Nitrate Adaptation Mechanism And Crp/Fnr Transcriptional Regulator In Desulfovibrio Vulgaris Hildenborough

Desulfovibrio vulgaris is a member of the dissimilatory sulfate-reducing bacteria(SRB)widely distributed in anaerobic environments,such as gas pipelines,oil reservoirs,sediments,and waster water treatment plants.SRBs are problematic in the oil industry because they cause corrosion of pipelines and equipment and souring of oil reservoirs,and the H2S produced by SRBs leads to hazards for personal working in the petroleum industry.Nitrate and nitrite have been used in the oil well and bioreactor models of the oil industry to prevent the problem.However,SRBs could survive inhibition by nitrate and nitrite.Survival in the nitrate could be beneficial in nitrate and heavy metal contaminated environmental sites where SRBs could be significant members involved in biomediation of heavy metals and radionuclides.Well studied Crp/Fnr family global transcriptional regulators make a significant contribution for DvH to survive the harsh conditions.Understanding of the nitrate adaptation mechanism and the function of Crp/Fnr family global transcriptional regulators in the DvH could facilitate the development of strategies to monitor and predict and performance of DvH in the petroleum industry and in bioremediatio.We used Desulfovibrio vulgaris Hildenborough(DvH)as a model organism to investigate the nitrate adaptation mechanism and function of Crp/Fnr regulator.Firstly,12 ancestral DvH populations,6 EC(evolved under control condition for 5000 generations)and 6 ES(evolved under salt stress for 5000 generatiosn)propagated in the nitrate stress LS4D(defined lactose/sulfate)medium for 1000 generations.To reveal the possible adaptation mechanism of DvH nitrate adaptation,we analyzed the physiological characters and whole genome resequencing data.Then,the in-frame deletion was applied to generate the Crp/Fnr regulators mutants to avoid potential polar effect.Physiological characters and gene expression profiling of the wild type strain and mutants under different stresses were investigated to understand the distinct and overlapped functions of the Crp/Fnr regulators,and main conclusions as following:(1)12 ancestral DvH populations,6 EC(evolved under control condition for 5000 generations)and 6 ES(evolved under salt stress for 5000 generatiosn)propagated in the nitrate stress LS4D(defined lactose/sulfate)medium for 1000 generations.Under 30 mM nitrate stress,the lag phase of EN populations was(16.30± 1.03)h,were shorted than lag phse of AN populations((27.33±1.15)h),and the growth rate of EN populations((0.167±0.018)h-1)increased compared to AN populations((0.118±0.014)h-1),the final biomass(OD600)were also increased for EN populations(1.176±0.048)in comparison of AN populations(0.924 ±0.039).Under 50 mM nitrate stress,the nitrate tolerence of 6 ECN and 6 ESN populations were increased,compared ECN population to EC populations,the lag phase was(30.58±6.93)%shortened,growth rate were increased(22.95±13.22)%,and final biomass were also increased(14.31±2.52)%.Compared ESN population to ES populations,the lag phase was(36.59±7.25)%shortened,growth rate were increased(9.97±7.46)%,and final biomass were also increased(12.33±3.72)%.In addition,the cell motility of the nitrate-evolved populations were significantly increased compared to the original populations.(2)Copared the whole genome sequences of the original populations and nitrate-evolved populations through the BLAST tools,SNPs and mutations were discovered in several gene clusters.The high mutation rates were found on the chemotaxis protein B glutamate methylesterase(CheB)gene and genes involved in interaction with CheB,which was consistence with the cell motility of the nitrate-evolved populations increased.Also,SNPs and mutations in the nitrate clusters(DVU0251-DVU0245)and HcpR(DVU2547,Crp/Fnr regulator)and its regulons were found in all the nitrate-evolved populations,which indicated these twe gene clusters were critical for nitrate adaptation of DvH.Through the secondary tertiary structure prediction of mutated HcpR,all the mutations caused the changes in the structure of HcpR,and this result confirmed the importance of HcpR on the regulation of nitrate resistence.In additional,several mutation were existed in general stress response,such as iron homeostasis regulated by Fur family regulator,LytS/LytR two component system,and actyl-carrier proteins gene which involved in lipid metabolism.Above all,the nitrate adaptation mechanism was linked to components of chemotaxis regulations,nitrate stress specific response and nitrite stress response,and several general stress response.