Cloning and transcriptional analysis of the nifUHDK genes of Trichodesmium sp. IMS101 reveals stable nifD, nifDK and nifK transcripts

Abstract

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.

Diurnal expression of hetR and diazocyte development in the filamentous non-heterocystous cyanobacterium Trichodesmium erythraeum

“…4), about 2 h before the maximum nitrogenase activity was reached (measured as acetylene reduction). These findings corroborate those reported previously for nifH expression in Trichodesmium IMS101 using Northern blots Dominic et al, 1998), immunolocalization of NifH (Fredriksson & Bergman, 1995) and acetylene reduction assays (Mulholland & Capone 1999). While NifH synthesis was below the detection level of Northern blots when the light was turned off (20 : 00), we were able to demonstrate by using the more sensitive realtime RT-PCR technique that initiation of nifH transcription commenced at about midnight and slowly increased until the light was again turned on at 08 : 00; it then increased rapidly (Fig.…”

RNA Processing of Nitrogenase Transcripts in the CyanobacteriumAnabaena variabilis

“…If a processing event places a stem-loop structure at the 5Ј end of the transcript, then that transcript will have increased stability (1). Processing of a nifHDK transcript in Rhodobacter (59) and of a nifH transcript in Heliobacterium chlorum (14) at the base of a stem-loop structure has been reported, and there appears to be processing of nifHDK transcripts in Trichodesmium as well (11). We investigated the 5Ј untranslated regions of the nifH1 and vnfH genes for potential secondary structure close to the 5Ј end of the transcript.…”

Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial

“…Promoter elements at Ϫ10 and Ϫ35, however, exhibited only weak similarity to the consensus E. coli 70 ( Table 2). The absence of typical promoter elements could indicate false predictions of transcription start sites due to incomplete processing of a larger transcript during RT or due to RNA cleavage (6,14). This may be true especially for the four transcription start sites detected for mcyE.…”