotene has 11 conjugated dou-
with the complex biological matrix. Significantly, ble bonds and is orange in color, while bacteri-
the resonance enhancement factor can be quite oruberin has 13 conjugated double bonds and is
large, in the order of 103 to 106 orders of magni- red in color (Fig. 2). Significantly, for Raman spec-
tude and, thereby, allow the analysis of chro- troscopic applications, when the wavelength of
mophore concentrations as low as 10(cid:3)4 to 10(cid:3)6 laser excitation coincides with an allowable (cid:3)-(cid:3)*
M. The chromophore vibrations completely dom- electronic transition of carotenoids, RR spectra
inate the spectrum. Consequently, RR spectros- are obtained.
copy could be used to identify and delineate po-
tential biomarker compounds, particularly at the
trace quantity level.
MATERIALS AND METHODS
Carotenoids are (cid:3)-electron-conjugated carbon-
chain molecules and are similar to polyenes with
Organisms and growth conditions
regard to their structure and optical properties.
Structurally, these molecules are a linear, chain- Halophilic archaea investigated in this study in-
like conjugated carbon backbone that consists of clude Halobacterium salinarum NRC-1, Halococcus
alternating carbon single (C–C) and double bonds morrhuae, and Natrinema pallidum. The strain Hbt.
(C¨C) with varying numbers of conjugated dou- salinarum NRC-1 was a gift from Professor Helga
ble bonds and a varying number of attached Stan-Lotter. Hcc. morrhuaeand Nnm. pallidumwere
methyl side groups. For example, the molecular gifts from Professor Masahiro Kamekura. All
structure of (cid:4)-carotene and bacterioruberin, strains, except Hbt. salinarumNRC-1, were cultured
which are the most important carotenoids in in DSM 97 medium (DasSarma et al., 1995) with 150
cyanobacteria and halophilic archaea, respec- g of NaCl and supplemented with 7.23 g MgCl (cid:6)
2
tively, are shown in Fig. 2. Carotenoids are 6 H O and 2.70 g CaCl (cid:6)2 H O per liter, pH 7.4
2 2 2
strongly colored as they have an allowed (cid:3)-(cid:3)* (or (DSM 97 modified). Hbt. salinarumNRC-1 was cul-
S -S ) transition that occurs in the visible region tured in ATCC 2185 medium as described at
0 2
of the electromagnetic spectrum. This color is de- http://www.atcc.org/mediapdfs/2185.pdf. Cul-
pendent on the number of conjugated double tures were incubated at 37 °C on a rocking plat-
bonds in the main linear chain. Red shifting of form for up to two weeks.
(cid:7)-carotene
OH
OH
OH
OH
Bacterioruberin
FIG. 2. A comparison of the molecular structures of (cid:2)-carotene and bacterioruberin carotenoids.
RAMAN SPECTROSCOPY OF ARCHAEA CAROTENOIDS 635
Carotenoid extraction backscattered radiation. The 488.0 and 514.5 nm
(cid:2)
line of a 5W Ar laser (Spectra-Physics Stabilite
The bacterioruberin carotenoid pigment was
2017 laser) orientated normal to the sample was
extracted from (cid:2)0.2 mg of Hbt. salinarum NRC-1
used to excite the sample. For both excitation
cells using 5 ml of cold acetone. The acetone-sol-
lines, the following spectra acquisition parame-
uble extract obtained from Hbt. salinarum NRC-1
ters were used for performing direct compar-
was identified by its electronic absorption spec-
isons: 10 seconds exposure time, 5 accumulations,
trum and compared with previously published
and (cid:2)1.2 mW of laser power at the sample. The
electronic spectra obtained from bacterioruberin scan ranges were 800–1800 cm(cid:3)1 in the finger-
(Britton, 1985; D’Souza et al., 1997). Bacterio-
print region of resonance-enhanced Raman spec-
rhodopsin (99% purity) isolated from the purple
tra obtained from carotenoids. The halophilic ar-
membranes of Hbt. salinarum was purchased from
chaea were smeared as a one-cell layer onto a
Sigma.
clean aluminum microscope slide and irradiated
with the laser to obtain spectra.
