ORGANIZATION OF THE nif GENES OF THE NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS101

An approximately 16-kb fragment of the Trichodesmium sp. IMS101 (a nonheterocystous filamentous cyanobacterium) "conventional" nif gene cluster was cloned and sequenced. The gene organization of the Trichodesmium and Anabaena variabilis vegetative ( nif 2 ) nitrogenase gene clusters spanning the region from nif B to nif W are similar except for the absence of two open reading frames (ORF3 and ORF1) in Trichodesmium . The Trichodesmium nif EN genes encode a fused Nif EN polypeptide that does not appear to be processed into individual Nif E and Nif N polypeptides. Fused nif  EN genes were previously found in the A. variabilis nif 2 genes, but we have found that fused nif EN genes are widespread in the nonheterocystous cyanobacteria. Although the gene organization of the nonheterocystous filamentous Trichodesmium nif gene cluster is very similar to that of the A. variabilis vegetative nif 2 gene cluster, phylogenetic analysis of nif sequences do not support close relatedness of Trichodesmium and A. variabilis vegetative ( nif 2 ) nitrogenase genes.     

GROWTH AND NITROGEN FIXATION OF THE DIAZOTROPHIC FILAMENTOUS NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS 101 IN DEFINED MEDIA: EVIDENCE FOR A CIRCADIAN RHYTHM1

Trichodesmium sp. IMS 101, originally isolated from coastal western Atlantic waters by Prufert-Bebout and colleagues and maintained in seawater-based media, was successfully cultivated in two artificial media. Its characteristics of growth, nitrogen fixation, and regulation of nitrogen fixation were compared to those of natural populations and Trichodesmium sp. NIBB 1067. Results indicate that the culture grown in artificial media had nitrogen fixation characteristics similar to those when the culture is grown in seawater-based medium and to those of Trichodesmium sp. in the natural habitat. The study provides practical artificial media to facilitate the physiological studies of these important diazotrophic cyanobacteria, as well as the cultivation of other Trichodesmium species in future studies. Manipulations of the light/dark cycle were performed to determine whether or not the daily cycle of nitrogen fixation is a circadian rhythm. Cultures grown under continuous light maintained the cycle for up to 6 days. We demonstrated that the daily cycle of nitrogen fixation in Trichodesmium sp. IMS 101 was at least partially under the control of a circardian rhythm.     

Light‐dependent oxygen consumption in nitrogen‐fixing cyanobacteria plays a key role in nitrogenase protection1

All colonial diazotrophic cyanobacteria are capable of simultaneously evolving O₂ through oxygenic photosynthesis and fixing nitrogen via nitrogenase. Since nitrogenase is irreversibly inactivated by O₂, accommodation of the two metabolic pathways has led to biochemical and/or structural adaptations that protect the enzyme from O₂. In some species, differentiated cells (heterocysts) are produced within the filaments. PSII is absent in the heterocysts, while PSI activity is maintained. In other, nonheterocystous species, however, a “division of labor” occurs whereby individual cells within a colony appear to ephemerally fix nitrogen while others evolve oxygen. Using membrane inlet mass spectrometry (MIMS) in conjunction with tracer ¹⁸O₂ and inhibitors of photosynthetic and respiratory electron transport, we examined the light dependence of O₂ consumption in Trichodesmium sp. IMS 101, a nonheterocystous, colonial cyanobacterium, and Anabaena flos‐aquae (Lyngb.) Bréb. ex Bornet et Flahault, a heterocystous species. Our results indicate that in both species, intracellular O₂ concentrations are maintained at low levels by the light‐dependent reduction of oxygen via the Mehler reaction. In N₂‐fixing Trichodesmium colonies, Mehler activity can consume ～75% of gross O₂ production, while in Trichodesmium utilizing nitrate, Mehler activity declines and consumes ～10% of gross O₂ production. Moreover, evidence for the coupling between N₂ fixation and Mehler activity was observed in purified heterocysts of Anabaena, where light accelerated O₂ consumption by 3‐fold. Our results suggest that a major role for PSI in N₂‐fixing cyanobacteria is to effectively act as a photon‐catalyzed oxidase, consuming O₂ through pseudocyclic electron transport while simultaneously supplying ATP in both heterocystous and nonheterocystous taxa.     

