The Novel Nitrogen Metabolic Pathways And Its Regulation Mechanism In Anaerobic Phototrophic Bacteria

With the rapid development of human socialization and urbanization activity,the elevated NH3-N and NO2-N pollution problems have destroyed the regional ecologic nitrogen equilibrium and imposed serious threats to the animal health and coastal/offshore environment.Studies showed that microorganisms play a crucial role in the nitrogen biogeochemical cycle.Recently,some new nitrogen metabolism pathways were subsequently reported,which provided a new perspective and effective strategies for the solutions to various nitrogen pollution problems.Anaerobic phototrophic bacteria(APB)are ubiquitous in natural habitat and possess diverse metabolism properties,which play an important role in driving the carbon,nitrogen and sulfur biogeochemical cycle.Despite of the extensively application of APB in wastewater treatment,agriculture and aquaculture etc.,they are limited in freshwater environment due to their salinity tolerance.Less information is available on true marine APB species as well as their functions in mariculture ecosystem.It remains unclear about the mechanisms involved in nitrogen pollution removal and their functions in marine ecosystem.Marichromatium gracile YL28,isolated from mangrove special habitat,efficiently removed nitrite and ammonium under aerobic or anaerobic condition.However,its mechanism of nitrogen removal,especially for high concentration of nitrite utilization and tolerance are still unclear.In this study,the metabolism pathways of C,N and S of APB were comprehensively analyzed by the comparative genome.The nitrogen metabolism pathways of 36 purple bacteria,the molecular mechanism of the high-efficiency nitrogen removal and nitrite tolerance were systematically elucidated.A new nitrogen metabolism pathway and new gene in APB were mined and verified,and a new nitrogen metabolism pathway was found and proposed.Furthermore,the response of YL28 to the disturbance of external nitrogen compounds,illumination and oxygen were clarified,and the coordination mechanisms of the three coexisting nitrogen metabolism pathways in YL28 were further elucidated.Finally,the in-situ bioremediation effects of YL28 in polluted shrimp mariculture ecosystem were investigated.The main results were as follows:1.The comparative genome in 36 purple bacteria including purple sulfur bacteria(PSB)and purple non-sulfur bacteria(PNSB)were comprehensively analyzed.New enzymes involved in assimilation nitrate reduction(ANR)and dissimilation nitrate reduction to ammonia(DNRA)pathway were mined,and a new ammonia oxidation pathway was found.Comparative genome analysis showed that more nitrogen metabolic-related genes were found in PNSB than those in PSB.The partial denitrification pathway was found in PSB for the first time.A new non-ammonia monooxygenase-dependent ammonia oxidation pathway was mined,which possibly widely exists in microorganisms.The three sulfur metabolism genes such as oxidation of sulfide,reversed dissimilatory sulfite reduction and sox system,some unique stress response genes and diverse carbohydrate metabolism-related genes such as involved in EMP,HMP,ED,TCA,glycosylate cycle,dicarboxylic acid/4-hydroxybutyric acid were revealed,which benefits YL28 to adapt the complex environment.2.To verify the new mining pathways of ANR,DNRA and non-ammonia monooxygenase dependent ammonia oxidation as well as their biological functions,the combined approaches of homology modeling,multiple structure alignment,physiological characteristics prediction,physiological experiment and heterologous expression were used to confirmed these pathways.It was found that NirA and NrfA had the identity of 27.1% and 19.2% with known protein respectively.They were to verify the new enzymes of ANR and DNRA pathway.The optimum temperature and pH of NirA and NrfA were at 40 ?,5.0 and 35 ?,6.0,respectively.Hcp could convert hydroxylamine into ammonium,the identity compared with known hcp protein was 45%.The optimal temperature and pH of the Hcp were at 35 ? and 6.The test of the non-dependent ammonia monooxygenase hydroxylamine formation pathway showed that the adding 2 mg/L Vc(hydroxyl radical