Abstract

We constructed an experimental apparatus that used lasers to provide the probe beams for measuring the transient absorption kinetics of bacterioplankton that contain proteorhodopsin, a microbial protein that binds retinal and is analogous to animal rhodopsin. With this approach we were able to observe photocycles characteristic of functioning retinylidene ion pumps. Using light from lasers instead of broadband sources as transmittance probe beams can be advantageous when examining optically dense, highly scattering samples such as concentrated microbial cultures. Such a laser-based approach may prove useful in shipboard studies for identifying proteorhodopsin in whole cell suspensions concentrated from seawater.

© 2007 Optical Society of America

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References

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  1. R. H. Lozier, "Rapid kinetic optical absorption spectroscopy of bacteriorhodopsin photocycles," Methods Enzymol. 88, 133-162 (1982).
    [CrossRef]
  2. J. K. Lanyi, "Understanding structure and function in the light-driven proton pump bacteriorhodopsin," J. Struct. Biol. 124, 164-178 (1998).
    [CrossRef]
  3. O. Béjà, L. Aravind, E. V. Koonin, M. T. Suzuki, A. Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates, R. A. Feldman, J. L. Spudich, E. N. Spudich, and E. F. DeLong, "Bacterial rhodopsin: evidence for a new type of phototrophy in the sea," Science 289, 1902-1906 (2000).
    [CrossRef] [PubMed]
  4. O. Béjà, E. N. Spudich, J. L. Spudich, M. Leclerc, and E. F. DeLong, "Proteorhodopsin phototrophy in the ocean," Nature 411, 786-789 (2001).
    [CrossRef] [PubMed]
  5. J. R. de la Torre, L. M. Christianson, O. Béjà, M. T. Suzuki, D. M. Karl, J. Heidelberg, and E. F. DeLong, "Proteorhodopsin genes are distributed among divergent marine bacterial taxa," Proc. Natl. Acad. Sci. USA 100, 12830-12835 (2003).
    [CrossRef] [PubMed]
  6. M. S. Rappé, S. A. Connon, K. L. Vergin, and S. J. Giovannoni, "Cultivation of the ubiquitous SAR11 marine bacterioplankton clade," Nature 418, 630-633 (2002).
    [CrossRef] [PubMed]
  7. S. J. Giovannoni, L. Bibbs, J.-C. Cho, M. D. Stapels, R. Desiderio, K. L. Vergin, M. S. Rappé, S. Laney, L. J. Wilhelm, H. J. Tripp, E. J. Mathur, and D. F. Barofsky, "Proteorhodopsin in the ubiquitous marine bacterium SAR11," Nature 438, 82-85 (2005).
    [CrossRef] [PubMed]
  8. B. R. Kelemen, M. Du, and R. B. Jensen, "Proteorhodopsin in living color: diversity of spectral properties within living bacterial cells," Biochim. Biophys. Acta 1618, 25-32 (2003).
    [CrossRef] [PubMed]
  9. D. Man, W. Wang, G. Sabehi, L. Aravind, A. F. Post, R. Massana, E. N. Spudich, J. L. Spudich, and O. Béjà, "Diversification and spectral tuning in marine proteorhodopsins," EMBO J. 22, 1725-1731 (2003).
    [CrossRef] [PubMed]
  10. M. Stomp, J. Huisman, F. de Jongh, A. J. Veraart, D. Gerla, M. Rijkeboer, B. W. Ibelings, U. I. A. Wollenzien, and L. J. Stal, "Adaptive divergence in pigment composition promotes phytoplankton biodiversity," Nature 432, 104-107 (2004).
    [CrossRef] [PubMed]
  11. K. S. Rowan, Photosynthetic Pigments of Algae (Cambridge University Press, 1989).
  12. G. Váró, L. S. Brown, M. Lakatos, and J. K. Lanyi, "Characterization of the photochemical reaction cycle of proteorhodopsin," Biophys. J. 84, 1202-1207 (2003).
    [CrossRef] [PubMed]
  13. A. K. Dioumaev, L. S. Brown, J. Shih, E. N. Spudich, J. L. Spudich, and J. K. Lanyi, "Proton transfers in the photochemical reaction cycle of proteorhodopsin," Biochemistry 41, 5348-5358 (2002).
    [CrossRef] [PubMed]
  14. R. A. Krebs, A. Alexiev, R. Partha, A. M. DeVita, and M. S. Braiman, "Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin," BMC Physiol. 2:5 (2002).

