| BackgroundDental caries is one of the most prevalent chronic human diseases throughout the world.Multiple bacteria,including Streptococcus mutans,Actinomyces viscosus,and Lactobacillus acidophilus,have been confirmed to initiate carious lesions.Several studies have found that when dental caries occurs,these oral cariogenic microorganisms will produce synergistic effects,further aggravating the occurrence of dental caries.Therefore,controlling the dental plaque formed by pathogens could effectively prevent and treat caries.Probiotics,defined as live microorganisms that confer a health benefit to the host,were also reported to have a positive effect on the prevention of the development of caries lesions.However,most of the recent probiotic strains for oral care are primarily used to obtain gastrointestinal benefits and thus are probably not ideal for the oral environment.A probiotic strain with an oral origin would be a better candidate to reduce the incidence of dental caries.Bacillus subtilis is a Gram-positive,rod-shaped and non-pathogenic bacterium that does not produce endotoxin or exotoxin.Previous studies have identified that B.subtilis has the ability to inhibit the growth of pathogens through the production of secondary metabolites.For example,B.subtilis GS67 isolated from the human gut was reported to inhibit growth of B.thuringiensis DB27 and Staphylococcus aureus by producing fengycin and plipastatin.Alternatively,B.subtilis SPB1 was shown to exhibit an important antimicrobial activity against microorganisms with multidrug-resistance by producing biosurfactants.Additionally,B.subtilis strains AS43.3 and AS43.4 were found to be antagonists of the plant pathogen Gibberellazeae by producing antifungal lipopeptides of the iturin and fengycin family.Given the safety evaluations by different authoritative bodies around the world,B.subtilis species have the potential to be used to inhibit the growth of cariogenic bacteria.Moreover,the human oral cavity is a highly dynamic and complex biological system in which bacteria must overcome a wide range of environmental conditions to survive,such as highly varied pH levels in dental plaque,oxygen gradients in the dental biofilm,and solute accumulation from different sources.Therefore,the ability to colonize the complicated oral habitat should be a prerequisite for the development of potential probiotic strains for the prevention of dental caries.In our study,we isolated a B.subtilis strain TLO3,from the dental plaque of a caries-free volunteer.This strain has been deposited at the China General Microbiological Culture Collection Center(CGMCC)under accession number 9952(named as B.subtilis zh78 previously).TLO3 had antagonistic effect on cariogenic species Streptococcus mutans,Actinomyces viscosus and Lactobacillus acidophilus.In addition,via untargeted metabolomics analysis,we found that this strain can produce 15 kinds of metabolites which may have antibacterial effects on S.mutans,L.acidophilus,and A.viscosus,such as xylitol,L-glutamic acid,and L-tyrosine(unpublished results).All these results confirmed that TLO3 could effectively inhibit the growth of cariogenic species.ObjectiveTo analyse the whole genome and molecular biological function of the B.subtilis TLO3,revealing the gene fragment that B.subtilis TLO3 can inhibit the growth of cariogenic species.And identify the characteristic genes of B.subtilis TLO3 by comparative genomics.MethodsThe genome of B.subtilis TLO3 was sequenced using a combination of the three-generation and second-generation sequencing technologies,and using relevant softwares for its genome assembly,gene prediction,functional annotation.And choosing B.subtilis subsp.spizizenii strain TU-B-10,B.subtilis subsp.inaquosorum strain DE111 and B.subtilis subsp.subtilis strain 168 whose complete genome sequences have been reported,to perform the comparative genome analysis with B.subtilis strain TLO3.Results1.The genome of B.subtilis TLO3 was sequenced using a combination of the PacBio RSII and Illumina MiSeq platforms.And draw the circular genome maps.2.The complete