| Nitrous oxide is one of the six greenhouse gases.Although nitrous oxide is a trace gas in the atmosphere,its ability to participate in photochemical reactions in the stratosphere of catalytic ozone depletion makes its warming potential of greenhouse effect 300 times greater than carbon dioxide and contributes significant influence on the global greenhouse effect.Microorganism denitrification in natural environment is the main source and also the sink of N2 O.And N2 O reductase gene(nos Z)in microorganisms is the only gene that can reduce N2 O to N2 among organisms.Therefore,the study of denitrifying microorganism N2 O reductase gene for mitigating N2 O emission is particularly important.In recent years,a new type of nitrous oxide reductase gene has been found in the existing gene pool.Sequence of this new type gene showed their difference with that of the traditional denitrifying bacteria.PCR primers designed based on the traditional nos Z gene sequence failed to capture this novel gene,resulting in a wide underestimation of the nos Z gene content in the environment as well as its ecological effects were not revealed.In particular,the physiological and ecological functions of two different nos Z genes are poorly understood.In this study,we analyzed the sequence of the N2 O reductase gene of the important denitrifying bacterium Thauera linaloolentis 47 Lol.It was found that the strain contains two copies of nos Z gene.By aligning sequence and analyzing gene encoding characteristics,we found the two copies belong to the traditional nos Z(nos ZI)and the new nos Z(nos ZII)respectively.In order to investigate the differences in the physiological characteristics of the two nos Z copies of the strain and the niche occupied by them,we used gene cloning technique to knock out nos Z in Thauera linaloolentis 47 Lol.We used SOE-PCR to construct target segments,constructed the recombinant plasmids to transform into intermediate strains,then cocultured with wild-type Thauera linaloolentis 47 Lol.After screening the mutants containing the correct knock-out sequence fragments,we obtained two mutants of Thauera linaloolentis 47 Lol successfully and each of them only contains one nos Z gene copy.In order to rapidly screen the possible environmental conditions that may influence physiological characteristics differences caused by the two types of nos Z genes,we used orthogonal experiments for different culture conditions and compared N2 O reduction ability with T.linaloolentis 47 Lol wild-type strain.The results showed that the medium type was the main reason for the differences between the two mutants.Through the refinement of the nutrient content,we further explored the conditions under which nos ZII exerts its N2 O reduction effects.Adequate carbon source and energy source are the key factors in the role of the novel nos Z gene in T.linaloolentis 47 Lol.In summary,this study showed that the N2 O reduction characteristics of nos ZI gene in Thauera linaloolentis 47 Lol was similar to that of the wildtype Thauera linaloolentis 47 Lol strain,which contains both two nos Z gene copies.Thauera linaloolentis 47Lol_nos ZI reduced N2 O to N2 rapidly under oligotrophic conditions,which showed its major role in the Thauera linaloolentis 47 Lol strain.However,the new nos ZII gene required more nutrient-rich culture conditions and N2 O reduction happened after a period of N2 O accumulation,indicating that the requirements for living environmental and anaerobic conditions between the two nos ZII genes are different as well as in physiological function. |
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