Russell A. Desiderio

Proteorhodopsin in the ubiquitous marine bacterium SAR11

Proteorhodopsins are light-dependent proton pumps that are predicted to have an important role in the ecology of the oceans by supplying energy for microbial metabolism. Proteorhodopsin genes were first discovered through the cloning and sequencing of large genomic DNA fragments from seawater. They were later shown to be widely distributed, phylogenetically diverse, and active in the oceans. Proteorhodopsin genes have not been found in cultured bacteria, and on the basis of environmental sequence data, it has not yet been possible to reconstruct the genomes of uncultured bacterial strains that have proteorhodopsin genes. Although the metabolic effect of proteorhodopsins is uncertain, they are thought to function in cells for which the primary mode of metabolism is the heterotrophic assimilation of dissolved organic carbon. Here we report that SAR11 strain HTCC1062 (‘Pelagibacter ubique’), the first cultivated member of the extraordinarily abundant SAR11 clade, expresses a proteorhodopsin gene when cultured in autoclaved seawater and in its natural environment, the ocean. The Pelagibacter proteorhodopsin functions as a light-dependent proton pump. The gene is expressed by cells grown in either diurnal light or in darkness, and there is no difference between the growth rates or cell yields of cultures grown in light or darkness.

The SAR92 Clade: an Abundant Coastal Clade of Culturable Marine Bacteria Possessing Proteorhodopsin

ABSTRACT Proteorhodopsin (PR) is a protein that is abundant in marine bacterioplankton. PR is hypothesized to be a light-dependent proton pump, thus creating a proton gradient that can be used for energy production without electron transport. Currently, the only culture that has been reported to possesses PR is the highly abundant alphaproteobacterium “Candidatus Pelagibacter ubique” (SAR11 clade), but surprisingly, its growth in batch culture was not enhanced by light. Here, we present the first cultured gammaproteobacterium that possesses a PR gene. Genome sequencing and analysis of HTCC2207 showed that the PR gene is present as a lone transcriptional unit directly followed by an operon containing genes that are presumably involved in the synthesis of retinal, the chromophore of PR. Half-time decay times of different PR intermediates in native HTCC2207 cells ranged between 2 and 15 ms, and the absorbance maximum of PR was determined to be 528 nm. Proteorhodopsin was identified in three additional strains, using a specific PCR assay on other cultured members of the SAR92 clade. Phylogenetic analyses of the PR genes determined that they form a deeply rooting cluster not closely related to any PR genes recovered so far. Fluorescence in situ hybridization and RNA blots showed that the SAR92 clade reaches up to 10% of the total bacterial population in surface waters close to the Oregon coast and decreases over depth and distance from the shore. Although the growth of HTCC2207 is limited by the amount of available carbon that is present in the medium applied, these cultures do not grow at higher rates nor do they have higher growth yields when incubated under light.

Comparison of some fiber optic configurations for measurement of luminescence and Raman scattering

Measurements are made for a number of dual fiber optic configurations to determine their relative sensitivity using bare fibers and graded-refractive-index lenses. An analysis of the background fiber emission for a typical silica-on-silica fiber (Diaguide, 200-microm core) is presented, and the origin (core or cladding) for several prominent Raman peaks is determined. Also, a forward-scattering fiber geometry is introduced, and the dependence of sensitivity on the type of optical termination and fiber separation is determined.

Multiple excitation fluorometer for in situ oceanographic applications

A new in situ fluorometer for the detection of oceanic photosynthetic pigment fluorescence is described. Emission spectra from 546 to 733 nm are recorded for each of three different visible excitation bands ten times a second. A Spectralon cell is used to improve the excitation coupling to and the collection efficiency from the sample volume. Laboratory tests demonstrated that the fluorescence emission spectra from the violet, blue, and green excitation can be used to discriminate among various algal species. The instrument was used at sea in extended in situ deployments on an undulating vehicle (SeaSoar).

