Analysis of cis-acting elements required for bfpA expression in enteropathogenic Escherichia coli

Abstract

bfpA expression in enteropathogenic Escherichia coli is regulated by growth medium, temperature, and ammonium concentration and requires the BfpT protein (also called PerA), a member of the AraC family of transcriptional activators. Site-directed and PCR random mutagenesis, as well as deletion analysis of the bfpA upstream regulatory region, supported assignment of the promoter elements and demonstrated that the cis-acting elements that mediate BfpT-dependent regulation of bfpA are located between positions -85 and -46. Interestingly, this region shares 73% identity with a 40-bp-long AT-rich tract located upstream of the bfpT gene, which is essential for bfpT autoregulation.

FIG. 1

Regulation of the bfpA 5′ regulatory region deletion fusions in response to environmental cues. The activities of plasmids pCAT232, pCAT85, pCAT77, and pCAT54 were tested in EPEC strain B171-8 grown in DME medium at 37, 25, and 39°C, in LB medium at 37°C, or in DME medium containing 15 mM ammonium sulfate at 37°C or in EPEC strain T::Gm^r, a bfpT mutant derived from strain B171-8 (27), grown in DME medium at 37°C. The graph shows the maximal CAT-specific activity reached late in growth. The data are representative of at least three different experiments.

FIG. 2

Nucleotide sequence alignment of the bfpA (upper line) and bfpT (lower line) 5′ regulatory regions. The upstream regulatory sequence up to position −85 contains all of the cis-acting elements required for the BfpT-dependent activation of bfpA (this work). The bfpA sequence between positions −85 and −46 shares a 73% identity over a 40-bp-long region (shaded bar) with the sequence between positions −65 and −26 of the bfpT regulatory region, which has been shown to be required for bfpT autoactivation (17). Brackets enclose the region bound by a T7-tag-BfpT fusion protein (27). Thin and thick broken arrows indicate the positions of each deletion and mutation, respectively, that affected CAT activity. The activities listed in parentheses are expressed as percentages of pCAT232 activity, which was assigned a value of 100% (see Fig. 1). Horizontal arrows above or below the sequences denote the inverted (IRS)- or direct-repeat elements. Promoter elements −35 and −10 and the transcription start sites are also indicated.

FIG. 3

Regulation of selected bfpA 5′ regulatory region mutants. The activities of bfpA-cat fusions containing down mutations (pMG51, pMG58, pMG60, and pMG65) (A) or promoter mutations (pMNC6 and pSNE10-232) (B) were tested EPEC strain B171-8 grown in DME medium at 37, 25, and 39°C, in LB medium at 37°C, or in DME medium containing 15 mM ammonium sulfate at 37°C or in strain T::Gm^r, a bfpT mutant EPEC strain, grown in DME medium at 37°C. The graph shows the maximal CAT-specific activity reached late in growth. The data are representative of at least three different experiments.

FIG. 4

Primer extension analysis of bfpA and bfpA-cat transcripts. (A) Total RNA samples extracted from EPEC strain B171-8 were hybridized to a 5′-³²P-end-labeled bfpA specific primer; primer extension was performed with avian myeloblastosis virus reverse transcriptase as described previously (19). Lanes G, A, T, and C correspond to the DNA sequence ladder obtained with the same primer. Arrow, the position of the extended products, which correspond to an A residue shown in boldface, 1 bp downstream from the G residue that was previously reported (19). The bfpA transcript is shown in lanes to the right and to the left of the sequence ladder. (B) Total RNA samples extracted from EPEC strain B171-8 carrying plasmid pCAT232 (wild type [lane 1]), pMNC6 (G-29T [lane 2]), or pSNE10-232 (G-12T [lane 3]) or from strain EPEC T::Gm^r carrying pSNE10-232 (lane 4) were hybridized to a 5′-³²P-end-labeled cat-specific primer.

FIG. 5

Regulation of the bfpA 5′ regulatory region mutant G-12T (contained in pSNE10-232) and its 5′ deletion derivative fused to the CAT reporter gene. The activities of pSNE10-232 and its 5′ deletion derivative pSNE10-40 were tested for EPEC strain B171-10 (EPEC-10), a spontaneous plasmid-cured derivative of B171-8 (this study), or for EPEC strain T::Gm^r, a bfpT mutant strain, grown in DME or LB medium at 37°C or in DME medium containing 15 mM ammonium sulfate at 37°C. For comparison, expression directed by the wild-type fusion pCAT232 in B171-10 is also shown. The graph shows the maximal CAT-specific activity reached during the culture period. The data are representative of at least three different experiments.