Characterization of BNT2, an intrinsically curved DNA of Escherichia coli O157:H7

Abstract

The gene regulation by intrinsically curved DNA is one way for bacterial sensing of and response to environmental changes. Previously, we showed that the genetic element BNT2 upstream of the ecf (eae-positive conserved fragment) operon in the Escherichia coli O157:H7 virulence plasmid (pO157) has characteristics typical of intrinsically curved DNA, including the presence of multi-homopolymeric adenine:thymine tracts (AT tracts) and electrophoretic anomaly at 4 degrees C. Here we report that a local intrinsic curvature induced by the two phased AT tracts within the unusual promoter sequence of BNT2 played a major role for its temperature-dependent promoter activity. The base substitution of the AT tract in the spacer DNA between the -35 and the unusual -10 regions of the BNT2 promoter with non-AT tract sequence reduced intrinsic curvature slightly at 4 degrees C, but greatly affected its transcriptional activity. This implies that such a local intrinsic curvature within the unusual promoter of BNT2 is important for thermoregulation of the ecf operon.

Fig. 1. Electrophoretic anomaly of BNT2 at 4°C

(A) One-dimensional polyacrylamide gel electrophoresis of BNT2. Each purified DNA was separated on 5 % polyacrylamide gel at 60°C or 4°C to examine intrinsic curvature. (B) Temperature dependency of the BNT2-driven transcription. The strain (YH2021) carrying a single-copy chromosomal BNT2∷lacZ transcriptional fusion construct in ATCC 43894 was grown in LB with (filled bar) or without 0.4% glucose (open bar) at various temperatures indicated and β-galactosidase activity was measured. The enzyme activities are represented as the means ± standard deviation from at least three independent experiments.

Fig. 2. Site-directed mutagenesis of the AT tract in the spacer DNA of the unusual promoter sequence of BNT2

The schematic diagram of the BNT2 containing region in pO157 is shown. The nucleotide base position in pO157 is indicated at the top left and right. The black boxes indicate the AT tracts and the arrow shows the transcriptional start site (+1 and the asterisk). The capitalized letter P in a circle represents the functional, but unusual promoter sequence of BNT2. The AT tract in the spacer DNA region between the -35 element and the unusual -10 element of BNT2 was replaced with non-AT tract sequences by PCR mutagenesis (see Materials and Methods). The resultant mutation was directly confirmed by DNA nucleotide sequencing.

Fig. 3. Prediction of intrinsic DNA curvature (A) and in silico modeling (B) of BNT2 and AT24M

Individual nucleotide sequence was analyzed for intrinsic curvature using the bend.it server. The 134-bp promoter regions containing the two phased AT tracts from BNT2 and AT24M were reconstituted by the model.it server. The arrow indicates the defined spacer DNA regions between the -35 element and the unusual -10 element of BNT2 or AT24M (panel A). In the panel B, changes in intrinsic DNA curvature were compared by measuring the angles produced by the two artificial lines (dotted lines).

Fig. 4

Involvement of a local intrinsic curvature within the unusual promoter sequence of BNT2 in its temperature-dependent transcription. (A) The HindIII-restricted fragments containing the 134-bp BNT2 or AT24M promoter regions were subjected to 2-D PAGE as indicated with 1.0 ug of the 100-bp ladder DNA marker (Gibco BRL). The 2-D gels were visualized with UV light after EtBr staining. The arrow indicates the DNA fragments containing the 134-bp BNT2 or AT24M promoter regions. The dash lines showed the migration status of the 200-bp DNA marker band on the 2-D gels and used to compare to the migration status of the DNA fragments containing the 134-bp BNT2 or AT24M promoter regions. (B) Temperature-dependent transcriptional activity of BNT2 and AT24M. The multi-copy transcriptional fusion plasmids of BNT2 (pSPBNT2) or AT24M (pSPAT24M) were constructed on a promoterless operon fusion vector (pSP471) and transformed into E. coli DH5α. The lacZ expression was measured as β-galactosidase activity after grown in LB at 37 or 24°C. The enzyme activities are represented as the means ± standard deviation from at least three independent experiments.