Restoration of the FunctionalnifGene Cluster by Seven Excision and Two Inversion Events during Heterocyst Development in the Nitrogen-Fixing CyanobacteriumCalothrixsp. NIES-4101

Abstract

AbstractIn the genome of the heterocystous cyanobacteriumCalothrixsp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB,nifH,nifD, andnifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intactnifBHDKgenes by the excision mechanism found in thenifDgene of theAnabaenasp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the completenifBHDKgenes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the completenifHDKandnifBgenes were found byde novoassembly of the sequencing reads. In addition, DNA fragments covering thenifBHDKoperon were successfully amplified by PCR. We propose that the process ofnifBHDKrestoration occurs as follows. First, thenifD-nifKgenes are restored by four excision events. Then the completenifHandnifBgenes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functionalnifgene cluster,nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of thenifBHDKgenes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.