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In the modern ocean, a significant amount of nitrogen fixation is attributed to filamentous, nonheterocystous cyanobacteria of the genus Trichodesmium. In these organisms, nitrogen fixation is confined to the photoperiod and occurs simultaneously with oxygenic photosynthesis. Nitrogenase, the enzyme responsible for biological N 2 fixation, is irreversibly inhibited by oxygen in vitro. How nitrogenase is protected from damage by photosynthetically produced O 2 was once an enigma. Using fast repetition rate fluorometry and fluorescence kinetic microscopy, we show that there is both temporal and spatial segregation of N 2 fixation and photosynthesis within the photoperiod. Linear photosynthetic electron transport protects nitrogenase by reducing photosynthetically evolved O 2 in photosystem I (PSI). We postulate that in the early evolutionary phase of oxygenic photosynthesis, nitrogenase served as an electron acceptor for anaerobic heterotrophic metabolism and that PSI was favored by selection because it provided a micro-anaerobic environment for N 2 fixation in cyanobacteria.

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.

The daily cycle of nitrogenase expression in the marine filamentous nonheterocystous cyanobacterium Trichodesmium spp. is controlled by a circadian rhythm. We evaluated the rhythm of two key photosynthesis genes, psbA of photosystem II and psaA of photosystem I, in Trichodesmium sp. IMS 101 using the 3 criteria for an endogenous rhythm. The transcript abundance of psbA and psaA transcripts oscillated with a period of ca. 24 h under a 12 h light/12 h dark regime. At 24 °C and 28 °C the cyclic pattern of transcript abundance was maintained for at least 58 h under constant light conditions, whereas the periods were about 24 h at 24 °C, and 26–30 h at the higher temperature. The cycles of psbA and psaA gene expression were entrained using light-dark cues. Transcription of nifHDK was initiated prior to the light period, followed by psbA and finally psaA. There was a 90° (6 h) phase difference between the net accumulation of nifHDK and psbA transcripts, as well as between that of psbA and psaA transcripts. Results of inhibitor experiments indicated that psbA and psaA transcription was regulated differently by initiation and degradation during the light period. Short-term changes of light conditions resulted in significant effects on psbA transcription and nitrogenase activity, but had less of an effect on psaA and nifHDK transcription.

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.

Analysis suggests that feedbacks between carbon (C), nitrogen (N), and oxygen (O) cycles helped prevent the oxidation of Earth in the Paleoproterozic. This stabilizing feedback, which was ultimately overridden, led to the contemporary nitrogen cycle where nitrate, rather than ammonium, was the stable form of fixed inorganic nitrogen in the oceans. Barring some minor changes in the trace element composition in nitrogenases, the core proteins remained essentially unchanged following the transition to an oxidized atmosphere. In the contemporary ocean, approximately 20%–30% of nitrogenase activity is inhibited at any moment in time by O 2 . This inhibition results in a negative feedback which constrains the upper level of O 2 on Earth. Three central aspects of cyanobacterial nitrogen fixation remain curious. First, although some trace elements have been altered in the evolution of nitrogenases, the core proteins have remained virtually unchanged. Second, while there is abundant evidence of lateral transfer of nitrogenase genes between prokaryotes, in the endosymbiotic appropriation of cyanobacteria into heterotrophic hosts to photosynthetic eukaryotes, nitrogenases were lost. Third, although free-living heterocystous cyanobacteria are abundant in lakes and brackish water ecosystems, they appear to be rare in the open ocean.