Electronic absorption spectroscopy
Electronic absorption spectra were obtained
from the acetone-soluble extract of Hbt. salinarum RESULTS AND DISCUSSION
NRC-1 biomass using a Cary 5 UV-Vis spec-
trometer in the 350–600 nm range. 1. Electronic absorption spectra
The representative electronic absorption spec-
Resonance Raman spectroscopy
trum shown in Fig. 3 was obtained from the ace-
A Renishaw InVia Reflex Raman microprobe tone-soluble extract of Hbt. salinarumNRC-1 bio-
with a multi-wavelength facility operating at mass. The red pigment was identified by its
488.0 and 514.5 nm was used to assess the effect characteristic absorption spectrum as bacterioru-
of excitation wavelength on the recording of spec- berin, with absorption maxima ((cid:6) ) at 388, 468,
max
tra from the halophilic archaea. The collection op- 495, and 530 nm in acetone. These results are in
tics are based on a Leica DMLM microscope. A accordance with the observations of Britton
refractive glass 50(cid:6)objective lens was used to fo- (1985) and D’Souza et al. (1997). Electronic ab-
cus the laser onto a 2 (cid:5)m spot to collect the sorption spectroscopy not only allows for pig-
FIG. 3. The electronic absorption spectrum of acetone-soluble extract of Hbt. salinarumNRC-1 cells.Absorptions
occur as broad bands in the blue/green spectral range with (cid:6) at 468, 495, and 530 nm characteristic for an ab-
max
sorption spectrum for bacterioruberin. The insert shows an energy level diagram for carotenoids.
636 MARSHALL ET AL.
ment identification, but it also delineates whether 2. Resonance Raman spectroscopy
the compound of interest exhibits absorption
bands due to electronic dipole transitions of the 2.1 Choice of excitation wavelength
molecules within a certain portion of the visible
wavelength range. When illuminated with mono- Figure 4 shows the representative RR spectra
chromatic light overlapping one of these absorp- in the region of 800–1800 cm(cid:3)1 acquired from a
tion bands, the Raman scattered light will exhibit one-cell-layer smear of Hbt. salinarumNRC-1 cells
a substantial resonance enhancement in the order recorded at 488.0 and 514.5 nm excitation wave-
of 105 and, therefore, allow judicious selection of lengths. It should be noted that a properly con-
the excitation wavelength to achieve this phe- figured Raman spectrometer for Mars explora-
nomenon. tion would be configured to encompass 50–4000
The electronic absorptions occur as broad cm(cid:3)1 Raman shift. This would enable, for exam-
bands ((cid:2)150 nm wide) in the blue/green visible ple, the observation in samples of bound and un-
spectral range. Carotenoid molecules feature an bound water ((cid:2)OH) stretching modes between
unusual even parity excited state (see insert, Fig. 3000 and 3800 cm(cid:3)1 if present. This is also sig-
3). Consequently, absorption transitions are elec- nificant in the astrobiological prospecting of
tronic-dipole allowed in these molecules, but Mars. However, the RR spectra shown here are
spontaneous emission is forbidden. The result- displayed in the region of 800–1800 cm(cid:3)1 since
ing absence of any strong fluorescence in bands occurring above 2000 cm(cid:3)1 are due over-
carotenoids is the main reason for the possibility tone and combination bands, which in this case
of applying RR spectroscopy for these analyses provide little to no information and obscure any
(shown as a downward pointing arrow—optical stretching vibrational modes associated with wa-
transition in the insert in Fig. 3). This absorption ter. Therefore, the fingerprint region for the res-
shows a clearly resolved vibronic substructure onance enhancement of carotenoids was chosen
due to a strong electron-phonon coupling. Strong for all spectra shown herein. The stacked RR spec-
electric-dipole allowed absorption transitions oc- tra in Fig. 4 contain major features at approxi-
cur between the molecules’ delocalized (cid:3)orbitals mately 1000, 1152, and 1505 cm(cid:3)1. The bands at
from the 1 1A singlet ground state to the 1 1B 1505 cm(cid:3)1 and 1152 cm(cid:3)1 are due to in-phase
g u
singlet excited state (insert, Fig. 3). Optical exci- C¨C ((cid:2) ) and C–C stretching ((cid:2) ) vibrations of
1 2
tation within the 1 1A (cid:2)1 1B absorption band the polyene chain in carotenoids. Additionally,
g u
led to only weak luminescence transitions in-plane rocking modes of CH groups attached
3
(Thrash et al., 1977; Shreve et al., 1991). It can be to the polyene chain coupled with C–C bonds oc-
noted from the insert in Fig. 3 that the extremely curred in the 1000 cm(cid:3)1 region.
