LETTER
The “Raman spectroscopic signature of life”
is closely related to haem function in budding yeasts
Liang-da Chiu
1and Hiro-o Hamaguchi *
;1; 21Department of Chemistry, the University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 Japan
2The Department of Applied Chemistry, National Chiao Tung University, 1001 University Road, Hsinchu, Taiwan 300 R.O.C.
Received 20 February 2010, revised 16 March 2010, accepted 24 March 2010 Published online 13 April 2010
Key words: The Raman spectroscopic signature of life, Haem protein, yeast
1. Introduction
Recent progress in lasers, optical multichannel detec-tors and other optoelectronic devices has facilitated the extensive use of Raman spectroscopy in studying biological systems [1]. In our previous work using living yeast cells as model organism, we have de-tected a still unassigned unique Raman signal at 1602 cm 1. We called it the “Raman spectroscopic
signature of life” because it was proven to be an in-dicator of the cell metabolic activity [2–6]. It is es-tablished by now that the intensity of this band de-creases after the cells are treated with potassium cyanide (KCN) [2] or sodium azide (NaN3) [7].
H2O2 treatment also causes the signal to vanish in
several minutes [4]. Besides yeasts, the signature is
found in rat liver mitochondria [8] and HELA cells [9] as well, suggesting it to be a Raman signal that universally exists in most kinds of cells.
The fact that the “Raman spectroscopic signature of life” diminishes after adding KCN or NaN3
sug-gests that it originates from a molecular species in-volved in a metabolic pathway that requires the utili-zation of molecular oxygen through haem proteins. To further discuss the origin of this signature, we ex-pect that yeast mutants deficient of haem synthesis such as HEM1 disrupted yeasts (hem1D strain) would be helpful. The HEM1 gene encodes d-ami-nolevulinate (ALA) synthase, the enzyme which cat-alyzes the first step of the haem synthetic pathway [10]. The disruption of this gene would lead to haem deficient and unviable cells unless the cells are
pro-# 2011 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Journal of
BIO
PHOTONICS
HEM1 gene encodes d-aminolevulinate synthase that is required for haem synthesis. It is an essential gene for yeast survival. The Raman spectra of HEM1 knockout (hem1D) yeast cells lacks a Raman band at 1602 cm 1 that has been shown to reflect cell metabolic activity. This result suggests that the molecule giving rise to the“Raman spectroscopic signature of life” is closely re-lated to haem functions in the cell. High amount of squalene is also observed in the hem1D strain, which is another new discovery of this study.
The image and Raman spectra of wild type and hem1D yeasts compared with squalene
* Corresponding author: e-mail: hhama@chem.s.u-tokyo.ac.jp, Phone: +81 3 5841 4327, Fax: +81 3 3818 4327
vided with d-aminolevulinate (ALA), the product of ALA synthase, or both ergosterol and unsaturated fatty acids [11]. Therefore, it is clear that hem1D yeasts are truly deficient in haem biosynthesis and no other metabolic pathways could compensate for it. In this study, we report the Raman spectroscopic study of the wild type and the hem1D yeasts to clar-ify how haem function affects the “Raman spectro-scopic signature of life” in yeasts.
2. Experimental
2.1 Yeast strains and culture conditions
The yeast strains used in this study are listed in Ta-ble 1. The wild type strains are cultured aerobically at 30C in 2% peptone, 2% D-glucose and 1% yeast
extract (YPD medium). All hem1D yeasts are cul-tured in the same condition except that 80 mg/ml ALA is supplied in the YPD medium. The hem1D yeasts not supplied with ALA are obtained by cul-turing hem1D yeasts in YPD medium without ALA for three days before Raman spectroscopy experi-ments.
2.2 Raman micro-spectroscopy experiment
The Raman micro-spectroscopy setup used in this study is the same as the one reported previously [2]. In brief, the pump wavelength is 785 nm and the la-ser power at the sample point is 18 mW. The expo-sure time is 100 seconds. Quartz coverslips and slides are used in order to avoid fluorescence from the glass. The Raman spectra of S. cerevisiae do not have strong spatial dependence within the cell, most prob-ably because of the laser trapping effect. Therefore, we always measure the cells at the place where the laser trapping of the whole cell occurs. It is also the place where we can obtain the highest signal-to-noise Raman spectrum of the cell.
