## 3 Results and Discussion
### Structure and Features of the Geosmin Operon  
Two putative cyclic nucleotide-binding protein genes (_cnb_) that are 1398 and 1407 bp in length were identified downstream of the _geo_ gene through genome walking (Figure 2). These two _cnb_ genes have the same transcriptional orientation and are homologous, with 73% DNA similarity. Further BLAST and CDD searches suggest that this type of cyclic nucleotide-binding gene belongs to the Crp-Fnr regulator family. The flanking regions of the geosmin synthase gene were also analyzed in the released genomic data (from NCBI) of _N. punctiforme_ PCC 73102, _Oscillatoria_ sp. PCC 6506, _Cylindosperm stagnale_ PCC 7417, four representative species of Actinomycetes and _Myxococcus xanthus_ DK 1622. As shown in Figure 3, the organization of the geosmin synthase genes in _N. punctiforme_ PCC 73102, _Oscillatoria_ sp. PCC 6506, _C. stagnale_ PCC 7417 and _M. xanthus_ DK 1622 are similar to that of the _A.__ucrainica_ strains. No _cnb_ genes were found in adjacent regions of the geosmin synthases of _S. coelicolor_ A3 (2) and _S.__avermitilis_ MA-4680. The array of the geosmin synthase with _cnb_ genes in _Saccharomyces erythraea_ NRRL2338 is different from that in _A.__ucrainica_, and only one _cnb_ gene is upstream of the geosmin synthase gene in _Frankia_ sp. Ccl3. Figure 3: Organization of genes that encode predicted geosmin/germacradienol synthases and flanking genes in different microorganisms. The filled, oblique-lined and unfilled arrows indicate the geosmin/germacradienol synthase gene, cyclic nucleotide-binding protein gene (_cnb_) and other unrelated genes, respectively. Line segments located in _A.__ucrainica_ CHAB 1432 show the positions of primers RNC1F(R) and RNC2F(R). A _rho_-independent transcriptional terminator was not identified in the interspacers of _geo_ and two _cnb_ genes (61 bp and 150 bp). This result suggests that these three genes are located in the same transcription unit and probably form an operon structure. Both reverse transcriptional-PCR and PCR using two pairs of primers (RNC1F with RNC1R, and RNC2F with RNC2R; see the positions in Figure 3) with cDNA as a template confirm that _geo_ and _cnb_ genes are located in one transcription unit (Figure 4). Furthermore, seven geosmin-producing cyanobacterial species that represent seven genera in Nostocales and Oscillatoriales were used to verify the organization of the geosmin operon in cyanobacteria. The results of regular-PCR with primer sets GTC1F(R) and GTC2F(R), gel electrophoresis and sequencing indicate a universal gene composition and arrangement of the geosmin operon (_geo/cnb/cnb_) in all investigated strains (Table 1). The above results revealed the pattern of geosmin synthase genes in an operon with two cnb genes that were predicted members of the Crp-Fnr regulator family, and this pattern was proven as the gene structure in most investigated cyanobacteria (except the _Phormidium_ sp.) (Table 1 and Figure 3). In the MIB biosynthesis genes identified in cyanobacteria, homologous _cnb_ genes form an operon structure with MIB cyclase [24,25]. The present study also showed that the Crp-Fnr family was highly conserved in different groups of microorganisms and was only found around terpene (geosmin/germacradienol and MIB) cyclase genes. This result suggests that this family of regulators is related to the corresponding cyclase genes, both in location and function. The Crp-Fnr family is a universally positive regulator that plays key roles in nitrogen fixation, photosynthesis and respiration in various microorganisms [26]. The functions of _cnb_ genes in the synthesis and evolution of sesquiterpenes (geosmin) and monoterenes (MIB) are interesting topics worthy of further study. Figure 4: Electrophoretic results of total RNA and reverse transcriptional-PCR. (**A**) Total RNA extracted from _A.