low quantum efficiency of the luminescence was Due to the absorption bands that occur as
caused by the existence of a second excited sin- broad bands in the blue/green spectral range, we
glet state, a 2 1A state, which lies below the 1 selected to illuminate with monochromatic light
g
1B state. Following excitation of 1 1B state, the overlapping these absorption bands with both
u u
carotenoid molecule relaxes very rapidly within 488.0 and 514.5 nm excitation lines. The mecha-
(cid:2)200 to 250 fs (Shreve et al., 1991), via non-ra- nism for resonance enhancement of carotenoids
diative transitions, to the lower 2 1A state from is attributable to the A-term with involvement of
g
which electronic emission to the ground state is a single excited electronic state ((cid:3)(cid:2)(cid:3)*). This
parity-forbidden. This is shown by the dashed process leads to a large intensity gain for those
downward pointing upper arrow in the insert of modes at 1505 and 1152 cm(cid:3)1 from stretching of
Fig. 3. The resulting low 1 1B (cid:2)1 1A lumines- the ((cid:2) ) C¨C and ((cid:2) ) C–C bonds, respectively,
u g 1 2
cence efficiency (10(cid:3)5 to 10(cid:3)4), and absence of 2 since these bond distances change appreciably in
1A (cid:2)1 1A fluorescence of the molecules, en- the (cid:3)* state. However, the C–H bonds show lit-
g g
abled the detection of the RR spectral response tle change when passing from the (cid:3)(cid:2)(cid:3)*. Con-
of the molecular vibrations clearly shown as a sequently, C–H stretching modes show minimal
solid downward pointing arrow in the insert of intensity variation as the resonance condition is
Fig. 3. Specifically, RR spectroscopy detects the approached. Most notably, when the excitation
stretching vibrations of the conjugated backbone line is changed from 488.0 to 514.5 nm, the (cid:2)(C–C)
as well as the methyl side groups (Thrash et al., and (cid:7)(C¨CH) decrease in intensity with a con-
1977; Shreve et al., 1991). comitant increase in the (cid:2)(C¨C) intensity. This
RAMAN SPECTROSCOPY OF ARCHAEA CAROTENOIDS 637
FIG. 4. Stacked resonance Raman spectra of a smear of bacterioruberin cells showing resonance enhancement
recorded at 488.0 and 514.5 nm excitations. Collection parameters for both 488.0 and 514.5 nm excitations are 10 s
exposure, 5 accumulations, and 1.2 mW laser power at the sample on an InViaReflex Renishaw Raman spectrome-
ter. RR-enhanced modes at 1505 cm(cid:3)1 and 1152 cm(cid:3)1 are due to in-phase C¨C ((cid:2) ) and C–C stretching ((cid:2) ) vibra-
1 2
tions of the polyene chain in carotenoids, and in-plane rocking modes of CH groups attached to the polyene chain
3
coupled with C–C bonds occur in the 1000 cm(cid:3)1region.
feature is not observed for botryoxanthin and ene chain coupled with C–C bonds occurred in
(cid:4)-carotene for RR spectra obtained from cyanobac- the 1000 cm(cid:3)1 region. Weaker features between
teria and micro-algae using 488.0 and 514.5 nm 920–980 and 1170–1450 cm(cid:3)1 were also noted.
