An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster

Abstract

Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication.

Figure 1. The EAEC sci1 T6SS gene cluster is regulated by iron levels and the Fur repressor.

(A) β-galactosidase activity of a promoterless lacZ fusion (open symbols) and of the sci1-lacZ reporter fusion (closed symbols) upon addition of the iron chelator 2,2′-dipyridyl (dip; 100 µM, squares) in a EAEC wild-type (WT) strain (triangles: no dip added). (B) β-galactosidase activity of a promoterless lacZ fusion (white bars) and of the sci1-lacZ reporter fusion (black bars) after 120 minutes of culture (OD_600nm = 0.8) upon a 30 min treatment with 2,2′-dipyridyl (+dip; 100 µM) or ethanol-carrier (-dip) in a WT strain or its isogenic fur mutant.

Figure 2. In silico analysis of the sci1 proximal promoter region.

(A) The proximal sci1 promoter region. The ATG translational codon of sciH is indicated as well as the Shine Delgarno (SD). The putative -10 and -35 elements of the σ⁷⁰ promoter (identified by the BProm algorithm) are indicated in blue, as well as Fur-binding sequences (red boxes) and GATC Dam-dependent methylation sites (yellow boxes). The two Fur-binding sequences and GATC sites are numbered from the start site. (B) Sequence alignment of the fur1 and fur2 sci1 boxes with the E. coli Fur box consensus sequence. Identical bases are framed in green.

Figure 3. Fur binds to the EAEC sci1 T6SS promoter in vivo and in vitro.

(A) Fur Titration assay (FURTA). H1717 reporter cells (fhuF-lacZ) carrying the empty vector or the vector bearing the sci1, sci2, or cir promoters, or the fur1 or fur2 sequences were spotted on MacConkey plates (upper panel) or on MacConkey plates supplemented with FeSO4 (30 µM; lower panel). A lacZ+ phenotype reports a derepression of the fhuF-lacZ reporter fusion by titration of the Fur protein bound to the fhuF promoter. (B) Electrophoretic mobility shift assay of the sci1 promoter (upper panel) or of the fur1 (middle panel) or fur2 (lower panel) sequences using purified Fur (lane 1, no protein; lane 2, 0.5 nM; lane 3, 2 nM, lane 4, 5 nM, lane 5, 20 nM) in presence of FeCl3 or in presence of EDTA (lane 6, Fur at 20 nM) or using purified NtrC transcriptional activator (lane 7, 50 nM). Controls include Fur shift assays of the Fur-dependent cir promoter (lane 8, no protein; lane 9, Fur at 5 nM) or of the Fur-independent sci2 promoter (lane 10, 20 nM). (C) Competition experiments for Fur binding (lane 1, no protein; lanes 2–6, Fur at 20 nM) with duplex consensus Fur- (lane 3, molecular ratio sci1:fur box 1∶2; lane 4, molecular ratio 1∶10) or σ54-binding sequence (lane 5, molecular ratio sci1:σ⁵⁴-box 1∶2; lane 6, molecular ratio 1∶10). (D) Binding of the Eσ⁷⁰ RNA polymerase holoenzyme (RNAP) (lanes 1, 4, 7 and 9, no RNAP; lanes 2 and 5, RNAP 0.5 unit; lanes 3, 6, 8 and 10, RNAP 2 units) on the sci1 or control cir promoter pre-incubated (+) or not (−) with Fur (20 nM). Fur-DNA, (Fur)₂-DNA, and RNAP-DNA complexes are indicated by *, **, and ǂ respectively.

Figure 4. Fur protects GATC-I from methylation in vitro.

A radiolabeled PCR product corresponding to the 596-bp sci1 promoter was digested by the restriction enzymes indicated on top (no, no digestion). Upper panel, untreated PCR product; middle panel, PCR product treated with the Dam methylase; lower panel, PCR product incubated with purified Fur (20 nM) prior to Dam methylation. The sizes of the digestion products (in bp) are indicated on the left. Red and blue frames emphasize the observation that incubation with Fur did not change the digestion profiles for GATC-II (-II) and GATC-III (-III) whereas the green frame emphasize the observation that GATC-I (-I) was not methylated upon Fur binding. Schematic representations of the conclusions of the left panels are shown on right. See Figure S1 for positions of restriction sites and sizes of DNA fragments.

Figure 5. Fur protects GATC-I from methylation in vivo.

The sci1 promoters purified from the EAEC wild-type strain (WT, upper panel) or its isogenic dam (second panel rom top) or fur (lower panel) mutant strains, or from the WT strain treated with 2,2′-dipyridyl (WT + dip; third panel from top) were digested by the restriction enzymes indicated on top (no, no digestion). The sizes of the digestion products (in bp) are indicated on the left. Arrows indicate the position of the digestion product obtained with the BclI restriction enzyme emphasizing the observation that GATC-I was not methylated in a WT strain but was methylated in a fur mutant strain (or in a WT strain treated with 2,2′-dipyridyl). Schematic representations of the conclusions of the left panels are shown on right. See Figure S1 for positions of restriction sites and sizes of DNA fragments.

Figure 6. GATC methylation influences Fur binding on fur1.

(A) Electrophoretic mobility shift assay of the non methylated or Dam-methylated (me-) sci1 or cir promoter using purified Fur (lanes 1 and 5, no protein; lanes 2 and 6, 2 nM; lanes 3 and 7, 5 nM; lanes 4 and 8-10, 20 nM). (B) Electrophoretic mobility shift assay of the non methylated or Dam-methylated (me-) fur1 sequence using purified Fur (lanes 1 and 6, no protein; lanes 2 and 7, 0.5 nM; lanes 3 and 8, 2 nM; lanes 4 and 9, 5 nM; lanes 5 and 10, 20 nM). Fur-DNA, (Fur)₂-DNA complexes are indicated by *, and ** respectively.

Figure 7. Schematic representation of the EAEC sci1 T6SS gene cluster epigenetic switch regulatory mechanism.

(A) In iron-replete conditions, Fur (red balls) represses the expression of the sci1 gene cluster by binding to specific boxes overlapping the putative -10 transcriptional element. The expression of the sci1 gene cluster is in the OFF phase. (B) In iron starvation conditions, Fur is relieved from the putative -10 element, leaving the promoter available for RNAP binding and transcription. Dam-dependent methylation at the GATC-I site prevents Fur binding. The expression of the sci1 gene cluster is in the ON phase. Transition to the OFF phase requires both iron replete conditions and hemi-methylation of GATC-I after DNA replication. Methylated GATC sites are indicated by CH₃.