摘要
To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially nfrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUML The nfrCgene in pSUM1 was then replaced by a /acZ-Kmr fragment resulted in the generation of a plasmid pSUM2. The lacZfragment in pSUM2 was further removed and a plasmid pSUMS produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially nfrC-deleted mutants A15CM1 (ntrC:: lacZ) and A15CM2 (ntrC-) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifA-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%
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.
