Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis
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To study the effect ofntrC gene product on the expression and regulation of other important nitrogen-fixing genes inAlcaligenes faecalis, partiallyntrC-deleted mutants ofA. faecalis have been generated. To start with, thentrC gene ofA. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. ThentrC gene in pSUM1 was then replaced by alacZ-Kmr fragment resulted in the generation of a plasmid pSUM2. ThelacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type ofA. faecalis A1501 by conjugation and two partiallyntrC-deleted mutants A15CM1 (ntrC::lacZ) and A15CM2 (ntrC -) were obtained. To understand the regulatory effect of the NtrC on the expression ofnifH andnifA, anifH-lacZ gene or anifA-lacZ gene was introduced into the ntrC- mutant by conjugation. The results indicated that: (i) although the ntrC- mutant was nif+, its nitrogen fixation activity was only 20% that of the wild type; (ii) the ntrC- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (iii) the regulation ofntrC gene expression did not require its own product; (iv) the expression ofnifH inA. faecalis was positively regulated by thentrC. Deletion of thentrC resulted in the reduction ofnifH expression or even totally inactivated nitrogen fixation; (v) there was no obvious influence on the expression ofnifA inA. faecalis if thentrC gene was deleted.
KeywordsAlcaligenes faecalis ntrC-mutant expression and regulation ofnif genes nitrogen fixation
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