Abstract
Type 1 fimbriae are filamentous structures on Escherichia coli. These structures are important adherence factors. Because binding to the host cells is the first step of infection, type 1 fimbria is an important virulence factor of pathogenic E. coli. Expression of type 1 fimbria is regulated by a phase variation in which each individual bacterium can alternate between fimbriated (phase-ON) and nonfimbriated (phase-OFF) states. The phase variation is regulated by the flipping of the 314-bp fimS fragment, which contains the promoter driving the expression of the genes required for the synthesis of type 1 fimbria. Thus, the bacterial proteins able to interact with fimS are likely to be involved in regulating the expression of type 1 fimbria. To identify novel type 1 fimbria-regulating factors, we used an E. coli K12 proteome chip to screen for the bacterial factors able to interact with a 602-bp DNA fragment containing fimS and its adjacent regions. The Spr protein was identified by the proteome chip-based screening and further confirmed to be able to interact with fimS by electrophoretic mobility shift assay. Deletion of spr in the neonatal meningitis E. coli strain RS218 significantly increased the ratio of the bacterial colonies that contained the type 1 fimbria phase-ON cells on agar plates. In addition, Spr interfered with the interactions of fimS with the site-specific recombinases, FimB and FimE, which are responsible for mediating the flipping of fimS. These results suggest that Spr is involved in the regulation of type 1 fimbria expression through direct interaction with the invertible element fimS. These findings facilitate our understanding of the regulation of type 1 fimbria.
Highlights
From the ‡Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli City, Taiwan; §Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan City, Taiwan; ¶Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan City, Taiwan; ʈCenter of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan City, Taiwan; **Department of Nutrition and Health Sciences, Kainan University
To identify novel bacterial factors that contribute to the regulation of type 1 fimbria phase variation, we initially used the E. coli K12 proteome chip, which contains at least 88% of entire E. coli K12 proteome [19], to screen for the proteins that may directly interact with fimS or its adjacent regions
The fimS-plus fragment was synthesized by PCR amplification of the chromosomal DNA of the neonatal meningitis E. coli strain RS218 with primers that were labeled with DIG at the 3Ј-ends
Summary
Proteome Microarray Construction—E. coli proteome microarrays were prepared as previously described [19]. Synthesis of the fimS-related DNA Fragments—The fimS-plus fragment was amplified by PCR using the primers, Ch1-F (5Ј-AGTAATGCTGCTCGTTTTGC-3Ј) and Ch1-R (5Ј-GACAGAGCCGACAGAACAAC-3Ј) as previously described [2]. The mixtures of the 6xHis-tagged recombinant proteins, fimS-plus, and 0.5% BSA (Sigma-Aldrich Co.) were incubated for 1 h at RT, and subjected to agarose gel electrophoresis (1.2% agarose, 4 °C). The SnaBI restriction fragments of the fimS-plus PCR products derived from the E. coli cells with fim promoter in the phase-ON orientation are 404 bp and 198 bp. The SnaBI restriction fragments of the fimS-plus derived from the phase-OFF cells are 160 bp and 442 bp. The fimS-plus PCR products derived from the E. coli cells cultivated in LB or on agar plates were purified and subjected to SnaBI (New England Biolabs) digestion. 10 l of 4Ј,6-diamidino-2phenylindol (DAPI) (0.2 l/ml) was applied to each sample to stain the nuclei of the bacteria
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