Study On The Related Functional Genes Of Nitrogen Metabolism In Halomonas Alkaliphila X3

With the acceleration of urbanization,and large-scale development of industries,agriculture and aquaculture,the discharge of wastewater with high concentration of nitrogen increased continuesly,which resulted in eutrophication and threatened the ecological balance of the environment.In the process of nitrogen removal from wastewater,biological methods are more economical and efficient than traditional physicochemical methods,also,it is simple to operate and does not cause the secondary pollution.In our laboratory,we ever isolated and purified a heterotrophic bacterium Halomonas alkalinephila X3,which has high effect on nitrogen removal.It is of great significance to study the mechanism of nitrogen metabolism in strain X3 for its application in waste water purification and to explore the contribution on the nitrogen recycle in marine eco-system of this kind of bacteria.In this paper,the existence and functions of the related functional genes in strain X3 and the possible nitrogen-metabolizing pathway were verified through PCR,qRT-PCR,enzyme activity detection and proctucts detection awere,and the secondary structure,3D structure and phylogeny of corresponding proteins were analyzed through bioinfermatic softwares.The main experimental results are as follows:(1)Through PCR,intermediate product and terminal products determinations,,the exist of nasA and narG were verified,and the non-predicted AMO,HAO,Nap,Nir,Nos,and NOR were failed to be amplified,and no hydroxylamine,the key intermediate product in nitrification,nor gas products was detected in the whole cultivating prosess,suggesting nitrification and denitrification not exist in the nitrogen metabolism of Halomonas alkalinephila X3.The utilization of inorganic nitrogen by this strain may be realized through the dissimilation and assimilation of nitrate reduction.,namely under the catalysis of dissimilary nitrate reductase(Nar)and assimilatory nitrate reductase(NasA),the nitrate was turned to nitrite,then was converted to ammonia nitrogen,and syntheticed cellular material.(2)There existed the Nar with express activity in the nitrogen metabolism pathway of Halomonas alkaliphila X3,and the reduction of nitrate to nitrite was catalyzed by Nar,and the enzyme activity was 21.415 U per gram of protein.Nar in Halomonas alkaliphila X3 consists of 4 subunits encoded by narG,narY,narJ,and narV,respectively.Of the predicted narGYJV-encoded protein functional regions,1-1246 amino acids belonged to the Nar G superfamily,formed the ? subunit of Nar while,1261-1752 amino acids belong to the DMSOR-beta-like superfamily and,formed the ? subunit of Nar,2061-2279 amino acids formed the ? subunit,and 1802-2018 amino acids formed the ? subunit.The 3D structures of NarG,NarY,and NarV are the closest to the 1Q16 3D structure of Escherichia coli in PDB,with the coverage rate of 96-100%.The phylogenetic trees showed that the genetic distance of NarG in X3 was closest to those in the same genus,but far from those in other genus although they possessed the same function.NarY was closely related to that of Escherichia coli,in amino acid sequence,but low in similarity.These findings proveds that the genetic evolution of the subunits in the NAR of alienated nitrate reductase was different.(3)There was a NasA in Halomonas basicphila X3,the full-length nasA sequence of 2739 bp was obtained,and the expression level of gene nasA under aerobic condition was higher than that under anaerobic condition.The presence of Mo in the most majority of nitrogen fixation were predicted in NasA of Halomonas alkaliphila X3,and no signal peptide was predicted in NasA,suggesting it is an intracellular enzyme.Of amino acids of NasA,13-587 amino acids belong to the Molybdopterin-binding superfamily;594-712 amino acids belong to the MopB_CT superfamily;847-896 amino acids belong to the NirB superfamily.Phylogenetic tree analysis showed that the nasA gene of strain X3 had the highest homology with Halomonas campaniensis and Halomonas sp.TD01.The nasA gene was different in different genera.The protein 3D structure of NasA is highly homologous to nitrate reductase(NapA)in Desulfovibrio desulfuricans.