Antiserum to Nitrogenase Generated from an Amplified DNA Fragment from Natural Populations of Trichodesmium spp

A fragment of the nifH gene was amplified from natural populations of Trichodesmium spp. and cloned into a maltose-binding protein (MBP) expression vector. The peptide product of the amplified 359-bp fragment of nifH was cleaved from the fusion protein, purified, and used to generate a specific antibody to the Fe protein of nitrogenase. The antiserum recognized the MBP-nitrogenase fusion protein and the cleaved nif peptide product but not MBP. The antibody cross-reacted with nitrogenase from natural populations of Trichodesmium spp. from the Caribbean Sea and with a cultured isolate from the Kuroshio waters (Trichodesmium sp. strain NIBB1067). The same nifH fragment was amplified, cloned, and sequenced from Trichodesmium sp. strain NIBB1067 and was found to be 98% identical at both the protein and DNA levels to nifH from the Caribbean populations. Three of the six nucleotide differences between the Trichodesmium sp. strain NIBB1067 and the Trichodesmium spp. nifH sequence had also been found in a second sequence from the natural populations, indicating either that there is more than one strain of Trichodesmium sp. in natural assemblages or that there are multiple copies of nifH in the genome. This DNA fragment, which is easily amplified with the polymerase chain reaction, may provide a good indicator of species relatedness without requiring extensive cloning or sequencing. Furthermore, the use of the polymerase chain reaction in combination with a MBP protein fusion vector provides a rapid method for production of highly specific sera, starting with a small amount of DNA.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  E. Carpenter,et al.  Basis for Diel Variation in Nitrogenase Activity in the Marine Planktonic Cyanobacterium Trichodesmium thiebautii , 1990, Applied and environmental microbiology.

[3]  Y. Fujita,et al.  THE EFFECT OF IRON NUTRITION ON PHOTOSYNTHESIS AND NITROGEN FIXATION IN CULTURES OF TRICHODESMIUM (CYANOPHYCEAE) 1 , 1990 .

[4]  H. Paerl,et al.  Immunochemical Localization of Nitrogenase in Marine Trichodesmium Aggregates: Relationship to N2 Fixation Potential , 1989, Applied and environmental microbiology.

[5]  J. Zehr,et al.  Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii , 1989, Applied and environmental microbiology.

[6]  P. Böger,et al.  Regulation of nitrogenase activity in Anabaena variabilis by modification of the Fe protein , 1989 .

[7]  B. Slatko,et al.  An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. , 1988, Gene.

[8]  Y. Fujita,et al.  Aerobic nitrogenase activity measured as acetylene reduction in the marine non-heterocystous cyanobacterium Trichodesmium spp. grown under artificial conditions , 1988 .

[9]  Y. Fujita,et al.  Cultures of the pelagic cyanophytes Trichodesmium erythraeum and T. thiebautii in synthetic medium , 1986 .

[10]  R. Haselkorn,et al.  Nucleotide sequence of a cyanobacterial nifH gene coding for nitrogenase reductase. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[11]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[12]  P. Ludden,et al.  Purification and properties of nitrogenase from Rhodospirillum rubrum, and evidence for phosphate, ribose and an adenine-like unit covalently bound to the iron protein. , 1978, The Biochemical journal.

[13]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[14]  H. Paerl,et al.  Immunofluorescence detection and characterization of N2-fixing microorganisms from aquatic environments , 1990 .

[15]  H Inouye,et al.  Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. , 1988, Gene.

[16]  J. Postgate The fundamentals of nitrogen fixation , 1978 .