Transcriptional Characterization Of Nitrate Assimilation Regulatory NasST/nasR Genes In Nitrogen-fixing Pseudomonas Stutzeri

Nitrate assimilation is an important part of nitrogen cycle.The reduction of nitrate to nitrous acid is the first redox reaction of assimilation.Environmental microbes play a key role in this process.Two typical nitrate assimilation pathways have been reported.They are the nitrate assimilation system regulated by the two-component NasST in A.vinelandii and the NasR regulation in K.oxytoca.In the nitrate assimilation system,the signal response and regulation mechanism of these two systems are obviously different.P.stutzeri A1501 is a model strain of rhizosphere associated nitrogen-fixing bacteria.The bacteria can not only fix nitrogen under micro-aerobic conditions,but also use nitrate as electron acceptor for denitrifying nitrogen fixation under anaerobic conditions,thus has strong nitrate assimilation ability.Genomic analysis showed that A1501 contains both NasST and NasR regulatory proteins,but the functions and regulatory mechanisms of these two sets of regulatory proteins are unclear.In this study,5'RACE,RT-qPCR and promoter fusion expression analysis were introduced to study the coding region and transcription characteristics of nasST and nasR genes.The main results are as follows:1.The growth curve of A1501 with nitrate or nitrite as the sole nitrogen source was measured under different temperature and pH gradient conditions.The results showed that A1501 had the best growth at 37?with pH8.0.In addition,the growth curve of A1501 under gradient nitrite concentrations as the sole nitrogen source were measured.The results showed that A1501 grew weaker in nitrite above6 mM.It is speculated that high concentration of nitrite may be toxic to cells;Comparing the nitrate assimilation capacity of wild-type and the mutants of key genes involved in nitrogen regulation ntrC and nifA,it was found that the growth of nifA mutant was slightly enhanced than wild-type,but ntrC mutant completely lost the ability of nitrate and nitrite assimilation.RT-qPCR analysis showed that the nitrate and nitrite transporter encoding genes nasA and nasF,and the nitrate reductase encoding gene nasB in the ntrC mutant were all down-regulated by more than 95%;in addition,the regulatory gene nasR was down-regulated by 70%,The two-component regulatory system gene nasS was down-regulated by 40%,indicating that the nitrate assimilation pathway of A1501 was regulated by NtrC protein.2.The 5'RACE method was introduced to determine the transcription start site of nasST and nasR respectively.On this basis,promoter-lacZ fusion expression vectors of DNA fragments with different lengths were constructed.By comparing the?-galactosidase activity of different vectors,we determined the promoter region of nasST and nasR:the promoter interval of nasST and nasR was determined,that is,the promoter of nasST is from-272 to+1,and the promoter of nasR is from-315 to+1;The sequence characteristics of the nasST and nasR promoters were analyzed,the results showed that there are three NtrC putative conserved binding sites and one RNA polymerase?factor RpoN putative binding site in the promoters of both nasST and nasR genes.3.The?-galactosidase activity of nasST and nasR gene promoter-lacZ fusion vectors under different nitrogen source conditions were measured.The results showed that The expression of nasST gene is not affected by nitrogen sources,while nasR was induced and expressed only under nitrate and nitrite treatment,and expressed at low levels under ammonium salt treatment.The effect of NtrC and RpoN binding site deletions?mutations?in nasST and nasR promoters was further determined.The results indicate that the NtrC binding site in the-39 to-19 region on the nasST promoter may be necessary for transcriptional activation,while the NtrC binding site in the-291 to-275 region of the nasR promoter is necessary for transcriptional activation;mutation of the RpoN conserved binding site does not affect the nasST promoter activity,but causes the nasR promoter to lose transcriptional activity.In summary,there are two different nitrate regulation systems NasST and NasR in A1501.The expression of nasST gene is not affected by nitrogen sources,its promoter is non-?⁵⁴ dependent type;the expression of nasR is only induced by nitrate and nitrite,its NtrC and RpoN dependent.This study shed light on the mechanism of nitrate assimilation regulation network in Pseudomomas stutzeri A1501.