Isolation And Identification Of Facultative Aerobic Mutant Of Coxiella Burnetii

Coxiella burnetii is the causative agent of human Q-fever.It's mainly transmitted by aerosols and is highly infectious to humans and animals.People infected with Q-fever are mainly characterized by acute influenza-like symptom,such as fever and headache,some of whom may turn into a chronic infection,endocarditis is the most common.Coxiella burnetii strain Nine Mile originated from the United States,is the reference strain in Q-fever research.it's prone to transform from virulent phase I to attenuated phase Crazy or avirulent phase ? when subcultured in vitro.In the past decades,C.burnetii has been considered to be an obligate intracellular microorganism and could not be cultured in vitro.The traditional culture method is to infect animals,chicken embryo,and cells.In 2009,a cell-free culture of C.burnetii appeared in the USA.Axenic cultivation of C.burnetii improves the efficiency of culture and leads to advances in genetic manipulation.Foreign studies have found that C.burnetii is a strict microaerobic bacteria and needs a certain concentration of oxygen which is more than 10%when cultured in a cell-free medium.In the first part of this study,we cloned and purified Nine Mile phase? strain and analyzed its whole genome sequence.C.burnetii has a long culture cycle and is prone to adaptive changes when cultured in vitro.Cross-contamination may occur in some cases when different isolates are cultured at the same time,thus misleading the results.So it is necessary to genetically identify the wild-type Nine Mile strain for research.At first,wild-type C.burnetii Nine Mile phase ? strain was purified with Semi-solid agar plate.The randomly selected monoclonal CB01 was cultured and sequenced.The genome of CB01 was compared with the genomes of different clones of C.burnetii to clarify the similarities and differences.The whole genome sequence of CB01 was obtained through next-generation sequencing and third-generation sequencing.The result showed that the CB01 genome contains a 1.987Mb circular chromosome and a 37.321Kb plasmid.Compared to the genome of Nine Mile phase ? clone RSA 493,CB01 has five insersionrepetitive sequences,a deletion region of 25997 bp,and 23 single nucleotide polymorphisms?SNP?.Compared to the genome of Nine Mile phase? clone RSA 439,CB01 has only 6 SNPs.CB01 and RSA439 share identical sequences of the CBU₀533 gene,which genetically explains the phase variation of CB01 due to the absence of O-antigen,The genome of CB01 clone is highly homologous with that of Nine Mile phase ? clone RSA 439.The results indicate that CB01 can be used as a reference strain for C.burnetii research in China.The second part of the study was to isolate and identify the facultative aerobic mutant from transformed Nine Mile phase ? strain,and to identify the growth of different strains under different media and different oxygen concentrations.In the process of culturing a transformed Nine Mile phase ? strain,we found that the transformant can grow normally when statically cultured under the condition of 20%oxygen concentration.To construct the transformant,a foreign plasmid was transferred into a wild-type Nine Mile phase ? strain by electroporation.Based on plasmid pMMB207,the plasmid transferred into C.burnetii contains an RSF1010 ori,a green fluorescent protein?GFP?gene,an antibiotic resistance marker,and a replication regulatory region and can passage stably in C.burnetii.Firstly,the transformed strain NM IIpMMGK was obtained by transformation and purified.And then,we tested the growth of C.burnetii in two culture media?ACCM-2 and ACCM-2 with 0.5 mM tryptophan?,two oxygen concentrations?20%and 2.5%?and three different inoculation concentrations.The wild-type Nine Mile phase ? strain and the wild-type Henzerling phase ? strain were set as controls.The initial inoculum and the product after 7 days of culture were collected separately,the genome was extracted,and the growth was measured by qPCR.The transformant NM ?pMMGK could normally grow at 20%oxygen concentration,but it was not stable and required a suitable initial concentration and fresh inoculum.And the transformant still needs 50%CO2 to grow at 20%oxygen concentration.Besides,limited growth could be detected in wild-type Nine Mile phase? strain at 20%oxygen concentration,with a suitable starting concentration,fresh inoculum,50%CO2,and tryptophan of 0.5 mM in ACCM-2.No growth could be detected on Henzerling phase ? under all the above conditions.The C.burnetii Nine Mile phase ? clone CB01 used in this experiment is highly homologous to the Nine Mile phase ? strain published abroad,the genomic sequences are only slightly different.Different strains have different tolerance to oxygen.Under certain conditions,the transformed strain NM ? pMMGK can grow normally under the condition of 20%oxygen concentration.The wild type Nine Mile phase ? strain can be limitedly grown under the condition of 20%oxygen concentration,while the wild type Henzerling phase ? can only grow under micro-oxygen concentration condition.The mechanism of the growth of C.burnetii at 20%oxygen concentration calls for further study.The results of our study showed that the genome of C.burnetii Nine Mile phase ?preserved in our laboratory has little difference with that of phase ? strain preserved abroad.It can be used as a standard strain for the study of C.burnetii,which would be helpful in further experiments.At the same time,facultative aerobic mutants were found and isolated,which provided a new idea for the cultivation of C.burnetii.In this study,the growth characteristics of C.burnetii at 20%oxygen concentration were identified,which has reference significance for exploring the mechanism of oxygen metabolism of C.burnetii.