With the help of a PCR-based screening method, the gene encoding squalene-hopene cyclase (SHC) of USDA 110 was isolated from a cosmid library. The SHC catalyses the cyclization of squalene to hopanoids, a class of triterpenoid lipids recently discovered in nitrogen-fixing, root-nodule-forming bacteria. Hybridization experiments showed that the gene is present in bacteria of all strains tested and in photosynthetic bacteria forming stem nodules on tropical legumes of the genus The gene is 1983 bp in length and encodes a protein of 660 amino acid residues with a calculated molecular mass of 73671 Da. Comparison of the deduced amino acid sequence with the sequences of other SHCs revealed highest similarity (70%) to the SHC from the Gram-negative and lower similarity (48%) to the SHCs from the Gram-positive and SHC also showed similarity (38-43%) to eukaryotic oxidosqualene cyclases. The gene was expressed in The recombinant SHC catalysed the cyclization of squalene to the hopanoids hopene and diplopterol However, the formation of the gammacerane derivative tetrahymanol, which is produced in addition to hopanoids in strains could not be detected Therefore, the presence of a second squalene cyclase in can be assumed. Sequence analysis of 0.5 kb upstream from the gene identified a partial ORF with significant similarity to the C-terminus of an ORF located immediately upstream from the gene in Both ORFs also showed similarity to phytoene desaturases from cyanobacteria and plants. The 3'-end of this ORF from overlaps with 13 bp at the 5'-end of The close proximity of this ORF to suggests that and this ORF may be part of an operon.


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