Microstructure Profiles of Laser-induced Chlorophyll Fluorescence Spectra: Evaluation of Backscatter and Forward-Scatter Fiber-Optic Sensors

Abstract The implementation and characterization of backscatter and forward-scatter fiber-optic fluorescence sensors attached to a microstructure profiling instrument are described. By using an optical multichannel array detector to record emission spectra, eight profiles per hour of chlorophyll fluorescence spectra at 2-cm intervals can be obtained throughout the upper 100 m of ocean. The advantages and disadvantages realized by the deployment of the two sensors are compared with respect to the determination of in situ chlorophyll concentrations and fluorescence microstructure in the ocean. It is concluded that the forward-scatter sensor has better signal-to-noise and signal variability characteristics than does the backscatter sensor and is less susceptible to nonlinear fluorescence responses of phytoplankton due to effects such as fluorescence induction.

In situ monitoring of ocean chlorophyll via laser-induced fluorescence backscattering through an optical fiber

The first seagoing test of a prototype laser/fiber-optic system for in situ detection of ocean chlorophyll fluorescence is described. Radiation at 488 nm originating from a shipboard argon laser was transmitted through 20 of 200-microm core optical fiber to the distal tip mounted on the microstructure profiler, the Rapid Sampling Vertical Profiler. The backscattered fluorescence emission signal was collected through the same fiber and processed on board ship. A series of measurements indicated that (1) successful isolation of shipinduced vibrations could be achieved using our optical bench framework to maintain optical alignments; (2) ambient chlorophyll concentrations could be detected in situ; (3) a Raman scattering signal from water could also be detected and should provide an internal standard against which chlorophyll fluorescence may be calibrated.

Measuring the Natural Fluorescence of Phytoplankton Cultures

A laboratory instrument, the Natural Fluorescence Chemostat, was developed to measure the natural fluorescence of phytoplankton cultures. With this instrument, the physical and chemical environment of a culture can be manipulated with respect to temperature, pH, nutrient delivery rate, and light intensity, while the natural fluorescence and a weak stimulated fluorescence are continuously recorded with high temporal resolution. The geometry and spectral distribution of the artificial light field minimize the contribution of scattering to the natural fluorescence signal. Preliminary investigations with the marine diatom T. weissflogii (Bacillariophyceae) indicate that the instrument can detect natural fluorescence signals in broadband artificial light fields as bright as 1250 mmol quanta m22 s 21. Since the influence of environmental factors on natural fluorescence is not well understood, laboratory experiments are essential for investigating how ocean physics and chemistry influence this signal. This instrument provides a quantitative means to examine how the magnitude and kinetics of phytoplankton natural fluorescence vary in response to changes in the physical and chemical environment.

Application of the Raman scattering coefficient of water to calculations in marine optics.

The relation between the total Raman scattering coefficient of water (b(r)) and the Raman scattering cross section of liquid water is clarified. Consideration of the experimental configurations used to derive the Raman scattering cross section of water relative to benzene leads to a revised value of b(r) for water. In certain applications in marine optics it is necessary to scale b(r) to effect a change from implicit units of energy to quanta.

Fluorescence microstructure using a laser/fiber optic profiler

Field tests were conducted during Aug/Sept 1989 of the second generation of a laser/fiber optic fluororneter attached to a microstructure proffling instrurnent (Rapid Sampling Vertical Profiler - RSVP). This instrumentation is designed to provide centirneter-scale biological measurements coincident with and at the same sampling frequency as those for temperature, conductivity, and horizontal velocity microstructure. The instrument used during the summer of 1989 employed a 200m dual fiber system, SMA optical fiber connectors used underwater, and an optrode (12mm diameter) containing two fibers, GRIN lenses, and filters. Shipboard laser excitation (488nrn or 514nm) was transmitted down a 2OOtm core excitation fiber, and the fluorescence emission spectrum returned via a 400pm core detection fiber to a shipboard multichannel array detector. Fluorescence emission spectra were acquired 30 times per second at an instrument drop rate of approximately 60 cm per second.

Using lasers to probe the transient light absorption by proteorhodopsin in marine bacterioplankton

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.