Elevated CO2 enhances nitrogen fixation and growth in the marine cyanobacterium Trichodesmium

The increases in atmospheric pCO₂ over the last century are accompanied by higher concentrations of CO₂(aq) in the surface oceans. This acidification of the surface ocean is expected to influence aquatic primary productivity and may also affect cyanobacterial nitrogen (N)‐fixers (diazotrophs). No data is currently available showing the response of diazotrophs to enhanced oceanic CO₂(aq). We examined the influence of pCO₂ [preindustrial∼250 ppmv (low), ambient∼400, future∼900 ppmv (high)] on the photosynthesis, N fixation, and growth of Trichodesmium IMS101. Trichodesmium spp. is a bloom‐forming cyanobacterium contributing substantial inputs of ‘new N’ to the oligotrophic subtropical and tropical oceans. High pCO₂ enhanced N fixation, C : N ratios, filament length, and biomass of Trichodesmium in comparison with both ambient and low pCO₂ cultures. Photosynthesis and respiration did not change significantly between the treatments. We suggest that enhanced N fixation and growth in the high pCO₂ cultures occurs due to reallocation of energy and resources from carbon concentrating mechanisms (CCM) required under low and ambient pCO₂. Thus, in oceanic regions, where light and nutrients such as P and Fe are not limiting, we expect the projected concentrations of CO₂ to increase N fixation and growth of Trichodesmium. Other diazotrophs may be similarly affected, thereby enhancing inputs of new N and increasing primary productivity in the oceans.     

PHOSPHATE UPTAKE AND GROWTH KINETICS OF TRICHODESMIUM (CYANOBACTERIA) ISOLATES FROM THE NORTH ATLANTIC OCEAN AND THE GREAT BARRIER REEF,AUSTRALIA1

We compared inorganic phosphate (P_i) uptake and growth kinetics of two cultures of the diazotrophic cyanobacterium Trichodesmium isolated from the North Atlantic Ocean (IMS101) and from the Great Barrier Reef, Australia (GBRTRLI101). Phosphate‐limited cultures had up to six times higher maximum P_i uptake rates than P‐replete cultures in both strains. For strain GBRTRLI101, cell‐specific P_i uptake rates were nearly twice as high, due to larger cell size, but P‐specific maximum uptake rates were similar for both isolates. Half saturation constants were 0.4 and 0.6 μM for P_i uptake and 0.1 and 0.2 μM for growth in IMS101 and GBRTRLI101, respectively. Phosphate uptake in both strains was correlated to growth rates rather than to light or temperature. The cellular phosphorus quota for both strains increased with increasing P_i up to 1.0 μM. The C:P ratios were 340–390 and N:P ratios were 40–45 for both strains under severely P‐limited growth conditions, similar to reported values for natural populations from the tropical Atlantic and Pacific Oceans. The C:P and N:P ratios were near Redfield values in medium with >1.0 μM P_i. The North Atlantic strain IMS101 is better adapted to growing on P_i at low concentrations than is GBRTRLI101 from the more P_i‐enriched Great Barrier Reef. However, neither strain can achieve appreciable growth at the very low (nanomolar) P_i concentrations found in most oligotrophic regimes. Phosphate could be an important source of phosphorus for Trichodesmium on the Great Barrier Reef, but populations growing in the oligotrophic open ocean must rely primarily on dissolved organic phosphorus sources.     

INTERACTIONS BETWEEN NITRATE UPTAKE AND NITROGEN FIXATION IN CONTINUOUS CULTURES OF THE MARINE DIAZOTROPH TRICHODESMIUM (CYANOBACTERIA)1

Diazotrophic cyanobacteria can take up combined nitrogen (nitrate, ammonium, amino acids, dissolved organic nitrogen) from solution, but the interaction between N₂ fixation and uptake of combined nitrogen is not well understood. We studied the effects of combined nitrogen ) additions on N₂ fixation rates in the cyanobacterium Trichodesmium erythraeum (IMS‐101) maintained in continuous culture in an N‐free medium (YBCII) and a 12:12‐h light:dark cycle. We measured acetylene reduction rates, nutrient concentrations, and biomass throughout the 12 h of illumination after the addition of nitrate (0.5–20 μM) at the start of the light period. Compared with unamended controls, Trichodesmium showed strong inhibition of acetylene reduction (up to 70%) in the presence of , with apparent saturation of the inhibition effect at an initial concentration of approximately 10 μM. The inhibition of acetylene reduction persisted through much of the light period as concentration in the culture vessel decreased. Recovery of N₂ fixation was observed late in the light period in cultures amended with low concentrations of (<5 μM) when ambient concentrations had decreased to 0.3–0.4 μM in the culture vessel. Nitrate uptake accounted for as much as 86% of total N uptake and, at the higher treatment concentrations, more than made up for the observed decrease in N₂ fixation rates. We conclude that Trichodesmium can obtain significant quantities of N through uptake of nitrate and does so in preference to N₂ fixation when sufficient is available.     