Cyanobacterial N2-fixation supplies the vast majority of biologically accessible inorganic nitrogen to nutrient-poor aquatic ecosystems. The process, catalyzed by the heterodimeric protein complex, nitrogenase, is thought to predate that of oxygenic photosynthesis. Remarkably, while the enzyme plays such a critical role in Earth's biogeochemical cycles, the activity of nitrogenase in cyanobacteria is markedly inhibited in vivo at a post-translational level by the concentration of O2 in the contemporary atmosphere leading to metabolic and biogeochemical inefficiency in N2 fixation. We illustrate this crippling effect with data from Trichodesmium spp. an important contributor of "new nitrogen" to the world's subtropical and tropical oceans. The enzymatic inefficiency of nitrogenase imposes a major elemental taxation on diazotrophic cyanobacteria both in the costs of protein synthesis and for scarce trace elements, such as iron. This restriction has, in turn, led to a global limitation of fixed nitrogen in the contemporary oceans and provides a strong biological control on the upper bound of oxygen concentration in Earth's atmosphere.

Summary: Trichodesmium spp. are marine filamentous, non-heterocystous cyanobacteria capable of aerobic nitrogen fixation. In this study, the nitrogenase structural genes (nifHDK) and nifU gene of Trichodesmium sp. IMS101 were cloned and sequenced. The Trichodesmium sp. IMS101 nifH, nifD and nifK amino acid sequences showed only 79%, 66% and 68% identity, respectively, to those of Anabaena sp. strain PCC 7120. A potential transcription start site for nifH was found 212 bases upstream of the nifH start codon. Promoter-like nucleotide sequences upstream of the transcription start site were identified that were very similar to those identified for the nitrogenase genes of Anabaena spp. Sequence analysis revealed regions of DNA that may form stem-loop structures in the intercistronic regions downstream of nifH and nifD. RNA analysis by Northern hybridization revealed the presence of transcripts corresponding to nifH, nifHD and nifHDK. Surprisingly, Northern hybridization also revealed the presence of transcripts that corresponded to nifD, nifDK and nifK, which have not been previously reported as transcripts in contiguous nifHDK genes of cyanobacteria. Transcription of the nifHDK genes was not significantly repressed in the presence of nitrate at a final concentration of 20 mM or at oxygen concentrations of up to 40%, whereas ammonium and urea inhibited nifHDK transcription. The transcription of the nifHDK genes was not affected by darkness, which suggests that transcription of these genes in Trichodesmium is not directly regulated by light.

Over the past decade, the marine cyanobacteria Trichodesmiumspp. have received increasing attention [1,2], because of their ability to fix nitrogen in the presence of oxygen, and the recognition of their importance in ocean N budgets [3,4]. Trichodesmiumspp. are distributed worldwide in tropical and subtropical seas [2]. Dugdale et al. [5] first demonstrated that nitrogen fixation occurred in association with Trichodesmiumaggregates in the sea, although it was many years before it became clear that the nitrogen fixation observed was indeed due to the cyanobacterium, and not associated heterotrophic bacteria. Evidence based on DNA sequences (nifH) amplified from natural populations, and immunological studies eventually confirmed that Trichodesmiumitself was the primary nitrogen fixer [6-8], even though other diazotrophs are sometimes found associated with Trichodesmiumaggregates [9,10].

Recent studies suggested that the daily cycle of nitrogen fixation activity in the marine filamentous nonheterocystous cyanobacterium Trichodesmium sp. is controlled by a circadian rhythm. In this study, we evaluated the rhythm of nitrogen fixation in Trichodesmium sp. strain IMS 101 by using the three criteria for an endogenous rhythm. Nitrogenase transcript abundance oscillated with a period of approximately 24 h, and the cycle was maintained even under constant light conditions. The cyclic pattern of transcript abundance was maintained when the culture was grown at 24 and 28.5°C, although the period was slightly longer (26 h) at the higher temperature. The cycle of gene expression could be entrained with light-dark cues. Results of inhibitor experiments indicated that transcript abundance was regulated primarily by transcription initiation, rather than by degradation. The circadian rhythm, the first conclusively demonstrated endogenous rhythm in a filamentous cyanobacterium, was also reflected in nitrogenase MoFe protein abundance and patterns of Fe protein posttranslational modification-demodification.