2005

S. J. Giovannoni, L. Bibbs, J.-C. Cho, M. D. Stapels, R. Desiderio, K. L. Vergin, M. S. Rappé, S. Laney, L. J. Wilhelm, H. J. Tripp, E. J. Mathur, and D. F. Barofsky, "Proteorhodopsin in the ubiquitous marine bacterium SAR11," Nature 438, 82-85 (2005).
[CrossRef] [PubMed]

2004

M. Stomp, J. Huisman, F. de Jongh, A. J. Veraart, D. Gerla, M. Rijkeboer, B. W. Ibelings, U. I. A. Wollenzien, and L. J. Stal, "Adaptive divergence in pigment composition promotes phytoplankton biodiversity," Nature 432, 104-107 (2004).
[CrossRef] [PubMed]

2003

G. Váró, L. S. Brown, M. Lakatos, and J. K. Lanyi, "Characterization of the photochemical reaction cycle of proteorhodopsin," Biophys. J. 84, 1202-1207 (2003).
[CrossRef] [PubMed]

B. R. Kelemen, M. Du, and R. B. Jensen, "Proteorhodopsin in living color: diversity of spectral properties within living bacterial cells," Biochim. Biophys. Acta 1618, 25-32 (2003).
[CrossRef] [PubMed]

D. Man, W. Wang, G. Sabehi, L. Aravind, A. F. Post, R. Massana, E. N. Spudich, J. L. Spudich, and O. Béjà, "Diversification and spectral tuning in marine proteorhodopsins," EMBO J. 22, 1725-1731 (2003).
[CrossRef] [PubMed]

J. R. de la Torre, L. M. Christianson, O. Béjà, M. T. Suzuki, D. M. Karl, J. Heidelberg, and E. F. DeLong, "Proteorhodopsin genes are distributed among divergent marine bacterial taxa," Proc. Natl. Acad. Sci. USA 100, 12830-12835 (2003).
[CrossRef] [PubMed]

2002

M. S. Rappé, S. A. Connon, K. L. Vergin, and S. J. Giovannoni, "Cultivation of the ubiquitous SAR11 marine bacterioplankton clade," Nature 418, 630-633 (2002).
[CrossRef] [PubMed]

A. K. Dioumaev, L. S. Brown, J. Shih, E. N. Spudich, J. L. Spudich, and J. K. Lanyi, "Proton transfers in the photochemical reaction cycle of proteorhodopsin," Biochemistry 41, 5348-5358 (2002).
[CrossRef] [PubMed]

R. A. Krebs, A. Alexiev, R. Partha, A. M. DeVita, and M. S. Braiman, "Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin," BMC Physiol. 2:5 (2002).

2001

O. Béjà, E. N. Spudich, J. L. Spudich, M. Leclerc, and E. F. DeLong, "Proteorhodopsin phototrophy in the ocean," Nature 411, 786-789 (2001).
[CrossRef] [PubMed]

2000

O. Béjà, L. Aravind, E. V. Koonin, M. T. Suzuki, A. Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates, R. A. Feldman, J. L. Spudich, E. N. Spudich, and E. F. DeLong, "Bacterial rhodopsin: evidence for a new type of phototrophy in the sea," Science 289, 1902-1906 (2000).
[CrossRef] [PubMed]

1998

J. K. Lanyi, "Understanding structure and function in the light-driven proton pump bacteriorhodopsin," J. Struct. Biol. 124, 164-178 (1998).
[CrossRef]

1982

R. H. Lozier, "Rapid kinetic optical absorption spectroscopy of bacteriorhodopsin photocycles," Methods Enzymol. 88, 133-162 (1982).
[CrossRef]

Biochemistry

A. K. Dioumaev, L. S. Brown, J. Shih, E. N. Spudich, J. L. Spudich, and J. K. Lanyi, "Proton transfers in the photochemical reaction cycle of proteorhodopsin," Biochemistry 41, 5348-5358 (2002).
[CrossRef] [PubMed]

Biochim. Biophys. Acta

B. R. Kelemen, M. Du, and R. B. Jensen, "Proteorhodopsin in living color: diversity of spectral properties within living bacterial cells," Biochim. Biophys. Acta 1618, 25-32 (2003).
[CrossRef] [PubMed]

Biophys. J.

