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The denitrification characteristics of Pseudomonas stutzeri SC221-M and its application to water quality control in grass carp aquaculture.

ABSTRACT: To reduce ammonium and nitrite in aquaculture water, an isolate of the denitrifying bacterium Pseudomonas stutzeri, SC221-M, was obtained. The effects of various nitrogen and carbon sources, the ratio of carbon to nitrogen and temperature on bacterial growth, denitrification rates and the expression levels of nirS and nosZ in SC221-M were studied. The following conditions were determined to be optimal for growth and denitrification in SC221-M: NaNO2 as the nitrogen source, sodium citrate as the carbon source, a carbon to nitrogen ratio range of 4-8, and a temperature range of 20-35°C. Subsequently, SC221-M and the Bacillus cereus BSC24 strain were selected to generate microbial preparations. The results showed that addition of the microbial preparations decreased various hydrochemical parameters, including total dissolved solids, ammonium, nitrite, total nitrogen and the chemical oxygen demand. Nitrogen removal rates were highest on day 9; the removal rates of BSC24, SC221-M, a mixed preparation and a 3× mixed preparation were 24.5%, 26.6%, 53.9% and 53.4%, respectively. The mixed preparation (SC221-M+BSC24) was more effective at removing nitrogen than either the SC221-M or BSC24 preparation. Roche 454 pyrosequencing and subsequent analysis indicated that the control and other groups formed separate clusters, and the microbial community structure in the water changed significantly after the addition of microbial preparations. These results indicate that the addition of microbial preparations can improve both the water quality and microbial community structure in an experimental aquaculture system. P. stutzeri strain SC221-M and its related microbial preparations are potential candidates for the regulation of water quality in commercial aquaculture systems.

SUBMITTER: Deng B

PROVIDER: S-EPMC4260960 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus of Pseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in a nirF strain, where NO generation from nitrite is prevented because of a defect in heme D(1) biosynthesis. NO-triggered responses were monitored from the nirSTB operon (encoding cytochrome cd(1) nitrite reductase), the norCB operon (encoding NO reductase), nosZ (encoding nitrous oxide reductase), and nosR (encoding a putative regulator). Transcription of nirSTB and norCB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell the transient appearance of nosZ and nosR transcripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N2O. NO at 250 nM stimulated in this cell type the expression of nos genes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression of dnrD in an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of the dnrD operon by inducing the transcription of dnrN and dnrO, located upstream of dnrD. Insertional mutagenesis of dnrN altered the kinetic response of the nirSTB operon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification in P. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by the narGHJI operon.

Project description:BackgroundMyxovirus resistance (Mx) proteins are crucial effectors of the innate antiviral response against a wide range of viruses, mediated by the type I interferon (IFN-I) signaling pathway. However, the antiviral activity of Mx proteins is diverse and complicated in different species.Methodology/principal findingsIn the current study, two novel Mx genes (CiMx1 and CiMx3) were identified in grass carp (Ctenopharyngodon idella). CiMx1 and CiMx3 proteins exhibit high sequence identity (92.1%), and low identity with CiMx2 (49.2% and 49.5%, respectively) from the GenBank database. The predicted three-dimensional (3D) structures are distinct among the three isoforms. mRNA instability motifs also display significant differences in the three genes. The spatial and temporal expression profiles of three C. idella Mx genes and the IFN-I gene were investigated by real-time fluorescence quantitative RT-PCR (qRT-PCR) following infection with grass carp reovirus (GCRV) in vivo and in vitro. The results demonstrated that all the four genes were implicated in the anti-GCRV immune response, that mRNA expression of Mx genes might be independent of IFN-I, and that CIK cells are suitable for antiviral studies. By comparing expression patterns following GCRV challenge or poly(I:C) treatment, it was observed that GCRV blocks mRNA expression of the four genes. To determine the functions of Mx genes, three CiMx cDNAs were cloned into expression vectors and utilized for transfection of CIK cells. The protection conferred by each recombinant CiMx protein against GCRV infection was evaluated. Antiviral activity against GCRV was demonstrated by reduced cytopathic effect, lower virus titer and lower levels of expressed viral transcripts. The transcription of IFN-I gene was also monitored.Conclusions/significanceThe results indicate all three Mx genes can suppress replication of grass carp reovirus and over-expression of Mx genes mediate feedback inhibition of the IFN-I gene.

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The denitrification characteristics of Pseudomonas stutzeri SC221-M and its application to water quality control in grass carp aquaculture.

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