inhibitor)could inhibit about 80% of hydroxylamine formation.The hydroxylamine information contents increased by 50% after the addition of 100 ?M hydrogen peroxide(hydroxyl radical promoter),the time of hydroxylamine production was shorter than that of the control group within 2 h,suggesting that hydroxyl radical is the key factor for hydroxylamine formation.3.The responses of the three coexisting nitrogen metabolism pathways to the change of nitrogen compounds,light and oxygen as well as the the coupling relationships among nitrogen,carbon and sulfur cycle were investigated.The results on transcription level showed that:(1)In the coexistence system of ammonia,nitrite or nitrate,ammonia exhibited a significant inhibition on the ANR pathway,the expression level of nirA was reduced by 50% with 2 mg/L of ammonia,but had less effect on DN.The presences of nitrate,hydroxylamine and nitrite also promoted the key gene expression of denitrification.As for DNRA,the four nitrogen compounds of ammonia,nitrate,hydroxylamine and nitrite promoted the gene expression of nrfA.Ammonia inhibited the gene expression of hydroxylamine reduction pathway,other three nitrogen sources had less effect on it.The presence of ANR and DNRA inhibited the expression of hcp due to the formation of ammonia produced from these two pathway.(2)Under the constant light and oxygen condition,the key gene expression level of three nitrogen metabolism pathways increased with increasing nitrate concentration;in the presence of high concentration of nitrate,the expression level of denitrifying genes was about 18 times(narI)and 37 times(norB),36 times(nirA)and 2.5 times(nrfA)higher than that in low concentration of nitrate.(3)The increasing light intensity promoted the gene expressions of nirA,norB and narI,but had no significant effect on nrfA(P < 0.05),the gene expression levels were approximately 20 times(narI),28 times(norB)and 24 times(nirA)higher than that of in the dark,indicating that light promote the denitrification and ANR activity.In the dark,the key gene expression levels were in the order of nirA > norB > nrfA.(4)The key genes involved in DN,ANR and DNRA were expressed both in oxic and anoxic condition,but high expression level were observed in anoxic condition and were about 37 times(norB),16 times(narI),20 times(nirA)and 3 times(nrf A)higher than that in oxic condition.In oxic condition,the gene expression levels were in the order of nirA > norB,narI > nrfA.(5)Under anaerobically in the light or dark and in the presence of nitrite and nitrate system,the gene expression level of DN was higher than that of DNRA,the gene expression level of ANR was significantly inhibited.The gene enrichment analysis showed that the gltb,glnA and cys E were the key coupling genes of carbon,nitrogen and sulfur cycle in purple sulfur bacteria,and the optimal ratio of C: N: S for nitrogen removal efficiently was 7.56: 6 :5.4.The bioremediation in-situ of YL28 and the ability to maintain nitrogen balance in marine aquaculture were investigated.In laboratory aquaculture system,approximately 99.96% of nitrite(1.0 mg/L)was removed within 7 d through denitrification coupled with assimilatory nitrate reduction.Ammonia(3.5 mg/L)of 95.6% was directly assimilated by YL28 within 7 d.Moreover,in zero exchange water from shrimp(Penaeus vannamei)aquaculture field trials(20 d),YL28 significantly reduced the ammonia accumulation(0.6 mg/L)and 99.3% of nitrite(1.25 mg/L).Toxicological studies with the Institute of Cancer Research(ICR)mice and Oryzias melastigma indicated that YL28 can be safely applied in marine aquatic ecosystems.Collectively,in this study,a new microbial ammonia oxidation pathway,two novel enzymes involved in ANR and DNRA pathways in PSB were mined and verified.The molecular mechanisms of the tolerance of high concentration nitrite and high efficient nitrogen removal of YL28 were linked to the combination of three nitrogen cycle pathways(DN,ANR and DNRA).This study provided a novel insight into the mechanisms of diverse nitrogen cycle,habitat-specificity and toxic nitrite utilization in YL28,which provided theoretical basis and effective strategy for the nitrogen pollution removal and the practical application of YL28.