3. Results and discussion
In most of our previous studies, we used the indus-trial W4 tetraploid yeast strain as our model system. However, tetraploid strains are not suitable for gene knockout experiments. Here we introduce haploid S. cerevisiae for our study. Figure 1 compares the opti-cal images and the Raman spectra of wild type tetra-ploid and hatetra-ploid yeast cells. Both the size of the cell
and the intensity of the “Raman spectroscopic signa-ture of life” show clear dependence on the cell ploi-dy. Tetraploid cells are generally larger in size and have a stronger 1602 cm 1 band than haploid cells.
The reason for this variance in the 1602 cm 1 signal could be the difference in the metabolic state of the cells. Most of the industrial yeast strains are tetra-ploid because they grow and ferment more actively than other yeast cells. This serves as another piece of evidence that the intensity of the “Raman spec-troscopic signature of life” is dependent on the me-tabolic state of the cells.
Since the spectra of a and a wild type haploid cells are almost identical, we have chosen only a type hap-loid cells for further experiments. Figure 2a–d shows the optical image and Raman spectra of wild type and hem1D yeasts cultured in YPD medium without any supplement. We have measured 30 different cells from 6 independent batches of culture and none of the hem1D yeasts showed the “Raman spec-troscopic signature of life” at 1602 cm 1. The strain
also displays growth arrest at early stage so that its OD600 never reaches the same level as wild type
strains or hem1D strain supplied with ALA (data not shown, refer to [11]). This result is consistent with our expectation that the 1602 cm 1signal is
clo-sely related to haem function.
Figure 1 (online color at: www.biophotonics-journal.org) The optical images and Raman spectra of (a, d) tetraploid cells, (b, e) a type haploid cells and (c, f) a type haploid cells. The asterix mark represents the laser focus and the bar below (c) is the 20 mm scale bar for the three optical images. The average size (g) and the average intensity of the 1602 cm 1 band (h) of 15 tetraploid and 13 haploid cells are also shown.
J. Biophotonics 4, No. 1–2 (2011) 31
LETTER
LETTER
# 2011 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Besides the 1602 cm 1 signal, the 1656 cm 1 Ra-man band in hem1D strain is no more visible and a new band appears at 1668 cm 1 as shown in
Fig-ure 2d. It is worth noting that besides the Raman band mentioned above, two new peaks appeared at 1380 cm 1and 1330 cm 1. The 1668 cm 1, 1380 cm 1
and 1330 cm 1 Raman bands correspond well with the Raman spectrum of squalene (Figure 2e, [12]). Therefore, it is clear that the 1656 cm 1Raman band
from C=C double bond stretch of unsaturated fatty acids [2] no more exists in hem1D cells, as haem de-ficient yeast could not synthesize unsaturated fatty acids [11], and the 1668 cm 1 signal comes from the C=C double bond stretch of squalene. Squalene is the precursor of many lipid structures in yeasts that requires haem protein in their synthetic process [13]. Since hem1D cells could not synthesize haem groups properly, it is reasonable that squalene is accumu-lated in the hem1D yeast cell. We believe this is the first in vivo observation of the accumulation of squa-lene in hem1D yeast cells. Similar results have been reported by Spanova et al. using the lipid extraction technique [14].
It is reported that the haem synthesis pathway of hem1D strains could be recovered by supplying ALA to the yeasts [11]. However, it has been
diffi-cult to determine whether hem1D strains supplied with ALA has indeed the same metabolic state as wild type cells. Here we propose Raman spectro-scopy as a useful method to analyze the general me-tabolic status of the two strains. As shown in Fig-ure 3, the Raman spectra of wild type and ALA supplied hem1D cells are basically identical, suggest-ing that the haem synthesis pathway has been fully recovered in the mutant strain.
4. Conclusion
In this paper we have shown the dependence of the “Raman spectroscopic signature of life” on the me-tabolic activity and the haem synthesis of the cell. It is the first gene knockout experiment for the eluci-dation of the 1602 cm 1 Raman band and has suc-cessfully confined the candidate molecular species that gives rise to the signal downstream of the haem synthetic pathway.
