Molecular characterization and expression of nif, glnA and ntcA genes in the aerobic nonheterocystous nitrogen-fixing cyanobacterium Trichodesmium sp. IMS101

Trichodesmium spp. are filamentous nonheterocystous diazotrophic marine cyanobacteria. Trichodesmium fixes nitrogen only during the day while evolving oxygen through photosynthesis. In this study, the nifBSUHDKENXWhesABfdxH, glnA and ntcA genes of Trichodesmium sp. IMS101 were cloned. A transcription start site for nifH was identified and promoter-like nucleotide sequences were characterized. 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 in cyanobacteria. In general, for the Trichodesmium and Anabaena variabilis vegetative (nifII ) nitrogenase gene clusters, the gene organization or arrangement on the chromosome for the region spanning from nifB to nifW is similar except for the absence of ORF3 and ORF1 in Trichodesmium. The absence of the fdxN gene between the nifB and nifS genes, the absence of the 11-kb insertion sequence that interrupts the nifD gene, and the presence of fused nifEN genes, are three characteristic features of the nifII gene cluster which are also found in the Trichodesmium nif gene cluster. The fused NifEN polypeptide does not appear to be processed into individual NifE and NifN polypeptides. The fused nifEN genes were found widespread in the nonheterocystous cyanobacteria. Even though the nif gene organization is very similar phylogenetic analysis of Nif sequences do not support close relatedness of Trichodesmium and Anabaena variabilis vegetative (nifII) nitrogenase genes. From the diel transcript abundance it appears that the nifHDK operon and the nifENXORF2 operon are not coordinately regulated in Trichodesmium . The diel periodicity of glnA transcription, with only two distinct phases, is apparently linked to nifHDK gene expression. The peak abundance of glnA mRNA coincided with, but persisted longer than, that of nifHDK mRNA. The ntcA transcription was constitutive, without any distinct phases and did not show any apparent relation to the intracellular nitrogen pools. The response of nifHDK and glnA to short term exogenous supply of ammonium and nitrate were similar to that of heterocystous cyanobacteria. However, ammonium failed to produce strong differences in ntcA transcript abundance while nitrate resulted in gradually decreasing ntcA transcript abundance.