The Specificity of Secretion through the Alpha and Beta Type II Secretion Systems of Escherichia coli
Miller, Mitchell B 1991-
The Type 2 Secretion System (T2SS) is a molecular apparatus that is found in many Gram negative bacteria. The T2SS system is used for the export of proteins from the cytosol to the extracellular space. A key element in secretion is the assembly of the secretin (GspD), during which monomeric GspD proteins must assemble into a multimeric structure in the outer membrane before secretion can occur. Escherichia coli possesses two different T2SS systems termed the alpha and the beta system. The beta T2SS system is normally active and has been shown to function in the secretion of heat-labile enterotoxin (LT) by Enterotoxigenic (ETEC) E. coli strains. The alpha T2SS system, present in both ETEC and K12 E. coli strains, is silenced during growth under standard laboratory conditions but has been used to study the secretion of LT when the T2SS system is overexpressed from plasmids. The goal of this study was to express the chromosomal copies of the cryptic alpha T2SS system of E. coli and to observe its function in the secretion of LT and Chitinase (ChiA). Mutant strains of E. coli K12 were created in strain BW25113, these mutations consisted of deletions of hns and stpA which are known gspα operon repressors. No expression of the GspDα secretin or secretion of LT or ChiA by the T2SS system were observed in the strains containing deletions in these genes. Additional mutant strains were created in E. coli K12 strain MG1655 in which the natural promoters of the gspα operons were replaced with inducible promoters, the lactosetryptophan fusion promoter (ptac) was used to control the gene expression of the gspABα operon while the arabinose promoter (paraBAD) was used to control the gene expression of the gspC-Oα operon. When the strains created were induced with IPTG and arabinose, expression and assembly of the secretin multimer was observed, but this did not result in the secretion of LT or ChiA. 4 The GspAB complex is required for the assembly of the secretin in Aeromonas hydrophila. The requirements for the GspAB complex in secretin assembly was investigated in E. coli K12 strain MG1655. Mutants were created with a kanamycin (kan) resistance cassette inserted into gspB, while the natural promoters of gspAB and gspC-O were replaced with the inducible promoters ptac and paraBAD, respectively. Induction of these strains showed that GspBα is not required for assembly of the secretin multimer.
DegreeMaster of Science (M.Sc.)
DepartmentMicrobiology and Immunology
ProgramMicrobiology and Immunology
CommitteeWhite, Aaron; Bull, Harold; Xiao, Wei; Chen, Jeffrey
Copyright DateSeptember 2016