Genomics and Physiology of a Marine Flavobacterium Encoding a Proteorhodopsin and a Xanthorhodopsin-Like Protein

Proteorhodopsin (PR) photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete β-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition.     

Specific Growth Rate Plays a Critical Role in Hydrogen Peroxide Resistance of the Marine Oligotrophic Ultramicrobacterium Sphingomonas alaskensis Strain RB2256

The marine oligotrophic ultramicrobacterium Sphingomonas alaskensis RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as Vibrio angustum S14. This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide. In addition to periods of starvation in the ocean, slow, nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time. In this study we examined the effects of growth rate on the resistance of S. alaskensis RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats. Glucose-limited cultures of S. alaskensis RB2256 at a specific growth rate of 0.02 to 0.13 h⁻¹ exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than cells growing at a rate of 0.14 h⁻¹ or higher. Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism. In contrast, V. angustum S14 did not exhibit growth rate-dependent stress resistance. The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium. Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance. More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h⁻¹. Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance. These studies demonstrated the crucial role that nutrient limitation plays in the physiology of S. alaskensis RB2256, especially under oxidative stress conditions.     

Ultradian Growth in Prochlorococcus spp.

Species of the widespread marine prokaryote Prochlorococcus exhibited ultradian growth (faster than 1 division per day) both in situ and in culture, even though cell division is strictly phased to the light-dark cycle. Under optimal conditions a second DNA replication and cell division closely followed, but did not overlap with, the first division. The timing of cell cycle events was not affected by light intensity or duration, suggesting control by a light-triggered timer or circadian clock rather than by completion of a light-dependent assimilation phase. This mode of ultradian growth has not been observed previously and poses new questions about the regulation of cellular rhythms in prokaryotes. In addition, it implies that conclusions regarding the lack of nutrient limitation of Prochlorococcus in the open ocean, which were based on the appearance that cells were growing at their maximal rate, need to be reconsidered.     

Effects of salinity on the immune response of an ‘osmotic generalist’ bird

Salt stress can suppress the immune function of fish and other aquatic animals, but such an effect has not yet been examined in air-breathing vertebrates that frequently cope with waters (and prey) of contrasting salinities. We investigated the effects of seawater salinity on the strength and cost of mounting an immune response in the dunlin Calidris alpina, a long-distance migratory shorebird that shifts seasonally from freshwater environments during the breeding season to marine environments during migration and the winter period. Phytohaemagglutinin (PHA)-induced skin swelling, basal metabolic rate (BMR), body mass, fat stores, and plasma ions were measured in dunlins acclimated to either freshwater or seawater (salinity: 0.3 and 35.0 ‰, respectively). Seawater-acclimated dunlins mounted a PHA-induced swelling response that was up to 56 % weaker than those held under freshwater conditions, despite ad libitum access to food. Freshwater-acclimated dunlins significantly increased their relative BMR 48 h after PHA injection, whereas seawater-acclimated dunlins did not. However, this differential immune and metabolic response between freshwater- and seawater-acclimated dunlins was not associated with significant changes in body mass, fat stores or plasma ions. Our results indicate that the strength of the immune response of this small-sized migratory shorebird was negatively influenced by the salinity of marine habitats. Further, these findings suggest that the reduced immune response observed under saline conditions might not be caused by an energy or nutrient limitation, and raise questions about the role of osmoregulatory hormones in the modulation of the immune system.     

Interaction between Light Intensity and NaCl Salinity and Their Effects on Growth, CO(2) Assimilation, and Photosynthate Conversion in Young Broad Beans

Seedings of Vicia faba were grown for four weeks at two different light intensities (55 and 105 watts per square meter) in a saline (50 millimolar NaCl) and nonsaline nutrient solution. NaCl salinity depressed growth and restricted protein formation, CO₂ assimilation, and especially the incorporation of photosynthates into the lipid fraction. Conversion of photosynthates in leaves was much more affected by salinity than was photosynthate turnover in roots. The detrimental effect of NaCl salinity on growth, protein formation, and CO₂ assimilation was greater under low than under high light conditions. Plants of the high light intensity treatment were more capable of excluding Na⁺ and Cl⁻ and accumulating nutrient cation species (Ca²⁺, K⁺, Mg²⁺) than plants grown under low light intensity. It is suggested that the improved ionic status provided better conditions for protein synthesis, CO₂ assimilation, and especially for the conversion of photosynthates into lipids.     

