Spectroscopy. Evidence of a very slight absorption peak between 500 and 600 nm was observed in suspensions of whole P. ubique cells. The flash photolysis technique was used because the PR transient signature can be identified on top of the steady-state absorption of the sample, even when the absorption of PR itself is too weak to be observed in a normal absorption spectrum.

Mass Spectrometry. At first, a single peptide (P3) was detected in the membrane fraction by LC/MS/MS. In-gel tryptic digestions of the whole seawater protein mixture followed by LC/MS/MS led to the identification of peptides P2 and P3 in the fraction of the gel that corresponded to the predicted molecular weight of the PR protein (27,700 Da).

Light-dependent pH shifts. Cloned proteorhodopsin from HTCC1062 (see methods for details) was grown in four separate 150 or 350 ml cultures containing LB broth and ampicillin for 1 hr at 37ºC and 250 rpm shaking. L-arabinose and retinal were added to the indicated cultures for final concentrations of 0.002% and 1 µM, respectively, and cultures were further incubated for four hours. Cultured cells were sedimented by centrifugation (3,400X g for 10 min in a JA-17 rotor at 4ºC) and resuspended in 0.5 vol of a cell suspension solution (10mM NaCl, 10 mM MgSO4, 0.1 mM CaCl2, and NaOH to pH 7.0). Cell sedimentation and resuspension were repeated as described two times except the final resuspension was in a volume resulting in a final OD600 between 3.9 and 4.4. pH was measured using a Denver Instruments model 215A pH meter (Fisher Scientific, Pittsburgh, PA) and a needle tip micro pH electrode (Thermo Orion, Waltham, MA). Cells were placed in an OX1LP water-jacketed cuvette, maintained at 18ºC, and stirred using a magnetic stir bar (Qubit Systems, Kingston, Ont., Can.). The beam from an argon laser (Omnichrome 532, Melles Griot, Carlsbad, California) emitting 20 mW of power at a wavelength of 514.5 nm was expanded to illuminate the entire cuvette sample volume to provide the light stimulus.

SupplementaryFigure Legends.

Fig. S1.A. Graphical representation of genes in a 9.71 kb region surrounding the Pelagibacter proteorhodopsin. Numbers indicate percent of synteny between the P. ubique genome and a set of 519 environmental contigs from the Sargasso Sea that contained PR genes. A, quinolinate synthetase A; B, glycine cleavage T protein; C, dihydroorotase; D, small multidrug resistance protein; E, acyl-coenzyme A synthetase/AMP-(fatty) acid ligase; PR – proteorhodopsin; F, ferredoxin, 2Fe-2S; G, thioredoxin-disulfide reductase; H, glutathione S-transferase; I, MOSC domain family protein; J, DnaK suppressor protein. B.The P. ubique PR aligned with its most similar homolog from public databases and the SAR86 proteorhodopsin discovered by Béjà, et al.1. The P. ubique PR differs at 46 positions from its closest neighbor, IBEA 2057454, from the Venter Sargasso Sea data set. Asterisks mark the positions of conserved residues involved in Schiff’s base transfer reactions. Position 105 is involved in spectral tuning. “R” and “N” indicate the positions of amino acid substitutions found in the proteorhodopsins from ten cultivated strains of P. ubique.

Fig. S2. Proton pump activation by light stimulus. Black – proteorhodopsin expressed, retinal added; Green – proteorhodopsin expressed, retinal not added; Red – proteorhodopsin not expressed, retinal added; Blue – proteorhodopsin not expressed, retinal not added. Cells were exposed to light and dark treatments for 3 minutes each as indicated by the arrows on the x axis.

Table S1. HTCC1062 proteorhodopsin peptides observed by mass spectrometry.

Peptide / Observed
m/z* / Mr (expt) (u)† / Mr(calc) (u)‡ / Delta
(u)¶ / ppm# / Ion
Score§ / Sequence / Culture / Seawater
P1:57-64 / 843.461 / 842.451 / 842.44 / 0.011 / 13 / 44 / ASVPAGWR / + / -
P2:86-99 / 1639.821 / 1638.811 / 1638.77 / 0.041 / 25 / 33 / DVWVMTGESPTVYR / + / +
P3:187-196 / 1036.581 / 1035.571 / 1035.55 / 0.021 / 20 / 40 / ALVTAFGAMR / + / +

*Mass to charge ratio observed in mass spectrum.

†Average mass determined from observed m/z in unified atomic mass units.

‡Average mass calculated from amino acid sequence in unified atomic mass units.

¶Delta = Mr (expt) - Mr (calc).

#ppm = Delta/Mr (calc).

§Probability-based ion score from the database search engine Mascot2 (Matrix Science Ltd., London, UK); in general, ion scores below 30 were not accepted.

Supplementary Table S2. Variation in proteorhodopsin
amino acid sequence among Pelagibacter isolates
HTCCStrain / Position 150* / Position 224*
1062 / G / S
1002 / G / N
1013 / G / S
1016 / G / S
1025 / G / N
1040 / G / N
1051 / G / N
1056 / G / N
1057 / R / N
1061 / G / N

* Sequence position numbering relative to strain HTCC1062

Fig. S1



Fig. S2.


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