The Molecular Identification And Study On Properties Of The Ammonium-excreting Mutants Of Alcaligenes Faeccalis

The Tn5-inserted mutant bank of A. frwcai/is A 1501 was constructed using Tn5 transposon mutagenesis and three ammonium-excreting mutants (A15M1~ A15M2. A15M3) were screened on minimal selective plates complemented with 50k?g/ml Kanamycin. The growth curve of the mutants were as same as that of the wild type in the minimal medium and in the 10mM NOQ medium. The ammonium excretion of the mutants was much higher than that of the wild type in both mediums. The maximum arnmontum-excreting amount of the mutant A15M3 is 3.96 mM..The homologous and heterologous nifH-lacZ gene fusion were introduced into mutants by conjugation. The f3 -galactosidase activity of mutants were higher than that of the wild type in the N-free medium. The 173 -galactosidase activity of Al 5M1 is 1400 units, A15M3 is 1600 units, but wild type strain A1501 is only 168.5 units. With increase of the concentration of NH4, the ~ -galactosidase activity of A 1501 got lower and lower. The 13 -galactosidase activity of mutants were almost not affected by ammonia.The acetylene-reducing method and the N tracer technology are used to detect the nitrogenase activity of mutants. The nitrogenase activity of AI5MI is 22.75 nmol C~H4/hr.tube. A15M3 is 23.27 nmol C2H4/hr.tube. The wild type strain A1501 is 18.33 nmol C,H4/hr.tube). The rate of nitrogen-fixation of AI5MI is 17.8 gN!10~CFU/day by ?N-tracer technology, AISM2 is 18.96 gN/IO7CFU day A15M3 is 19.14 gN/10 CFU/day. The mutants increased Ndfa% by l5.9%~ 23.40/0 and 24.6% compared with that ofAl5Ol.Population of three mutants colonized on root surface were estimated at 61 .53%, 70.2%, 60% of the total inoculation with co-culture of wild type strain Al501 each of three mutants.respectively. In situ straining of the bacteria with 5-bromo-4-chloro-3indolyi- fB -D-galactopyranoside(X-gal) as a chromogenic substrate and examination by light microscopy revealed that bacteria could localized on the surface of the rice root and also within the cortex. The scanning electron microscopic observation revealed the stimulating effect of mutants on formation of root hairs were stronger than that of wild type strain AlSO I.A SOObp specific band could be amplified by PCR from total DNA ot Al 5M1 and A15M3 using a pair of PCR primer specific to nptll. It indicated that Tn5 sequence had insert into chromosome DNA of wild type Al 501. The SouthernhybridiZaian of the total DNA digisted by different restrichon enzymes Were cAnedout using DG-labeled nptII DNA probe. The restilt indicated the lpeus of Tn5insertion wes locared on the l0 kb SacI-EcdRI DNA ?fragm.nt from A l 5Ml genome.The locus of Tn5 insertion was located in the' l8 kb SacI-EcoRI fragment fromA15M3'...