Organization and architecture of AggR-dependent promoters from enteroaggregative Escherichia coli

Abstract

Enteroaggregative Escherichia coli (EAEC), is a diarrhoeagenic human pathogen commonly isolated from patients in both developing and industrialized countries. Pathogenic EAEC strains possess many virulence determinants, which are thought to be involved in causing disease, though, the exact mechanism by which EAEC causes diarrhoea is unclear. Typical EAEC strains possess the transcriptional regulator, AggR, which controls the expression of many virulence determinants, including the attachment adherence fimbriae (AAF) that are necessary for adherence to human gut epithelial cells. Here, using RNA-sequencing, we have investigated the AggR regulon from EAEC strain 042 and show that AggR regulates the transcription of genes on both the bacterial chromosome and the large virulence plasmid, pAA2. Due to the importance of fimbriae, we focused on the two AAF/II fimbrial gene clusters in EAEC 042 (afaB-aafCB and aafDA) and identified the promoter elements and AggR-binding sites required for fimbrial expression. In addition, we examined the organization of the fimbrial operon promoters from other important EAEC strains to understand the rules of AggR-dependent activation. Finally, we generated a series of semi-synthetic promoters to define the minimal sequence required for AggR-mediated activation and show that the correct positioning of a single AggR-binding site is sufficient to confer AggR-dependence.

Figure 1

AggR‐regulated genes in EAEC strain 042. The figure shows the differential gene expression observed between wild‐type EAEC 042 and its aggR mutant on A. the chromosome and B. plasmid pAA2, as determined by RNA‐seq. A. The data are displayed in rings from the outside inwards. The outermost red lines identify some of the differentially expressed genes (which are labelled with their gene name or number), followed by the base coordinates of the chromosome (labelled in Mb). The annotated genes of EAEC 042 are indicated in the forward and reverse orientation (light blue and dark blue respectively). The EAEC 042 chromosomal regions of difference (RODs) as identified by Chaudhuri et al. (2010) are presented in orange. The inner most circle shows the log₂ fold difference for each gene compared between wild‐type EAEC 042 and the aggR mutant. Positively differential expressed genes are presented in green and negatively differentially expressed genes are in red. B. The rings depicting the data for plasmid pAA2 are the same as for the EAEC 042 chromosome in A. Note that base numbering for pAA2 is in Kb.

Figure 2

Analysis of the aafD100 promoter fragment from EAEC strain 042. A. The panel shows the base sequence of the EAEC 042 aafD100 regulatory region fragment, which includes the start of the aafD coding sequence. The sequence is flanked by upstream EcoRI and downstream HindIII sites and is numbered from the base immediately upstream of the HindIII site. The limits of the aafD99, aafD98, aafD97, aafD96, aafD95 and aafD94 nested deletions are indicated by flags. The proposed promoter −10 hexamer element is underlined, the experimentally determined transcript start sites are indicated by bent horizontal arrows and the initiating ATG codon is in bold. Potential AggR‐binding sites are indicated by horizontal arrows, with functional and non‐functional sites denoted by dark and light shading respectively. Each site is aligned with the AggR‐binding consensus (Morin et al., 2010). The locations of the 65C and 92C/90C substitutions, which disrupt the −10 element and the functional AggR‐binding site, respectively, are shown. B. The panel illustrates measured β‐galactosidase activities in E. coli K‐12 BW25113 ∆lac cells, containing pRW50 carrying the aafD100 fragment, shortened derivatives or no insert. Cells also carried either pBAD/aggR (grey bars) or pBAD24 (black bars). C. The panel shows an autoradiogram of a denaturing polyacrylamide gel run to determine the primer extension products from RNA synthesis initiating at the aafD promoter in BW25113 cells carrying pRW50/aafD96. AggR (+) and AggR (‐) indicates cells carried pBAD/aggR or pBAD24. Reactions are calibrated with the M13mp18 phage reference sequence (A, C, G and T), which serves as sequence ladder. Primer extension products, produced in the presence of AggR, are indicated by arrows. D. The panel shows the β‐galactosidase activities of BW25113 cells, containing pRW50 carrying either the aafD96 fragment or mutant derivatives. Cells also carried either pBAD/aggR (grey bars) or pBAD24 (black bars). In panels B. and D. cells were grown in LB medium in presence (+) or absence (−) of 0.2% arabinose. β‐galactosidase activities are expressed as nmol of ONPG hydrolysed min^–1 mg^–1 dry cell mass. Each activity is the average of three independent determinations and standard deviations are shown for all data points.

Figure 3

Analysis of afaB100 promoter fragment from EAEC strain 042. A. The panel shows the base sequence of the EAEC 042 afaB100 regulatory region fragment flanked by upstream EcoRI and downstream HindIII sites. The sequence is numbered from the base immediately upstream of the HindIII site. The limits of the afaB99, afaB98 and afaB97 nested deletions are indicated by flags. The proposed −10 hexamer element is underlined and the experimentally determined transcript start sites are indicated by bent horizontal arrows. Potential AggR‐binding sites are indicated by horizontal arrows, with functional and non‐functional sites denoted by dark and light shading respectively. Each site is aligned with the AggR‐binding consensus (Morin et al., 2010). The location of the 293C and 320C/318C substitutions, which disrupt the −10 element and the functional AggR‐binding site, respectively, is shown. B. The panel illustrates measured β‐galactosidase activities in E. coli K‐12 BW25113 cells containing pRW50, carrying the afaB100 fragment, shortened derivatives, or no insert. Cells also carried either pBAD/aggR (grey bars) or pBAD24 (black bars). C. The panel shows an autoradiogram of a denaturing polyacrylamide gel run to determine the primer extension products from RNA initiating from the afaB promoter in BW25113 cells, carrying pRW50/afaB100. AggR (+) and AggR (–) indicates cells carried pBAD/aggR or pBAD24. Reactions are calibrated with the M13mp18 phage reference sequence (A, C, G and T), which serves as sequence ladder. Primer extension products, produced in the presence of AggR, are indicated by arrows. D. The panel shows the β‐galactosidase activities in BW25113 cells containing pRW50 carrying either the afaB100 fragment or mutant derivatives. Cells also carried either pBAD/aggR (grey bars) or pBAD24 (black bars). In panels B. and D. cells were grown in LB medium in presence (+) or absence (−) of 0.2% arabinose. β‐galactosidase activities are expressed as nmol of ONPG hydrolysed min^–1 mg^–1 dry cell mass. Each activity is the average of three independent determinations and standard deviations are shown for all data points.