G. Váró, L. S. Brown, M. Lakatos, and J. K. Lanyi, "Characterization of the photochemical reaction cycle of proteorhodopsin," Biophys. J. 84, 1202-1207 (2003).
[CrossRef] [PubMed]

BMC Physiol.

R. A. Krebs, A. Alexiev, R. Partha, A. M. DeVita, and M. S. Braiman, "Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin," BMC Physiol. 2:5 (2002).

EMBO J.

D. Man, W. Wang, G. Sabehi, L. Aravind, A. F. Post, R. Massana, E. N. Spudich, J. L. Spudich, and O. Béjà, "Diversification and spectral tuning in marine proteorhodopsins," EMBO J. 22, 1725-1731 (2003).
[CrossRef] [PubMed]

J. Struct. Biol.

J. K. Lanyi, "Understanding structure and function in the light-driven proton pump bacteriorhodopsin," J. Struct. Biol. 124, 164-178 (1998).
[CrossRef]

Methods Enzymol.

R. H. Lozier, "Rapid kinetic optical absorption spectroscopy of bacteriorhodopsin photocycles," Methods Enzymol. 88, 133-162 (1982).
[CrossRef]

Nature

M. S. Rappé, S. A. Connon, K. L. Vergin, and S. J. Giovannoni, "Cultivation of the ubiquitous SAR11 marine bacterioplankton clade," Nature 418, 630-633 (2002).
[CrossRef] [PubMed]

S. J. Giovannoni, L. Bibbs, J.-C. Cho, M. D. Stapels, R. Desiderio, K. L. Vergin, M. S. Rappé, S. Laney, L. J. Wilhelm, H. J. Tripp, E. J. Mathur, and D. F. Barofsky, "Proteorhodopsin in the ubiquitous marine bacterium SAR11," Nature 438, 82-85 (2005).
[CrossRef] [PubMed]

O. Béjà, E. N. Spudich, J. L. Spudich, M. Leclerc, and E. F. DeLong, "Proteorhodopsin phototrophy in the ocean," Nature 411, 786-789 (2001).
[CrossRef] [PubMed]

M. Stomp, J. Huisman, F. de Jongh, A. J. Veraart, D. Gerla, M. Rijkeboer, B. W. Ibelings, U. I. A. Wollenzien, and L. J. Stal, "Adaptive divergence in pigment composition promotes phytoplankton biodiversity," Nature 432, 104-107 (2004).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. USA

J. R. de la Torre, L. M. Christianson, O. Béjà, M. T. Suzuki, D. M. Karl, J. Heidelberg, and E. F. DeLong, "Proteorhodopsin genes are distributed among divergent marine bacterial taxa," Proc. Natl. Acad. Sci. USA 100, 12830-12835 (2003).
[CrossRef] [PubMed]

Science

O. Béjà, L. Aravind, E. V. Koonin, M. T. Suzuki, A. Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates, R. A. Feldman, J. L. Spudich, E. N. Spudich, and E. F. DeLong, "Bacterial rhodopsin: evidence for a new type of phototrophy in the sea," Science 289, 1902-1906 (2000).
[CrossRef] [PubMed]

Other

K. S. Rowan, Photosynthetic Pigments of Algae (Cambridge University Press, 1989).

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Figures (3)

Fig. 1
Fig. 1

Schematic of the laser-probe transient absorption apparatus. Green 532   nm pump laser (Nd:YAG), right-skew-angle periscope (PER), mirror (M) beam expanding telescope (TELE), cylindrical lens (CYL), blue 488   nm probe laser ( Ar + ) , 488   nm spike filter (SF), red 633   nm probe laser (HeNe), 1   m focal length lens (L), polarizer (POL), dichroic beam splitter (DBS), magic angle polarizer (MAP), shutter (SH), fluorescence submicrocuvette (CUV), achromatic collection lens (ACHR), filter sets (FS), diffuser (DIF), photomultiplier tube (PMT), digital storage oscilloscope (DSO), data acquisition computer (CMPTR), and custom-designed system controller (CTRL). The transmission of the filter sets FS, as measured on the Cary spectrophotometer, are as follows:  488   nm FS, 44% T at 488   nm , 25   nm FWHM, out of passband blocking 200 800   nm , OD > 6; 633   nm FS, 48% T at 633   nm , 10   nm FWHM, out of passband blocking 200 800   nm , OD > 6 . Solid lines indicate electrical connections. Thin dashed lines indicate probe laser beams; slanted double lines indicate that the lasers were placed as far away from the detection electronics as practicable. Thick dashed lines indicate pump laser beam.

