SIMPLE approach for isolating mutants expressing fimbriae.
Genomes of members of the family Enterobacteriaceae contain large repertoires of putative fimbrial operons. Since many of these operons are poorly expressed in vitro, a convenient method for inducing elaboration of the encoded fimbriae would greatly facilitate their functional characterization. Here we describe a new technique for identifying fimbriated bacteria from a library of transposon mutants by screening with immunomagnetic particles for ligand expression (SIMPLE). The SIMPLE method was applied to identify the T-POP mutants of Salmonella enterica serotype Typhimurium carrying on their surfaces filaments composed of PefA, the major subunit product of a fimbrial operon (pef) that is not expressed during growth in Luria-Bertani broth. Four such mutants were identified from a library of 24,000 mutants, each of which carried a T-POP insertion within the hns gene, which encodes a global silencer of horizontally acquired genes. Our data suggest that the SIMPLE method is an effective approach for isolating fimbriated bacteria, which can be readily applied to fimbrial operons identified by whole-genome sequencing.
Nuccio
Sean-Paul
SP
Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA 95616-8645, USA.
Chessa
Daniela
D
Weening
Eric H
EH
Raffatellu
Manuela
M
Clegg
Steven
S
Bäumler
Andreas J
AJ
D004251:DNA Transposable Elements; D036781:Fimbriae Proteins / Q000096:biosynthesis / Q000276:immunology; D010861:Fimbriae, Bacterial / Q000235:genetics* / Q000276:immunology; D018189:Immunomagnetic Separation / Q000379:methods*; D016253:Microscopy, Immunoelectron; D016254:Mutagenesis, Insertional; D009154:Mutation; D012486:Salmonella typhimurium / Q000235:genetics* / Q000302:isolation & purification*
D004251:DNA Transposable Elements; C101015:pefA protein, Salmonella enterica; D036781:Fimbriae Proteins