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SIMPLE approach for isolating mutants expressing fimbriae.

ABSTRACT: 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.

SUBMITTER: Nuccio SP

PROVIDER: S-EPMC1932825 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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SIMPLE approach for isolating mutants expressing fimbriae.

Nuccio Sean-Paul SP Chessa Daniela D Weening Eric H EH Raffatellu Manuela M Clegg Steven S Bäumler Andreas J AJ

Applied and environmental microbiology 20070525 14

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 a ...[more]

PMID: 17526787

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Project description:Bacterial biofilm plays a pivotal role in bioremediation of heavy metals from wastewaters. In this study, we isolated and identified different biofilm producing bacteria from wastewaters. We also characterized the biofilm matrix [i.e., extracellular polymeric substances (EPS)] produced by different bacteria. Out of 40 isolates from different wastewaters, only 11 (27.5%) isolates (static condition at 28°C) and 9 (22.5%) isolates (agitate and static conditions at 28 and 37°C) produced air-liquid (AL) and solid-air-liquid (SAL) biofilms, respectively, only on salt-optimized broth plus 2% glycerol (SOBG) but not in other media tested. Biomass biofilms and bacteria coupled with AL biofilms were significantly (P ? 0.001) varied in these isolates. Escherichia coli (isolate ENSD101 and ENST501), Enterobacter asburiae (ENSD102), Enterobacter ludwigii (ENSH201), Pseudomonas fluorescens (ENSH202 and ENSG304), uncultured Vitreoscilla sp. (ENSG301 and ENSG305), Acinetobacter lwoffii (ENSG302), Klebsiella pneumoniae (ENSG303), and Bacillus thuringiensis (ENSW401) were identified based on 16S rRNA gene sequencing. Scanning electron microscope (SEM) images revealed that biofilm matrix produced by E. asburiae ENSD102, uncultured Vitreoscilla sp. ENSG301, A. lwoffii ENSG302, and K. pneumoniae ENSG303 are highly fibrous, compact, and nicely interlinked as compared to the biofilm developed by E. ludwigii ENSH201 and B. thuringiensis ENSW401. X-ray diffraction (XRD) results indicated that biofilm matrix produced by E. asburiae ENSD102, uncultured Vitreoscilla sp. ENSG301, and A. lwoffii ENSG302 are non-crystalline amorphous nature. Fourier transform infrared (FTIR) spectroscopy showed that proteins and polysaccharides are the main components of the biofilms. Congo red binding results suggested that all these bacteria produced proteinaceous curli fimbriae and cellulose-rich polysaccharide. Production of cellulose was also confirmed by Calcofluor binding- and spectrophotometric assays. E. asburiae ENSD102, Vitreoscilla sp. ENSG301, and A. lwoffii ENSG302 were tested for their abilities to form the biofilms exposure to 0 to 2000 mg/L of copper sulfate (for Cu), zinc sulfate (for Zn), lead nitrate (for Pb), nickel chloride (for Ni), and potassium dichromate (for Cr), several concentrations of these metals activated the biofilm formation. The polysaccharides is known to sequester the heavy metals thus, these bacteria might be applied to remove the heavy metals from wastewater.

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SIMPLE approach for isolating mutants expressing fimbriae.

Publications

SIMPLE approach for isolating mutants expressing fimbriae.

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