Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2.

After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella.

A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity.

Heat-stable nucleoid structuring protein (H-NS) plays a crucial role in gene silencing within prokaryotic cells and is important in pathogenesis. It was reported that H-NS silences nearly 5% of the genome, yet the molecular mechanism of silencing is not well understood. Here, we employed a highly-sensitive single-molecule counting approach, and measured the dissociation constant (KD) of H-NS binding to single DNA binding sites. Charged residues in the linker domain of H-NS provided the most significant contribution to DNA binding affinity. Although H-NS was reported to prefer A/T-rich DNA (a feature of pathogenicity islands) over G/C-rich DNA, the dissociation constants obtained from such sites were nearly identical. Using a hairpin unzipping assay, we were able to uncouple non-specific DNA binding steps from nucleation site binding and subsequent polymerization. We propose a model in which H-NS initially engages with non-specific DNA via reasonably high affinity (∼60 nM KD) electrostatic interactions with basic residues in the linker domain. This initial contact enables H-NS to search along the DNA for specific nucleation sites that drive subsequent polymerization and gene silencing.

Locus of enterocyte effacement-encoded regulator (Ler) of pathogenic Escherichia coli competes off histone-like nucleoid-structuring protein (H-NS) through noncooperative DNA binding.

The locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) functions to activate transcription of virulence genes silenced by the histone-like nucleoid-structuring protein (H-NS). Despite its important role in the bacterial gene regulation, the binding mode of Ler to DNA and its mechanism in alleviating genes repressed by H-NS are largely unknown. In this study, we use magnetic tweezers to demonstrate that Ler binds extended DNA through a largely noncooperative process, which results in DNA stiffening and DNA folding depending on protein concentration. We also show that Ler can replace prebound H-NS on DNA over a range of potassium and magnesium concentrations. Our findings reveal the DNA binding properties of Ler and shed light to further understand the anti-silencing activity of Ler.