excitation (Marshall et al., 2006a). This phenome- Carotenoids range in color from pale yellow to
non does not, however, have any significant im- bright orange to deep red; color is directly related
pact on excitation choice for obtaining biomarker to their structure. As the number of conjugated
data pertaining to bacterioruberin biosynthesised (C¨C) bonds increases, the wavelength of the ab-
by halophilic archaea. The 514.5 nm excitation sorbed light also increases, which gives the pig-
line was chosen for the RR spectroscopic investi- ment an increasingly red appearance. The Raman
gation of the other halophilic archaea. shift (cm(cid:3)1) of the (cid:2) band is strongly dependent
1
on the length of the carotenoid chain. It has been
shown (Thomas et al., 1990; Veronelli et al., 1995;
2.2 Comparison between different
Withnall et al., 2003; Schulz et al., 2005) that a cor-
halophilic archaea
relation between (cid:2) Raman shift (cm(cid:3)1) and ef-
1
Spectra acquired from the three Halobacteria fective conjugated chain length occurs. These in-
cultures, namely Hbt. salinarum NRC-1, Hcc. mor- vestigators used this correlation to determine the
rhuae, and Nnm. pallidum, contained major fea- number of double bonds in the polyconjugated
tures at approximately 1000, 1152, and 1505 cm(cid:3)1 main chains of carotenoids, including dode-
(Fig. 5). It can be seen that there were no spectral capreno-(cid:4)-carotene, decapreno-(cid:4)-carotene, lyco-
differences between RR analyses of the three dif- pene, (cid:4)-carotene, crocetin, and retinal. Conse-
ferent halophilic archaea. The bands at 1505 cm(cid:3)1 quently, the (cid:2) (C¨C) stretching mode is an
1
and 1152 cm(cid:3)1are due to in-phase C¨C ((cid:2) ) and important marker band by which to elucidate the
1
C–C stretching ((cid:2) ) vibrations from the polyene structure of carotenoids. Application of this to
2
chain of carotenoids. Additionally, in-plane rock- our data acquired from the various halophilic ar-
ing modes of CH groups attached to the poly- chaea that show a Raman shift (cm(cid:3)1) of 1505
3
638 MARSHALL ET AL.
FIG. 5. Stacked resonance Raman spectra of a smear of Hbt. salinarum NRC-1, Hcc. morrhuae, and Nnm. pallidum
cells. Collection parameters for the three different archaea are 514.5 nm excitation, 10 s exposure, 5 accumulations,
and 1.2 mW laser power at the sample on an InVia Reflex Renishaw Raman spectrometer. RR-enhanced modes at
1505 cm(cid:3)1 and 1152 cm(cid:3)1 are due to in-phase C¨C ((cid:2) ) and C–C stretching ((cid:2) ) vibrations of the polyene chain in
1 2
carotenoids, and in-plane rocking modes of CH groups attached to the polyene chain coupled with C–C bonds oc-
3
cur in the 1000 cm(cid:3)1region.
cm(cid:3)1 for the (cid:2)1 (C¨C) band indicated a number ish-red carotenoid and this C50carotenoid can be
of 13 double bonds in the polyconjugated main used as a halophilic archaeal biomarker using
chain for bacterioruberin. This result is in agree- RR spectroscopy. Since bacteriorhodopsin is an-
ment with the structure for bacterioruberin (Fig. other possible biomarker for hypersaline envi-
2). Resonance Raman spectroscopy reveals that ronments, we undertook a comparison of the RR
the different halophilic archaea analyzed in this spectra produced by both bacterioruberin and
investigation all biosynthesize bacterioruberin. bacteriorhodopsin (Fig. 6).
Figure 6 shows representative stacked RR spec-
tra acquired from a culture of Hbt. salinarum
2.3 Comparison between bacterioruberin
NRC-1 and the isolated bacteriorhodopsin. The
and bacteriorhodopsin
spectrum acquired for bacterioruberin is less
The differences between bacteriorhodopsin complex than that acquired for bacteriorhodop-
and bacterioruberin are somewhat unclear. For sin. RR spectra of carotenoproteins such as bac-
example, Ellery and Wynn-Williams (2003) noted teriorhodopsin generally exhibit more vibrational
that halobacteria biosynthesize a red pigment bands than the free carotenoids. No bands as-
bacteriorhodopsin, which is based on a C signable to the protein component are observed,
50
carotenoid that could be detected by Raman spec- though. The RR spectrum acquired from the bac-
troscopy. Our electronic absorption and RR spec- teriorhodopsin standard contains several modes
tral results show that bacterioruberin is the pink- that can be divided into four groups: the C¨C
RAMAN SPECTROSCOPY OF ARCHAEA CAROTENOIDS 639
FIG. 6. Stacked resonance Raman spectra of bacterioruberin and bacteriorhodopsin acquired from Hbt. salinarum
NRC-1.Collection parameters for both spectra are 514.5 nm excitation, 10 s exposure, 5 accumulations, and 1.2 mW
laser power at the sample on an InViaReflex Renishaw Raman spectrometer. RR-enhanced modes at 1505 cm(cid:3)1and
1152 cm(cid:3)1are due to in-phase C¨C ((cid:2) ) and C–C stretching ((cid:2) ) vibrations of the polyene chain in carotenoids, and
1 2
in-plane rocking modes of CH groups attached to the polyene chain coupled with C–C bonds occur in the 1000 cm(cid:3)1
3
region and can be observed for bacterioruberin. While four vibrational modes can be observed for bacteriorhodopsin:
the C¨C stretches (1600–1500 cm(cid:3)1), the CCH in-plane rocks (1400–1250 cm(cid:3)1), the C–C stretches (1250–1100 cm(cid:3)1),
and the hydrogen out-of-plane wags (1000–700 cm(cid:3)1).