Specific growth rate plays a critical role in hydrogen peroxide resistance of the marine oligotrophic ultramicrobacterium sphingomonas alaskensis strain RB2256

The marine oligotrophic ultramicrobacterium Sphingomonas alaskensis RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as Vibrio angustum S14. This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide. In addition to periods of starvation in the ocean, slow, nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time. In this study we examined the effects of growth rate on the resistance of S. alaskensis RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats. Glucose-limited cultures of S. alaskensis RB2256 at a specific growth rate of 0.02 to 0.13 h(-1) exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than cells growing at a rate of 0.14 h(-1) or higher. Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism. In contrast, V. angustum S14 did not exhibit growth rate-dependent stress resistance. The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium. Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance. More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h(-1). Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance. These studies demonstrated the crucial role that nutrient limitation plays in the physiology of S. alaskensis RB2256, especially under oxidative stress conditions.     

Phenotypic Plasticity of Southern Ocean Diatoms: Key to Success in the Sea Ice Habitat?

Diatoms are the primary source of nutrition and energy for the Southern Ocean ecosystem. Microalgae, including diatoms, synthesise biological macromolecules such as lipids, proteins and carbohydrates for growth, reproduction and acclimation to prevailing environmental conditions. Here we show that three key species of Southern Ocean diatom (Fragilariopsis cylindrus, Chaetoceros simplex and Pseudo-nitzschia subcurvata) exhibited phenotypic plasticity in response to salinity and temperature regimes experienced during the seasonal formation and decay of sea ice. The degree of phenotypic plasticity, in terms of changes in macromolecular composition, was highly species-specific and consistent with each species’ known distribution and abundance throughout sea ice, meltwater and pelagic habitats, suggesting that phenotypic plasticity may have been selected for by the extreme variability of the polar marine environment. We argue that changes in diatom macromolecular composition and shifts in species dominance in response to a changing climate have the potential to alter nutrient and energy fluxes throughout the Southern Ocean ecosystem.     

Light Enhances Survival of Dinoroseobacter shibae during Long-Term Starvation

Aerobic anoxygenic phototrophs (AAPs) as being photoheterotrophs require organic substrates for growth and use light as a supplementary energy source under oxic conditions. We hypothesized that AAPs benefit from light particularly under carbon and electron donor limitation. The effect of light was determined in long-term starvation experiments with Dinoroseobacter shibae DFL 12^T in both complex marine broth and defined minimal medium with succinate as the sole carbon source. The cells were starved over six months under three conditions: continuous darkness (DD), continuous light (LL), and light/dark cycle (LD, 12 h/12 h, 12 µmol photons m⁻² s⁻¹). LD starvation at low light intensity resulted in 10-fold higher total cell and viable counts, and higher bacteriochlorophyll a and polyhydroxyalkanoate contents. This coincided with better physiological fitness as determined by respiration rates, proton translocation and ATP concentrations. In contrast, LD starvation at high light intensity (>22 µmol photons m⁻² s⁻¹, LD conditions) resulted in decreasing cell survival rates but increasing carotenoid concentrations, indicating a photo-protective response. Cells grown in complex medium survived longer starvation (more than 20 weeks) than those grown in minimal medium. Our experiments show that D. shibae benefits from the light and dark cycle, particularly during starvation.     

