Applied and Environmental Microbiology, November 1999, p. 4935-4942, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Institute of Physical and Chemical Research
(RIKEN)1 and Japan Science and
Technology Corporation (JST),
Received 12 July 1999/Accepted 27 August 1999
Expression of the nitrogen fixation gene, nifH, in the
gut of the termite Neotermes koshunensis was characterized
without cultivation. nifH cDNA was directly amplified from
mRNA of the mixed microbial population in the gut by reverse
transcription (RT)-PCR. Analyses of the RT-PCR products revealed that,
among the diverse nifH sequences, only a few corresponding
to an alternative nitrogenase (encoded by the anf gene)
were preferentially transcribed in the termite gut. Expression of the
anf gene was further investigated quantitatively under
several termite feeding conditions by competitive PCR. The levels of
expression of the anf gene were largely congruent with the
nitrogen fixation activity displayed by the termite. The amounts of the
genomic anf gene in the population showed no significant
change, indicating that the level of expression was critical for
nitrogen fixation activity. Interestingly, no significant decrease in
the expression level was observed when the diet contained molybdenum
(Mo), which represses ordinary anf genes. A 3.6-kb DNA
region downstream of the anf gene was isolated and found to contain reading frames homologous to anfH,
anfD, and anfG of the Bacteria
domain which encode subunits of an alternative nitrogenase having no Mo
as a cofactor. This DNA region also contained reading frames encoding
glnB-like proteins, which is a common feature of the
nitrogenase genes of the Archaea domain. These results indicate that the anf group of nitrogenase genes is the
most important group of genes responsible for nitrogen fixation in
N. koshunensis and that the anf gene possesses
novel features with respect to the regulation of its expression and its
gene organization.
*
Corresponding author. Mailing address: Microbiology
Laboratory, RIKEN (The Institute of Physical and Chemical Research),
Hirosawa 2-1, Wako, Saitama 351-0198, Japan. Phone: 81-48-462-1111, ext. 5724. Fax: 81-48-462-4672. E-mail:
mohkuma{at}mailman.riken.go.jp.
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