CHARACTERIZATION OF GENES FOR AN ALTERNATIVE NITROGENASE IN THE CYANOBACTERIUM ANABAENA-VARIABILIS

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cya nobacterium that has been reported to fix nitrogen and reduce acetylen e to ethane in the absence of molybdenum. DNA from this strain hybridi zed well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azot obacter vinelandii. The hybridizing region was cloned from a lambda EM BL3 genomic library of A. variabilis, mapped, and sequenced. The deduc ed amino acid sequences of the vnfD and vnfK genes of A. variabilis sh owed only about 56% similarity to the nifDK genes of Anabaena sp. stra in PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabil is was similar to that of A. vinelandii. However, in A. variabilis, th e vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a sou rce of fixed nitrogen in the presence of molybdenum but grew poorly wh en vanadium replaced molybdenum. In contrast, the parent strain grew e qually well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA fr om Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nos toc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing Cla I fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates o f Anabaena sp. from the Azolla symbiosis.