Figure 4

Analysis of aggD100 promoter fragment from EAEC strain 17‐2. A. The panel shows the base sequence of the EAEC 17‐2 aggD100 regulatory region fragment, which includes the start of the aggD coding sequence. The sequence is flanked by upstream EcoRI and downstream HindIII sites and is numbered from the HindIII site. The limits of the aggD99, aggD98 and aggD97 nested deletions are indicated by flags. The proposed −10 hexamer element is underlined and the initiating ATG codon is in bold. Potential AggR‐binding sites are indicated by horizontal arrows, with functional and non‐functional sites denoted by dark and light shading respectively. Each site is aligned with the AggR‐binding consensus (Morin et al., 2010). The location of the 60C and 86C substitutions, which disrupt the −10 element and the functional AggR‐binding site, respectively, is shown. B. The panel illustrates measurements of β‐galactosidase expression in E. coli K‐12 BW25113 ∆lac cells, containing pRW50 carrying the aggD100 fragment, shortened derivatives or no insert. The cells also carried either pBAD/aggR (grey bars) or pBAD24 (black bars). C. The panel shows the β‐galactosidase activities of BW25113 cells containing pRW50 carrying either the aggD98 fragment or mutant derivatives. Cells also carried either pBAD/aggR (grey bars) or pBAD24 (black bars). In panels B. and C. cells were grown in LB medium in presence (+) or absence (−) of 0.2% arabinose. β‐galactosidase activities are expressed as nmol of ONPG hydrolysed min^–1 mg^–1 dry cell mass. Each activity is the average of three independent determinations and standard deviations are shown for all data points.

Figure 5

Analysis of EAEC 55989 agg3D100 and EAEC C1010‐00 agg4D100 promoter fragments. A. The panel shows the sequences of AggR‐dependent fimbrial promoters investigated in this study. The AggR‐binding sites are bold type and the −10 hexamer elements are indicated by grey lines. The underline double arrowheads mark the distance between AggR‐binding sites and −10 hexamer elements. B. The panel illustrates the β‐galactosidase activities of BW25113 cells containing pRW50 carrying various agg3D100 and agg4D100 promoter derivatives, from EAEC strains 55989 and C1010‐00. Cells also carried either pBAD/aggR (grey bars) or pBAD24 (black bars) and were grown in LB medium in presence (+) or absence (−) of 0.2% arabinose. β‐galactosidase activities are expressed as nmol of ONPG hydrolysed min^–1 mg^–1 dry cell mass. Each activity is the average of three independent determinations and standard deviations are shown for all data points. The 307C and 331C/333C substitutions disrupt the −10 element and AggR‐binding site, respectively, in the EAEC 55989 agg3D100 promoter fragment, whilst the 186C and 211C/213C substitutions disrupt the corresponding sequences in the EAEC C1010‐00 agg4D100 fragment (see Fig. S4).

Figure 6

Construction and analysis of semi‐synthetic AggR‐dependent promoters. A. The panel ilustrates the DNA sequence of the CCmelR promoter region and the DAM20, DAM21, DAM22 and DAM23 promoter constructs. In these promoters, the AggR‐binding site from the aafD promoter has been transplanted at different distances from the −10 elements (20 bp to 23 bp). The CRP‐binding half‐sites in the CCmelR promoter are italicized and underlined. Thick black lines indicate the aafD promoter sequence transplanted, with the AggR‐binding site in bold, and the −10 elements are indicated by grey lines. Sequence is numbered from the CCmelR promoter transcript start site (+1). B. The panel illustrates the measured β‐galactosidase activities in BW25113 cells, containing pRW50 carrying the CCmelR and various DAM promoter derivatives. Cells also carried either pBAD/aggR (grey bars) or pBAD24 (black bars). Cells were grown in LB medium in presence (+) or absence (−) of 0.2% arabinose. β‐galactosidase activities are expressed as nmol of ONPG hydrolysed min^–1 mg^–1 dry cell mass. Each activity is the average of three independent determinations and standard deviations are shown for all data points.

Figure 7

The AggR‐binding site consensus. The figure shows motifs for: A. the AggR‐binding site consensus sequence and B. AggR‐dependent promoter organization. Motifs were generated using the WebLogo server (Crooks et al., 2004) with sequences from the EAEC 042 aafD and afaB promoters, the EAEC 17‐2 aggD promoter, the EAEC 55989 agg3D promoter and the EAEC C1010‐00 agg4D promoter identified by experiments in Figs. 2, 3, 4, 5, the aap, aatP and aaiA promoters identified by similar experiments by Yasir (2017) and the AggR‐binding site at the aggR promoter (Morin et al., 2010).