stretches (1600–1500 cm(cid:3)1), the CCH in-plane gated chain length for the chromophore portion
rocks (1400–1250 cm(cid:3)1), the C–C stretches (1250– probed by 514.5 nm consists of 5 conjugated dou-
1100 cm(cid:3)1), and the hydrogen out-of-plane wags ble bonds.
(1000–700 cm(cid:3)1). For more detailed assignments
in this region refer to Marshall et al. (2006b).
3. Astrobiological implications of resonance
These results highlight the difference between
Raman spectroscopy of carotenoids
the two biomarker compounds from hypersaline
environments. This confusion is surprising given Currently, there are a number of analytical
the macromolecular complexity and protein-reti- techniques employed for carotenoid identifica-
nal nature of bacteriorhodopsin (MW 26,000 Dal- tion and structural elucidation. Unfortunately,
ton) in comparison with bacterioruberin, a C most of these techniques must rely on tedious
50
carotenoid. The differences between Raman spec- separation methods (Patzlaff and Barry, 1996;
tra acquired from bacterioruberin and bacteri- Wilhelm et al., 1995; Berkaloff et al., 1990). Typi-
orhodopsin were due to molecular structure and cally, carotenoids need to be isolated by centrifu-
composition (Fig. 7). Application of the well-es- gation and solubilisation. Separation techniques
tablished relationship of the Raman shift (cm(cid:3)1) such as high performance liquid chromatogra-
position of the marker (cid:2) (C¨C) band, the Ra- phy, reverse-phase liquid chromatography, or
1
man shift of 1536 cm(cid:3)1, indicated a polyconju- thin layer chromatography are also required in
640 MARSHALL ET AL.
FIG. 7. A comparison of the molecular structure of bacterioruberin and the structure of the retinal chromophore
of the retinal-protein complex of bacteriorhodopsin.
order to purify the carotenoids (Wilhelm et al., various cellular components, including the cyto-
1995). Additionally, a large biomass is required plasm (dominated by proteins and nucleic acids),
since the pigments are in trace quantities within the cytoplasmic cell membrane (composed pre-
the cell. The purified carotenoid extracts are sub- dominantly of phospholipids), and the cell wall
sequently analyzed by techniques that range (composed predominantly of peptidoglycan). On
from mass spectrometry to optical spectroscopy average, the composition of microbial cells are
and fluorescence excitation and emission (Faw- fairly homogenous: (cid:2)40–60% proteins, (cid:2)15–20%
ley, 1989). Apart from the large amount of bio- nucleic acids, (cid:2)10–15% polysaccharides, (cid:2)10–
mass required, another problem is that most sep- 15% lipids, and (cid:2)3% or less other organic and in-
aration and detection methods are slow. organic components. Therefore, within a typical
Moreover, the extraction process can be destruc- mixture, it is nearly impossible to extract con-
tive to the carotenoid and retinal-protein com- ventional infrared or Raman vibrational spectra
plexes, which may be the only unique biomark- of biological markers. From an astrobiological
ers present. From an astrobiological perspective, perspective, it is desirable to target organic mol-
none of the methods that require sample prepa- ecules that are clearly distinguished from abio-
ration allow for remote detection. genic compounds that are widely distributed
Vibrational spectroscopy as a means for the de- throughout the cosmos. For example, an infrared
tection of potential martian biomolecules is es- spectrum of protein does not necessarily indicate
pecially attractive because of its speed, non- biogencity. A more useful spectroscopy for the
destructive nature, and very small (single cell) identification of molecules that are only synthe-
sample size. However, certain limitations arise sized by biological processes would be one that
due to the complex chemical composition of mi- can selectively and sensitively excite biogenic
croorganisms. For example, microbial cells con- markers, such as carotenoids. Since potential
sist of macromolecules that are distributed in martian microbes could conceivably contain pho-