Growth and toxin production in batch cultures of a marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea

Nutritional and environmental conditions were characterized for a batch culture of the marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea for its growth (cells ml⁻¹), cellular toxin content (Qt in fmol cell⁻¹) and toxin composition (mol%). Under a nutrient replete condition, Qt increased with cell growth and peaked at the late stationary phase. Toxin content increased with the nitrate concentration in the culture while it reached a maximum at 5 μM phosphate. When nitrate was replaced with ammonia, Qt decreased by 4.5-fold. Salinity and light intensity were important factors affecting Qt. The latter increased two-fold over the range of salinity from 15 to 30‰, while decreased 38% as light intensity increased from 80 to 220 μE m⁻² s⁻¹. Toxin composition varied with growth phase and culture conditions. In nutrient replete cultures, toxin composition varied greatly in the early growth phase (first 3 days) and then C1/C2, C3/C4 and GTX1 remained relatively constant while GTX4 increased from 32 to 46% and GTX5 decreased from 28 to 15%. In general, the composition of GTXs was affected in a much greater extent than C toxins by changes in nutrient conditions, salinity and light intensity. This is especially true with GTX4 and GTX5. These data indicate that the cellular toxin content and toxin composition of A. tamarense HK9301 are not constant, but that they vary with growth phase and culture conditions. Use of toxin composition to identify a toxigenic marine dinoflagellate is not always valid. The data also reveal that high salinity and low light intensity, together with high nitrate and low phosphate concentrations, would favor toxin production by this species.     

Gas-Exchange Properties of Salt-Stressed Olive (Olea europea L.) Leaves

The effects of two levels of salinity on photosynthetic properties of olive (Olea europea L.) leaves were observed either in low or in high H₂O vapor pressure deficit (vpd). Under moderate salt stress, stomata were found to be less open and responsive both to light and vpd, but the predominant limitation of photosynthesis was due to the mesophyll capacity of CO₂ fixation. We elaborate a procedure to correlate mesophyll capacity and liquid phase diffusive conductance. The estimated liquid phase diffusive conductance was reduced by salt and especially by high vpd; morphological and physiological changes could be responsible for this reduction. As a result, the chloroplast CO₂ partial pressure was found to decrease both under salt and vpd stress, thus resulting in a ribulose-1,5-bisphosphate carboxylase limitation of assimilation. However, under combined salt and vpd stress, O₂ sensitivity of assimilation increased, as would be expected under conditions of limiting ribulose 1,5-bisphosphate regeneration. Fluorescence induction measurements indicated that, under these conditions, energy supply may become limiting. When Cl⁻ concentration exceeded 80 millimolar in tissue water, zero growth and 50% leaf drop was observed. Fluorescence induction showed irreversible damage at Cl⁻ levels higher than 200 millimolar and basal leaves reached this concentration earlier than the apical ones.     

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species (Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation; cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms caused by two species in the Yangtze River Estuary.     

DISSOLVED ORGANIC PHOSPHORUS EXCRETION BY MARINE PHYTOPLANKTON1,2

The amounts of extracellular dissolved organic phosphorus (DOP) produced by 8 species of marine planktonic algae under various conditions of light, salinity, and nutrition were compared. Large amounts, more than 20% of the total P in the system, were excreted by Cyclotella cryptica, Thalassiosira fluviatilis, Dunaliella tertiolecta, and Synechococcus under 1 or more of the experimental conditions. Excretion of DOP was proportional to light intensity in Dunaliella, Rhodomonas, Chlorella sp., and Coccolithus huxleyi. Phosphorus limitation reduced DOP production by Cyclotella and Thalassiosira, nitrogen limitation reduced DOP production by Phaeodactylum, Dunaliella, and Rhodomonas, and lack of iron reduced DOP levels in Cyclotella cultures. Salinity affected growth, but no clear relationship to DOP excretion was evident. The DOP eliminated during growth was reassimilated by the species that produced it and by other species, but lack of alkaline phosphatase reduced the amount of DOP that was available to certain algae.     

STATE TRANSITIONS AND NONPHOTOCHEMICAL QUENCHING DURING A NUTRIENT‐INDUCED FLUORESCENCE TRANSIENT IN PHOSPHORUS‐STARVED DUNALIELLA TERTIOLECTA1