OSCILLATORIA (TRICHODESMIUM) THIEBAUTII (CYANOPHYTA) IN THE CENTRAL NORTH ATLANTIC OCEAN12

Physiological rate measurements were made with Oscillatoria thiebautii (Gom.) Geitler in the subtropical north Atlantic Ocean between Spain and Bermuda during May and June of 1975. The near surface C:N fixation ratios averaged 6.5, and the cellular composition ratio was 6.2, suggesting that N₂ fixation is the major path of nitrogenous nutrition for this alga. Compared to other oceanic phytoplankters, it has a low affinity for orthophosphate at oceanic concentrations (k_s= 9.0); however, it has a high potential for utilizing phosphomonoesters (170–300 ng atoms P ·μg chl a^?1· h^?1). Maximal photosynthesis occurred at 450–700 μ Einstein · m^?2· s^?1, and was inhibited by full sunlight. Calculated cell division rates (ca. 180 days) suggest that relative to other phytoplankters in this oceanic region, O. thiebautii must be subjected to negligible grazing pressure. No major differences in C, N, chl a or ATP were observed between the tuft (fusiform) and puff (spherical) colonies. ATP concentrations relative to other cellular constituents varied greatly between colonies, suggesting a general inter-colony physiological variability in the open Atlantic. With increasing depth in the euphotic zone, there was no evidence for chromatic adaption. The observations that O. thiebautii represents only a small fraction of total phytoplankton biomass and that its growth rate is 10–100 times slower than that of the other indigenous phytoplankton, strongly suggest that N₂ fixation by this alga is a virtually insignificant component of the nitrogenous nutrition for the phytoplankton of the North Atlantic central gyre in late Spring.     

NITROGENASE CONFINED TO RANDOMLY DISTRIBUTED TRICHOMES IN THE MARINE CYANOBACTERIUM TRICHODESMIUM THIEBAUTII1

Nitrogenase reductase (Fe-protein) was detected in the marine planktonic cyanobacterium Trichodesmium. The molecular weight was about 38 kD, as shown by western blotting using anti -Rhodospirillum rubrum nitrogenase reductase antiserum. The enzyme was confined to a limited number (ca. 10–40%) of randomly distributed trichomes in the Trichodesmium colonies, as shown by immunogold localization and transmission electron microscopy. Associated microorganisms had little or no nitrogenase. Nitrogenase showed a diel cycle in localization: present throughout the cytoplasm of cells in N₂-fixing (daytime) colonies but at the periphery of non-N₂-fixing (nighttime) colonies. This structural arrangement of N₂-fixing trichomes and nitrogenase is novel and different from the previously held paradigm for this and other diazotrophic cyanobacteria.     

NITROGEN UTILIZATION AND METABOLISM RELATIVE TO PATTERNS OF N2 FIXATION IN CULTURES OF TRICHODESMIUM NIBB1067

We measured uptake kinetics for four combined N sources, ambient rates of N uptake and N₂ fixation, glutamine synthetase activity (transferase and biosynthetic), and concentrations of intracellular pools of glutamate (glu) and glutamine (gln) in cultures of Trichodesmium NIBB1067. N dynamics and metabolism were examined to assess the relative importance of N₂ fixation and N uptake to Trichodesmium nutrition. Comparisons were made between cultures grown on medium without added N, with excess NO, or with excess urea. Of the combined N sources tested, Trichodesmium NIBB1067 had the highest affinity for NH; high uptake capacities for NH, urea, and glu; and little capacity for NO uptake. In cultures grown on medium without added N, NH accumulated in the medium during growth, resulting in high NH uptake rates relative to rates of N₂ fixation. Glu uptake rates were low but consistent throughout the diel period. In cultures grown on excess NO or urea, uptake of these compounds supplied the majority of the daily N demand, although some N₂ fixation occurred during the light period. NO uptake rates were reduced when N₂-fixation rates were high. In all of the cultures, the highest gln/glu ratios and the lowest glutamine synthetase transferase/biosynthetic ratios were observed during the period when rates of total N uptake were highest. In cultures growing exponentially on medium without added N, N₂ fixation accounted for 14%– 16% of the total daily N uptake. Uptake of NH and glu, presumably regenerated within the culture vessels, represented 84%–86% of the daily N uptake. Because these systems were closed, net growth was constrained by the rate at which N₂ could be fixed into the system. However, total daily N turnover was greater than that necessary to accommodate the observed increase in culture biomass. The rapid N turnover rates observed in these cultures may support gross productivity and balance the high rates of C fixation observed in natural populations of Trichodesmium.