Isolation of nifH and part of nifD by modified capture polymerase chain reaction from a natural population of the marine cyanobacterium Trichodesmium sp.

A modified capture polymerase chain reaction (CPCR) technique was used to isolate the entire sequence of the nifH gene and its flanking regions from a natural population of Trichodesmium sp. A set of specific CPCR primers derived from a known 72-bp DNA segment of the nifH sequence permitted isolation of both the upstream and the downstream region of Trichodesmium sp. nifH. The 882-bp nifH gene presented here is the first full-length gene isolated from Trichodesmium sp. A sequence similar to a nif-like promoter was found in front of nifH. The nifH open reading frame of Trichodesmium sp. encoded 294 amino acids. Comparative analysis of the Trichodesmium sp. NifH sequence revealed strong similarity with 23 known NifH proteins. Amino acids postulated to be involved in binding of the 4Fe:4S cluster and those subjected to ADP-ribosylation were present. An open reading frame for the nifD gene was identified 189 bp downstream of nifH. A sequence similar to the consensus of the nif-like promoter was also found in front of nifD.