genome of B.subtilis TLO3 consists of one 4,072,939 bp chromosome and one 6610 bp plasmid,with a G+C content of 43.61 and 39.30%,respectively.The complete genome chromosome and plasmid sequences of B.subtilisstrainTLO3 have been deposited in the GenBank database with accession numbers CP023257 and CP023428 respectively.3.A total of 4444 open reading frames(ORFs)are predicted,of which chromosome ORF are 4434,with an average length of 806 bp,accounting for 87.73% of the whole genome,and can encode 4406 CDs,the number of rRNA are 30,tRNA are 85,accounting for 1.125% and 0.161% respectively.The ORF number of the plasmid are 10,average length 456 bp,accounting for 69.03% and 8 CDs were predicted.4.The preliminary analysis of the B.subtilis TLO3 complete genome showed: no CRISPRs in B.subtilis TLO3,2 incmplete prophages,1 questionable prophage;700 genes involved in the formation of 30 genomics islands;54 antibiotic-related genes were predicted,of which 34 genes associated with antibiotic resistance,2 genes were associated with antibiotic biosynthesis;155 CAZy were predicted in the chromosome.And the signal peptide could be found in 259 CDs of chromosome,and 1CDs of plasmid.5.There are 43 genes identified contributing to the synthesis of 14 antibacterial compounds(Phenylpropanoid,Isoquinoline alkaloid,Tropane,Piperidine and Pyridine alkaloid,Caffeine metabolism,Glucosinolate,Penicillin and Cephalosporin,Carbapenem,Monobactam,Streptomycin,Neomycin,Kanamycin and Gentamicin,Acarbose and Validamycin,Novobiocin,Prodigiosin,Phenazine.)6.Neighbor-joining tree based on 16 S rRNA gene sequences of B.subtilis TLO3 showing that strain TLO3 was closely related to Bacillus tequilensisKCTC13622,B.subtilis subsp.spizizenii NRRLB23049,B.subtilis subsp.inaquosorum KCTC 13429 and B.subtilis subsp.subtilis NCIB 3610 with a similarity of 99.86%,99.71%,99.86% and 99.79%,respectively.The OrthorANI algorithm was further used to assess the whole genome similarity between the five strains and indicated that strain TLO3 was most similar to the B.subtilis subsp.subtilis strain NCIB 3610(98.43%)and the B.tequilensis KCTC13622 showed the lowest similarities to the other strains7.The comparative genome analysis of B.subtilis strain TLO3,B.subtilis subsp.subtilis strain 168,B.subtilis subsp.spizizenii strain TU-B-10 and B.subtilis subsp.inaquosorum strain DE111 indicated:(1)Evolutionary analysis showed that the strain TLO3 was most similar to the strain 168,followed by TU-B-10,and far away from DE111.(2)The collinear analysis showed that except for a certain degree of rearrangement,inversion and deletion,the whole genome sequences of four Bacillus subtilis were highly similar and their evolutionary distances were relatively short.The genomes of TLO3 and 168 were the highest.(3)Comparative analysis of the gene familily and the eggNOG-Mapper functional annotation indicated that the number of protein sequences in replication,recombination and repair were 93 in strain TLO3,which were higher than the other three strains by 41%,31%,and 52%,respectively.8.There are a total of 27 secondary metabolite gene clusters in the B.subtilis TLO3 complete genome,of which 7 secondary metabolite gene clusters have antibacterial properties,like as Surfactin,Fengycin,Bacillibactin,Bacilysin,Subtilosin_A,Zwittermycin_A.ConclusionThe novel oral B.subtilis strain TLO3 contains genes that are involved in the synthesis of antimicrobial compounds(surfactin,tridecaptin,fengycin,bacillibactin,terpenes,and sactipeptide)and is believed to be more resistant and adapted to the complicated environment of the oral cavity.The double beneficial effects of B.subtilis strain TLO3,together with its dental colonization capacity,make it a potential probiotic for the prevention or treatment of dental caries.Further research on the genome presented here will contribute to the understanding of the biosynthesis of antimicrobial secondary metabolites produced by B.subtilis and will provide more information on a potential tool for the prevention of dental caries. |
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