Cloning and characterization of an iron regulated iroA locus in Salmonella choleraesuis

• Main Authors: Wen-Sheng Wu, 吳玟軒
• Other Authors: Chao-Fu Chang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/14026819499705162952

碩士 === 國立臺灣大學 === 獸醫學研究所 === 89 === To identify genes belonging to the Fur regulon of Salmonella choleraesuis, a plasmid gene bank consisting of 10,000 independent clones was screened for Fur promoters by a Fur titration assay (FURTA) and resulted in 15 positive clones. DNA sequence analysis from one of these FURTA positive clones, pSC4, showed homologous to the iroB gene of the iroA locus of S. typhi. The iroA locus of S. choleraesuis was cloned and sequenced. The complete nucleotide sequence of 9,848 bp of iroA locus of S. choleraesuis comprises iroBCDE cluster and iroN genes, which were 97% identical to that of S. typhi. The iroA locus consists of 2 operons with a putative Fur box each. IroN mediated the uptake of the catechol siderophores and also showed homologous to the family of TonB-dependent outer membrane siderophore receptors and a putative virulence gene, iroNE.coli, of the extraintestinal pathogenic E. coli. The expression of rIroN was by using T7 RNA polymerase and belong to partial insoluble protein while cultured at 20℃. IroN was located on the outer membrane fractions when probed with rabbit anti rIroN serum. The convalescent porcine sera contained antibodies against the three major iron regulated outer membrane proteins, FepA, IroN, and FhuE of S. choleraesuis. A S. choleraesuis iroN insertion inactivation mutant generated by allelic exchange resulted in the loss of expression of the 80 kDa protein. Introducing of a plasmid containing the iroN coding sequence with its upstream Fur box into this mutant had restored its expression. The results of LD50 did not show any different between wild type and mutant strains via intraperitoneal inoculation in BALB/c mice. It indicates the iroN gene of S. choleraesuis did not the virulence factor.