Download 14 Nitrogen Fixation: Nitrogenase Genes and

14 Nitrogen Fixation: Nitrogenase
Genes and Gene Expression
JP Zehr and PJ Turner
Department of Ocean Sciences,University of California-Santa Cruz, Santa Cruz, California 95064, USA
CONTENTS
Introduction
Methods overview
PCR amplification of nitrogenase genes and phylogenetic analysis
Primers and controls for PCR
Genomic DNA extraction
Polymerase chain reaction (PCR)
Nitrogenase gene expression
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Problems, limitations and caveats
Applications
Future directions and alternative approaches
INTRODUCTION
Biological nitrogen fixation is the enzymatic reduction of atmospheric dinitrogen to ammonium. This process, a key component of the nitrogen cycle,
is important in many ecosystems when biologically more available forms,
such as nitrate or ammonium, are present in small amounts relative to biological demand for growth. The capability for nitrogen fixation is widely
dispersed among prokaryotic taxa. Very divergent, distantly related organisms are able to fix nitrogen. On the other hand, not all taxa within a specific group are able to fix nitrogen. For example, there are nitrogen-fixing
and non-nitrogen-fixing species of unicellular cyanobacteria.
The nitrogenase enzyme is a multi-component enzyme that typically
consists of the iron (Fe) protein and the molybdenum iron (MoFe) protein
(Howard and Rees, 1996). This nitrogenase is termed 'conventional'.
Alternative forms of the protein exist that replace Mo with vanadium
('alternative') or Fe ('second alternative'). The conventional nitrogenase is
encoded by the nifHDK genes, which are often found in contiguous
arrangement within the genome. Alternative nitrogenases (alternative
and second alternative) also contain nifH, but contain a third protein in the
counterpart to the Mo protein, which is encoded by nifG (nifDGK). The
METHODS IN MICROBIOLOGY, VOLUME 30
ISBN 0-12-521530-4
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