The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium

Abstract

Integration of horizontally acquired genes into transcriptional networks is essential for the regulated expression of virulence in bacterial pathogens. In Salmonella enterica, expression of such genes is repressed by the nucleoid-associated protein H-NS, which recognizes and binds to AT-rich DNA. H-NS-mediated silencing must be countered by other DNA-binding proteins to allow expression under appropriate conditions. Some genes that can be transcribed by RNA polymerase (RNAP) associated with the alternative sigma factor σ^S or the housekeeping sigma factor σ⁷⁰ in vitro appear to be preferentially transcribed by σ^S in the presence of H-NS, suggesting that σ^S may act as a counter-silencer. To determine whether σ^S directly counters H-NS-mediated silencing and whether co-regulation by H-NS accounts for the σ^S selectivity of certain promoters, we examined the csgBA operon, which is required for curli fimbriae expression and is known to be regulated by both H-NS and σ^S . Using genetics and in vitro biochemical analyses, we found that σ^S is not directly required for csgBA transcription, but rather up-regulates csgBA via an indirect upstream mechanism. Instead, the biofilm master regulator CsgD directly counter-silences the csgBA promoter by altering the DNA-protein complex structure to disrupt H-NS-mediated silencing in addition to directing the binding of RNAP.

Figure 1. Depletion of H-NS using an inducible asHNS construct

H-NS was quantified from exponential and stationary phase cultures of SLN90 containing pHN1009 (asVector) and pHN1009asHNS, which encodes an asRNA complimentary to the region flanking the ribosome binding site of hns. asRNA expression was induced with IPTG for 4 hrs, cells were harvested, and H-NS levels visualized via immunoblot analysis using an anti-HA antibody. σ^S is absent in log phase but increases in abundance during stationary phase. GroEL was detected as a loading control.

Figure 2. CsgD and σ^S up-regulate csgB in stationary phase

csgB transcript levels were quantified by qRT-PCR during A) exponential or B) stationary phase in wild-type, rpoS, csgD, and rpoS csgD S. Typhimurium strains containing pHN1009 or pHN1009asHNS, with IPTG added to deplete H-NS. Data are presented as the mean +/− SD of three replicates normalized to gyrB; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 by ratio-paired t-test.

Figure 3. Constitutive csgD expression in trans stimulates σ^S-independent csgB transcription

A) csgD transcript levels were quantified using qRT-PCR in wild-type and rpoS S. Typhimurium containing pCsgD, which contains csgD downstream of a constitutive promoter. B) csgB transcript levels were quantified in response to csgD constitutive expression in strains containing both pCsgD and pHN1009asHNS. Data are presented as the mean +/− SD of three replicates normalized to gyrB expression; ** indicates p<0.01, and *** indicates p<0.001 by ratio-paired t-test.

Figure 4. Depletion of H-NS restores biofilm formation in σ^S-deficient S. Typhimurium

A) Wild-type and rpoS strains of S. Typhimurium were spotted on Congo Red plates and incubated at 30 or 37°C to determine their RDAR phenotypes. B) rpoS strains containing pCsgD exhibited enhanced RDAR morphology in an rpoS mutant when compared to an rpoS strain containing the control vector.

Figure 5. CsgD counter-silences the csgB promoter in vitro

The csgB transcriptional circuit was reconstituted in vitro using purified, supercoiled DNA template and purified protein components. A) Purified supercoiled plasmid containing a 3kb-region of DNA surrounding the csgB promoter was incubated in the presence of increasing concentrations of H-NS and either Eσ^S or Eσ⁷⁰ to assay transcriptional output. Transcript levels are normalized to the RNAP-only control. B) Either Eσ^S or Eσ⁷⁰ can initiate transcription of the csgB promoter in vitro. CsgD-mediated counter-silencing was assayed by incubating the csgB template in the presence of 50 nM CsgD before adding 130 nM H-NS. Transcript levels are expressed as relative copy number. Data are presented as the mean +/− SD of three replicates; * indicates p<0.05 by ratio-paired t-test.

Figure 6. DNaseI Differential DNA Footprint Analysis (DDFA) of the csgB promoter region

In vitro DNase I footprinting of the anti-sense strand at the csgB promoter was performed with 130 nM H-NS and 50 nM CsgD, as indicated. Peaks are regions of hypersensitivity, which are suggestive of distorted or bent DNA, whereas valleys indicate sites of protection. Base positions are indicated relative to the TSS. A) H-NS, B) CsgD, or E) CsgD and H-NS were added to the csgB promoter and the fluorescent peak height following DNase I cleavage determined relative to the protein-free control in relative fluorescent units (RFU). The differences compared to the H-NS control (C) and the CsgD control (D) were also analyzed. Data are presented as the mean +/− SD of three replicates.

Figure 7. KMnO₄ Differential DNA Footprint Analysis (DDFA) at the csgB promoter region

KMnO₄ footprinting reactions, which detect regions of single-stranded DNA generated during open-complex formation, were performed on the csgB promoter region with RNAP, CsgD, and H-NS, as indicated. Peaks, in relative fluorescent units (RFUs), indicate regions of single stranded DNA present in the experimental samples that are not present in control (no RNAP) reactions. Base positions are indicated relative to the TSS. a) KMnO₄ footprinting analysis indicates that the location of RNAP open complex formation on the naked DNA promoter is dependent on the presence or absence of CsgD. RNAP alone is capable of forming a weak open complex at an upstream site, but the addition of CsgD directs RNAP to the previously identified transcriptional start site. b) RNAP is unable to form an open complex in the presence of H-NS, but the addition of CsgD restores open complex formation at the csgB promoter. Data are presented as the mean +/− SD of three replicates.