UV-induced Mutation Of Vibrio Harveyi GDH11385 And Comparative Genomics Analysis Of Avirulent Strains

Vibrio harveyi is a bacterial pathogen that seriously harms mariculture animals in South China in recent years.In order to explore the molecular pathogenic mechanism of this pathogen,we adopted a ultraviolet mutagenesis approach to obtain mutant strains with different virulence.The genomic comparative analysis technique was used to deeply mine the mutated genes in order to identify the major genes related to the virulence of Vibrio harveyi.Using the UV mutagenesis technique,2361 mutant strains were obtained.However,a large number of mutant strains could not be normally cultured and were very likely to enter the VBNC(viable but non-culturable)state.Finally,we successfully recovered 1222 mutant strains after ultraviolet mutagenesis with the optimal medium(2216E medium with additional of Tween 20(0.18 m L/m L)and vitamin B(2mg/m L))determined by single factor tests.The juvenile prawn of L.vannamei were used as test animals.The infection assays were conducted by intramuscular injection.The results showed that the probability of generation of low virulent strain could reach up to 63% at the following condition: 15 W UV lamp power,30 cm irradiation distance,and 270 s irradiation time.Therefore,using this condition as an ideal condition for obtaining low virulent strains of Vibrio harveyi GDH11385,a total of 378 avirulent strains(virulence was lower than that of wild strains,and the mortality rate of artificial infection within 24 hours was less than 50%)and 41 virulent strains(virulence was greater than that of wild strains,all prawns died in 2h after artificial infection)were obtained.After all the attenuated strains were continuously subcultured,89 attenuated strains that could inherit stably were obtained,including 31 avirulent strains.The third generation sequencing technology was used to re-sequence four mutagenized avirulent strains(s11,s31,s41,and s51),and the results of genome sequencing of the wild-type strain GDH11385 were taken as the reference for analysis.The results showed that the total length of the genome sequence of the four mutant strains was 5,046,212 bp～5,126,831 bp,and the GC content was between 45.20% to45.31%.The cumulative length of the coding gene fragments of the four mutant strains accounted for between 84.39% to 85% of the total genome length;the tandem repeat sequences of strains s11,s31,s41 and s51 accounted for 0.2743,0.2375,0.2908,and 0.346 of their genomes,respectively;the minisatellite DNA of strains s11,s31,s41,and s51 accounted for 0.0601,0.0364,0.0399,and 0.0447 of their genomes,respectively;the microsatellite DNA of strains s11,s31,s41,and s51 accounted for0.0077,0.0091,0.0091,and 0.0128 of their genome,respectively.By comparing the genomes with wild-type strains,we found that the four strains all lacked the T1SS-encoding gene.The virulence factor database(VFDB)of the bacterial pathogen was used to annotate the genome sequences of wild strains and four avirulent strains.The results showed 28 virulence-related genes,such as acf A and acf B,are not to be virulence-related genes of Vibrio harveyi,and determined that the gene encoding T1 SS is not a virulence related gene of Vibrio harveyi.But the 221virulence-related genes were related to the strong pathogenicity of wild strains.