Recent studies suggested that the daily cycle of nitrogen fixation activity in the marine filamentous nonheterocystous cyanobacteriumTrichodesmium sp. is controlled by a circadian rhythm. In this study, we evaluated the rhythm of nitrogen fixation inTrichodesmium 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.
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. I M S l O l were cloned and sequenced. The Trichodesmium sp. I M S l O l 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 a t a final concentration of 20 mM or a t 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.
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 degrees C and 28 degrees 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 degrees 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 degrees (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.
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.
Trichodesmium spp. are marine filamentous nitrogen-fixing cyanobacteria which play an important role in the nitrogen budget of the open ocean. Trichodesmium is unique in that it is nonheterocystous and fixes nitrogen during the day, while evolving oxygen through photosynthesis, even though nitrogenase is sensitive to oxygen inactivation. The sequence of the gene encoding the Fe protein component of nitrogenase from the recently cultivated isolate Trichodesmium sp. IMS 101 was used to construct a 3-dimensional model of the Fe protein, by comparison to the X-ray crystallographic structure of the Fe protein of the gamma-proteobacterium Azotobacter vinelandii. The primary differences in amino acid sequences of the Fe protein from diverse organisms do not impact the critical structural features of the Fe protein. It can be concluded that aerobic nitrogen fixation in Trichodesmium spp. is not facilitated by unique structural features of Trichodesmium Fe protein.
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