Assessments of nutrient‐limitation in microalgae using chl a fluorescence have revealed that nitrogen and phosphorus depletion can be detected as a change in chl a fluorescence signal when nutrient‐starved algae are resupplied with the limiting nutrient. This photokinetic phenomenon is known as a nutrient‐induced fluorescence transient, or NIFT. Cultures of the unicellular marine chlorophyte Dunaliella tertiolecta Butcher were grown under phosphate starvation to investigate the photophysiological mechanism behind the NIFT response. A combination of low temperature (77 K) fluorescence, photosynthetic inhibitors, and nonphotochemical quenching analyses were used to determine that the NIFT response is associated with changes in energy distribution between PSI and PSII and light‐stress‐induced nonphotochemical quenching (NPQ). Previous studies point to state transitions as the likely mechanism behind the NIFT response; however, our results show that state transitions are not solely responsible for this phenomenon. This study shows that an interaction of at least two physiological processes is involved in the rapid quenching of chl a fluorescence observed in P‐starved D. tertiolecta: (1) state transitions to provide the nutrient‐deficient cell with metabolic energy for inorganic phosphate (P_i)‐uptake and (2) energy‐dependent quenching to allow the nutrient‐stressed cell to avoid photodamage from excess light energy during nutrient uptake.     

EFFECT OF IRON NUTRITION ON THE MARINE CYANOBACTERIUM SYNECHOCOCCUS GROWN ON DIFFERENT N SOURCES AND IRRADIANCES1

The effects of Fe deficiency on the marine cyanobacterium Synechococcus sp. were examined in batch cultures grown on nitrate or ammonium as a sole nitrogen source under two different irradiances. Fe-stressed cells showed lower chlorophyll a content and cellular C and N quotas. Light limitation increased the critical iron concentration below which both suppression of growth rate and changes in cellular composition were observed. At a limiting irradiance (26 μmol.m⁻².s⁻¹), this critical value was ∼10 nM, a 10 times increase compared to high-light cultures. Moreover, at low light the cellular chlorophyll a concentration was higher than at saturating light (110 μmol.m⁻².s⁻¹), this difference being most pronounced under Fe-stressed conditions. Cells grown on ammonium showed a lower half-saturation constant for Fe (K_s) compared to cells grown on nitrate, indicating Synechococcus sp. has the ability to grow faster on ammonium than on nitrate in a low Fe environment at high light. Consequently, in high-nutrient and low-chlorophyll regions where Fe limits new production, cyanobacteria most likely grow on regenerated ammonium, which requires less energy for assimilation. The K_s for growth on Fe at low light was significantly higher than at high light compared with the cells grown on the same N source, suggesting the cells require more Fe at low light. Therefore, if cells that are already Fe-limited also become light-limited, their iron stress level will increase even more. For cyanobacteria this is the first report of a study combining the interactions of Fe limitation, light limitation, and nitrogen source (NO₃⁻ vs. NH₄⁺).     

Factors controlling the production of domoic acid by Pseudo-nitzschia (Bacillariophyceae): A model study

A mechanistic model has been developed to explore the factors controlling the production of domoic acid (DA) by the pennate diatom Pseudo-nitzschia. The idealized model allows consideration of the uncoupling between photosynthesis and growth, while DA production has been set as a secondary metabolism sharing common precursors with growth. Under growth limitation, these precursors can accumulate, resulting in an increased DA production. The model was first evaluated based on its ability to simulate the observed DA production by either silicon (Si) or phosphorus (P) limited batch cultures of Pseudo-nitzschia available in the literature. Sensitivity tests were further performed to explore how the ambient nutrients and the light regime (intensity and photoperiod length) are possibly directing the Pseudo-nitzschia toxicity. The general pattern that emerged is that excess light, in combination with Si or P limitation, favours DA production, provided nitrogen (N) is sufficient. Model simulations with varying nutrient stocks supporting Pseudo-nitzschia blooms under non-limiting light suggest two potential ways for nutrients to control DA production. First, N excess in comparison to available Si and P relieves DA production from its limitation by N, an absolute requirement of the DA molecule. Second, increased nutrient stocks amplify the DA production phase of the blooms (in addition to enhancing Pseudo-nitzschia biomass) which leads to an even more toxigenic bloom. Simulations investigating the light regime suggest a light threshold below which an important delay in DA production could be expected in Pseudo-nitzschia cultures. In the natural environment, the monitoring of light conditions during Pseudo-nitzschia blooms might help to anticipate the magnitude of the toxic event. Pseudo-nitzschia toxicity is indeed linked to the excess of primary carbon that accumulates during photosynthesis under growth limitation by nutrients.