Fig. 2
Fig. 2

Static attenuances as a function of wavelength of (a) a membrane preparation of recombinant PR in E. coli, (b) a dark-grown HTCC1062 culture, (c) light-grown HTCC1062 culture I, (d) light-grown HTCC1062 culture II, and (e) a control sample of Synechococcus bacillus. Solid traces represent total sample attenuance (absorption plus scattering, left ordinate) and dashed traces represent the residual attenuance after subtracting a quintic baseline (right ordinate). Vertical dashed lines are located at 532   nm , the wavelength of the transient-inducing pump beam.

Fig. 3
Fig. 3

(Color online) Transient absorption traces for (a) a membrane preparation of recombinant PR in E. coli, (b) a dark-grown HTCC1062 culture, (c) light-grown HTCC1062 culture I, (d) light-grown HTCC1062 culture II, and (e) a control sample of Synechococcus bacillus. Red (HeNe: 633   nm ) absorbance transients are positive, blue ( Ar + : 488   nm ) transients are negative. Dashed curves drawn through the data indicate nonlinear regressions (Table 1) to the kinetic model of Appendix A.

Tables (1)

Tables Icon

Table 1 Nonlinear Regression Coefficients and Their 95% Confidence Intervals from Fitting the Data in Fig. 3 to Eqs. (4) and (5) in Appendix A

Equations (130)

Equations on this page are rendered with MathJax. Learn more.

24 μ mol
m 2 s 1
8.37 × 10 5
1.24 × 10 6   cells   ml 1
1.02 × 10 6   cells   ml 1
0.35   ml
1   cm
0.16   ml
488   nm
Ar +
Ar +
488   nm
1   m
1.5   mm
633   nm
2   mm
1   mm
633   nm
488   nm
7.5 μ W
488   nm
3.0 μ W
633   nm
1   cm
50.8   mm
150   mm
0.25 μ W
532   nm
254   mm
12   mm
12   mm
2 3   mm
1 0   Hz
1 0 mJ / pulse
3 0 mJ / pulse
1 0   mJ
3 0 mJ / pulse
3 0 mJ / pulse
532   nm
532   nm
1 0 mJ / pulse
600   V
100   k Ω
900   mV
3   dB
30 μ s
532   nm
5 10   ns
530   nm
10   nm
( 22   s )
10   Hz
10   Hz
532   nm
633   nm
530   nm
532   nm
530   nm
60   Hz ( Ar + )
120   Hz ( HeNe )
633   nm
60   Hz
Ar +
60   Hz
488   nm
Ar +
60   Hz
Ar +
Δ A 633
Δ A 488
τ O
τ O
Δ A 488
τ O
633   nm
488   nm
488   nm
488   nm
633   nm
488   nm
633   nm
k M
k O
τ M
τ O
PR k fast M k M O k O PR .
488   nm
633   nm
488   nm
633   nm
488   nm
633   nm
Ar +
488   nm
k fast
Δ A 488 ( t ) = C 488 ( k M e k O t k O e k M t ) k M k O ,
C 488
k M
k O
Δ A 488 ( t ) = C 488 [ e k O t ( k O / k M ) e k M t ] ,
k M
Δ A 488 ( t )
e k M t
( k O / k M )
Δ A 488 ( t ) = C 488 e k O t ,
488   nm
633   nm
( τ M )
633   nm
τ O
Δ A 633 ( t ) = k M k M k O C 633 ( e k O t e k M t ) ,
C 633
532   nm
488   nm
( Ar + )
488   nm
633   nm
1   m
488   nm
488   nm
25   nm
200 800   nm
OD > 6; 633   nm
633   nm
10   nm
200 800   nm
OD > 6
532   nm
633   nm
( Ar + : 488   nm )

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