The discovery of proteorhodopsin, a new form of prototrophy in the sea

OLD Audio recording

Video recording (.mov format, 2.0Gbytes)
Video recording (480p .mp4 format, 0.3Gbytes)
Video recording (1080p .mp4 format, 1.6Gbytes

 

Beja O, et al. 2000 Bacterial rhodopsin: Evidence for a new type of phototrophy in the sea. Science 289:1902-1906.


Question : What is the ecological role of the SAR86 rDNA sequence group?
.

This paper addresses the problem of linking microbial processes to specific organisms using the genomic approach; fishing out big pieces of DNA that contain both phylogenetic information (a copy of the ssu-rRNA gene) and phenotypic information (in this case, a gene for phototrophy). Because both types of genes come from single pieces of DNA, they must be from the same organism.

In this paper, the authors already know from molecular phylogenetic analysis of ocean water that members of the "SAR86" group of gamma-proteobacteria are abundant worldwide, but had no idea what their role in the ecology of the ocean might be. So, again, the question is linking a particular phylogenetic group (the known in this case) to a particular ecological process (the unknown in this case).

In attempt to find out what SAR86 is doing in the ocean, they made a cosmid bank of DNA isolated from ocean water. These cosmid clones contain DNA fragments more than 100Kbp in length. They screened these cosmids by hybridization to identify those that contained a gene for ssu-rRNA, so they could identify the organism the DNA fragment came from. One such cosmid clone (EBAC31A08) proved to be a member of the SAR86 group, based on phylogenetic analysis of this ssu-rRNA sequence, and the authors therefore sequenced the entire 130Kbp DNA fragmen, hoping to find genes that would provide clues about the metabolism of the organism. This isn't as hopeless as you might think at first - most gamma-proteobacteria have a genome size of 2- 4Mbp and have about 4-7 rRNA operons, so that on average about 20% of the genome is within 130Kbp of an rRNA gene. Another way top look at it is that the average size of a gene is about 1Kbp, so they expected to get about 130 genes - the chances are good that 130 genes chosen randomly from a bacterial genome will give you clues about it's phenotype.

(As an aside, if you want to look at this chunk of sequence and all the genes it contains, here's a link to it: http://www.ncbi.nlm.nih.gov/nuccore/AF279106.2 )

All the same, they were successful beyond any reasonable expectation. One of the genes they found on their cosmid seemed to be a gene encoding a rhodopsin, presumably originating by horizontal transfer from a halophilic archaeon. Halophilic Archaea have 3 types of rhodopsin. The main one is bacteriorhodopsin, the light-driven proton pump they use to capture energy (via the proton motive force) from light. Another is halorhodopsin, a light driven chloride pump used to bailing Cl- out of the cell (cells use organic anions like glutamate rather than harsher salt anions like chloride). They also have a sensory rhodopsin, used along with a signal transducer protein to signal the presence of sufficient light to justify turning on the genes for the other rhodopsins and retinal (the cofactor for all of these rhodopsins). Rhodopsin genes have also been transfered to eukaryotes at least once - the mold Neurospora has an archaeal-derived rhodopsin it uses for light sensing to control its diurnal cycle. It may (may!) also be that the opsins used in vision in animals originated from one of the archaeal genes. Could it be that these organisms are using this rhodopsin to grow phototrophically? Is primary production in the ocean based on two kinds of photosynthesis instead of just one?

Figure 1 is a pair of trees based on the ssu-rDNA (panel A) and the rhodopsin gene (panel B). The rDNA tree is just shown to demonstrate that the genome fragment comes from an organism of the SAR86 group. The rhodopsin tree is an attempt to determine the likely function of the rhodopsin - is it related to proton-pumping, chloride-pumping, or sensory rhodopsins? The tree suggests that it might be in the sensory rhodopsin group, but this is not a strong association. We'll get back to this issue - the biochemical properties of the protein expressed in E.coli are not those of a sensory rhodopsin but those of a proton-pumping rhodopsin. Also note that the sequence is not related to the Neurospora rhodopsin gene NOP1.

In figure 2, they show that the predicted secondary structure of the "proteorhodopsin" is consistent with that of a bona fide opsin, and contains the conserved amino acids needed to bind it's cofactor, retinal.

In figure 3, they show that if they express this protein in E.coli and add retinal (E.coli doesn't make retinal), the cells quickly turn a hue of red (Absorbance max of 520nm) consistent with a rhodopsin. In other words, the protein as expressed by E.coli is correctly folded and inserted into the membrane in a form that can correctly bind the cofactor.

But does it pump protens? Figure 4 shows that E.coli with both rhodopsin and retinal pumps protons from inside to outside (as measured by the change in pH of the media) when and only when provided with light. They use TTP uptake by rhodopsin/retinal containing vesicles to measure the electrical potential generated : -90mV, which is consistent with a strong proton pump.

In figure 5 they dissect the reaction cycle photometrically to show that this looks like a proton or chloride pump rather than a sensory rhodopsin. Sensory opsins have a slow reaction cycle, >300msec. The longer the recycling time, the longer the signal is sent to its associated transducer/regulator protein. This is why the rod cells in your eyes allow you to see better in the dark - their opsins have a longer reaction cycle than those of the cone cells. Proton or chloride pump rhodopsins, however, have short reaction cycles, <20msec, so they're ready to absorb another photon & pump another ion as quickly as possible. Panel A shows absorbance changes in rhodopsin-containing E.coli over time after being pulsed with a 532nm laser. Absorption increased at 400nm for a very short time, <5mec - this is the activated retinal (the "M" intermediate). The increase in absorbance at 590nm, which increases at the same time scale as the M-intermediate decays and decays with a halflife of 15msec, is another intermediate in the light cycle of retinal, the "O" intermediate. The decrease in absorbance at 520 returns to normal in the 15msec timescale too, so the O-to-groundstate transition seems to be the rate-limiting step of the photocycle, as it is in proton-pumping bacteriorhodopsins. The timescale of the cycle is clearly that of a pump rather than sensory opsin.

So, this seems to be a functional, light-driven proton-pumping rhodopsin in a group of uncultivated gamma-proteobacteria that make up as much as 10% of the biomass of the ocean surface water. In a subsequent paper, Ed Delong's lab shows that they can readily detect this rhodopsin (spectroscopically) in bacteria from ocean water, show that it actually works in cells isolated directly from the ocean, that the rhodopsin is present in large enough amounts to provide the energy a cell needs, and showed that deepwater and surface water organisms produce rhodopsins that are tuned to the differences in the wavelengths of light that are available to them. Furthermore, they also showed by cloning and sequencing other SAR86 genome chunks in cosmids that different SAR86 types have different proteorhodopsin genes, presumably tuned by wavelength.

So, the SAR86 organisms seem to be phototrophic using rhodopsin! As abundant as they are in the ocean, this represents a huge ecological impact. But are they also photosynthetic? That is, can they fix carbon, are they primary producers? So far, the answer seems to be "no". No cultivated SAR 11 organisms (there aren't many, and they're not easy to work woth) can fix carbon. So they must be getting organic carbon for growth, but using light and proteorhodopsin for energy. This means, of course, that they are photoheterotrophs, like most purple non-sulfur Bacteria.

But this isn't the end of the story. It turns out a lot of marine Bacteria from many phylogenetic groups have proteorhodopsin genes, apparently acquired by horizontal transfer. This gene seems to move around a lot. Phototrophy via rhodopsins may turn out to be an important part of energy collection, and perhaps also primary production, in the oceans. Given that many of these other organisms are probably capable of carbon fixation, this may represent an important, and previously unsuspected, component of primary production in the ocean.