(3)Four Crp/Fnr homologs(DVU0379,DVU2097,DVU2547,and DVU3111)in DvHare considered global transcriptional regulators in responses to a variety of environmental changes.In this study,we generated all possible deletions of those four genes,including four single,six double,four triple and one quadruple markerless mutants,and then their growth under different environmental stressors,such as elevated nitrate,nitrite,NaCl,H2O2,and chromate conditions.Our results indicated that the four Crp/Fnr homologs played distinct roles in stress response and functional redundancy was observed.Specifically,DVU0379 and DVU3111 played major roles in response to elevated NaCl,DVU2097 to elevated H2O2,DVU2547 to elevated nitrite and nitrate,and DVU3111 to elevated chromate.More interestingly,the quadruple deletion mutant showed an increased growth in response to elevated H202 although a decreased growth was observed under other environmental stressors compared to the wild-type,suggesting that other regulators may also play a role 'in response to environmental stressors when those four reported Crp/Fnr homologs were absence.(4)The feasi-bility of new Agilent pure culture microarray(Version 5)for the transcriptional assay for DvH was tested,and considerd high hybridization rate of gDNA,less number of probes with saturated value of cDNA,and less false positive rate,the concentration of formamide should be 16.5%.Then,the gene expression profiling of wild type strain JW710 and Crp/Fnr regulators deleted mutant JWQ0123 under NaCl,NaNO3 and NaNO2 stresses were analyzed using microarray.The expression of formate dehydrogenase gene was increased under all the stresses,which suggested the formate metabolism played a role in stress response.The expression of genes involved in glycerol transport and metabolism were changed,indicated the osmotic stress contribute to inhibition of DvH by elevated nitrate.And expression of regulons of HcpR were upregulated under nitrate and nitrite stress,indicated the positive regulation of HcpR for nitrate adaptation,which was consistence with the result that the nitrate resistence of DVU2547 deleted mutants decreased.And gene expression of cell motility increased under nitrate stress was correspondent with the increased cell motility of nitrate-evolved populations.Compared the gene expression profiling of JW710 and JWQ0123,the expression of the genes involved in energy production and conversion,amino acid transport and metabolism,transcription and translation,and replication,recombination and repair increased in JWQ0123.And changes of expression of genes involved in coenzyme transport and metabolism only existed in JWQ0123.In additional,the expression of genes involved in cell wall and membrane biogenesis,post-translational modification,protein turnover and chaperones increased in JWQ0123 under nitrate and nitrite stress.Finally,expression of genes involved in tryptophan metabolisms was upregulated under all the stresses in JWQ0123,which suggested genes involved in tryptophan metabolisms played a rote in stress response when Crp/Fnr family global transcriptional regulators were absence.Above all,the nitrate adaptaion mechanism was linked to component of increasing cell motility,and positive regulation of HcpR regulator,nitrate resistence of nitrate cluster(DVU0251-DVU0245),osmotic stress response and general stress response,such as iron homeostasis regulated by Fur family regulator,LytS/LytR two component system,actyl-carrier proteins gene which involved in lipid metabolism,and energy metabolism such as formate metabolism.Four Crp/Fnr homologs(DVU0379,DVU2097,DVU2547,and DVU3111)in DvH are considered global transcriptional regulators in responses to a variety of environmental changes.Specifically,DVU0379 and DVU3111 played major roles in response to elevated NaCl,DVU2097 to elevated H2O2,DVU2547 to elevated nitrite and nitrate,and DVU3111 to elevated chromate.More interestingly,the quadruple deletion mutant could survived under multiple stresses.And the transcriptional assay suggesting that genes involved in tryptophan metabolisms played a role in stress response when Crp/Fnr family global transcriptional regulators were absence.