Vibrio anguillarum is a common bacterial pathogen in fish.However,little is known about its pathogenic mechanism,in part,because the entire genome has not been completely sequenced.We constructed a fosmid library for V.anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage.We characterized the library by end-sequencing 50 randomly selected clones.This generated 93 sequences with a total length of 57 485 bp covering 1.4% of the whole genome.Of these sequences,58(62.4%) were homologous to known genes,30(32.3%) were genes with hypothetical functions,and the remaining 5(5.3%) were unknown genes.We demonstrated the utility of this library by PCR screening of 10 genes.This resulted in an average of 6.2 fosmid clones per screening.This fosmid library offers a new tool for gene screening and cloning of V.anguillarum,and for comparative genomic studies among Vibrio species.
We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar,lower protease activity against azocasein,and lower activity for four glycosidases.This prtV mutant strain also had increased activity for two esterases in its extracellular products,as analyzed by the API ZYM system.In addition,the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes.Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish.We propose that prtV plays an important role in the pathogenesis of V.anguillarum.
EmpA is an extracellular metalloprotease secreted by Vibrio anguillarum.For better understanding its role in the patho-genicity of V.anguillarum strain M3,empA insertion mutant was constructed.In the mutant it decreased in extracellular proteolytic activity,swarming motility,hemolytic activity and virulence on turbot(Scophthalmus maximus).Significant decline(by 5-fold)of extracellular proteolytic activity and similar growth curve between mutant and wild type strains indicated that EmpA was the major extracellular protease of M3.LD50 of mutant increased by 38-fold compared with wild type.No pro-EmpA was detected in the su-pernatant of culture,indicating that EmpA autolyzed to mature protein after 24 h.Secretion of EmpA in M3 was similar to that in NB10 strain.Attenuated virulence of mutant was similar to that of M93Sm strain.It was demonstrated that specific operation of EmpA was different from that in previous studies and EmpA contributed to the swarming motility and hemolytic activity in V.an-guillarum strain M3.The results provides insight into understanding the function of EmpA and its potential application in vaccine development.
HAN YifanMO ZhaolanXIAO PengHAO BinLI JieYANG Guanpin