Together with our previous results, in which the signal was inhibited by KCN [2] and NaN3[7], it is
clear that the haem-oxygen reaction is necessary for the 1602 cm 1 band to exist. Isotope substitution
ex-periments also showed that the signal originates from a C=C double bond structure [15]. The infor-mation above has helped us to finally propose
sev-Table 1 The yeast strains used in this study.
Strain Name Species Name Ploidity Source
W4*;** Saccharomyces cerevisiae Saccharomyces bayanus Tetraploid Suntory Ltd. FY1679 a* Saccharomyces cerevisiae Haploid Prof. Ferreira FY1679 a* Saccharomyces cerevisiae Haploid Prof. Ferreira Hem1D a Saccharomyces cerevisiae Haploid Prof. Ferreira [11] * Wild type strains. ** Industrial strain.
Figure 2 (online color at: www.biophotonics-journal.org) The optical images and Raman spectra of (a, c) wild type a strain and (b, d) hem1D a strain without any supple-ment. The asterix mark represents the laser focus and the bar below (b) is the 20 mm scale bar for the three optical images. Spectra (e) is the spectrum of squalene.
Figure 3 (online color at: www.biophotonics-journal.org) The optical image and Raman spectra of (a, c) wild type a strain and (b, d) hem1D a strain supplied with ALA. The asterix mark represents the laser focus and the bar below (b) is the 20 mm scale bar for the three optical images.
L.-da Chiu and H.-o Hamaguchi: The “Raman spectroscopic signature of life” is related to haem function in yeasts 32
Journal of
BIO
PHOTONICS
eral specific candidates of the “Raman spectroscopic signature of life” and we hope that the true origin of the signal will be elucidated soon.
Acknowledgements The authors would like to thank Prof. Ferreira in Universite´ de Poitiers and Ms. Yomo in Suntory Co., Ltd. for providing the yeast strains. They are also grateful to Mr. Onogi for pointing out the spectrum of squalene and Dr. Ling in RIKEN for his helps and ad-vices in yeast culturing.
References
[1] P. Carey, J. Biol. Chem. 274, 26625–26628 (1999). [2] Y. S. Huang, T. Karashima, M. Yamamoto, T. Ogura,
and H. Hamaguchi, J. Raman Spectrosc. 35, 525–536 (2004).
[3] Y. S. Huang, T. Karashima, M. Yamamoto, and H. Ha-maguchi, Biochemistry-US 44, 10009–10019 (2005). [4] Y. S. Huang, T. Nakatsuka, and H. Hamaguchi, Appl.
Spectrosc. 61, 1290–1294 (2007).
[5] Y. Naito, A. Toh-e, and H. Hamaguchi, J. Raman Spectrosc. 36, 837–839 (2005).
[6] C. Onogi and H. Hamaguchi, J. Phys. Chem. B 113, 10942–10945 (2009).
[7] L.-D. Chiu, M. Ando, and H. Hamaguchi, J. Raman Spectrosc. 41, 2–3 (2010)
[8] H. Tang, H. Yao, G. Wang, Y. Wang, Y. Q. Li, and M. Feng, Opt. Exp. 15, 12708–12716 (2007).
[9] V. V. Pully and C. Otto, J. Raman Spectrosc. 40, 473– 475 (2009).
[10] E. G. Gollub, K. Liu, J. Dayan, M. Adlersberg, and D. B. Sprinson, J. Biol. Chem. 252, 2846–2854 (1977). [11] L. Pineau, L. Bonifait, J. M. Berjeaud, P.
Alimardani-Theuil, T. Berges, and T. Ferreira, Mol. Biol. Cell 19, 807–821 (2008).
[12] V. Baeten, P. Dardenne, and R. Aparicio, J. Agricult. Food Chem. 49, 5098–5107 (2001).
[13] E. Rosenfeld and B. Beauvoit, Yeast 20, 1115–1144 (2003).
[14] M. Spanova, T. Czabany, G. Zellnig, E. Leitner, I. Hapala, and G. Daum, J. Biol. Chem. 285, 6127–6133 (2010).
[15] C. Onogi, H. Torii, and H. Hamaguchi, Chem. Lett. 38, 898–899 (2009).
J. Biophotonics 4, No. 1–2 (2011) 33
LETTER
LETTER
# 2011 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
