As an environment-dependent pleiotropic gene regulator in Gram-negative bacteria, the H-NS protein is crucial for adaptation and toxicity control of human pathogens such as Salmonella, Vibrio cholerae or enterohaemorrhagic Escherichia coli. Changes in temperature affect the capacity of H-NS to form multimers that condense DNA and restrict gene expression. However, the molecular mechanism through which H-NS senses temperature and other physiochemical parameters remains unclear and controversial. Combining structural, biophysical and computational analyses, we show that human body temperature promotes unfolding of the central dimerization domain, breaking up H-NS multimers. This unfolding event enables an autoinhibitory compact H-NS conformation that blocks DNA binding. Our integrative approach provides the molecular basis for H-NS–mediated environment-sensing and may open new avenues for the control of pathogenic multi-drug resistant bacteria.

Sepsis is the third leading cause of mortality in Western countries and one of the Global Health Threats recognized by the WHO since 2017. Despite Escherichia coli constituting the most common cause of bloodstream infections (BSI), its epidemiology is not fully understood, in part due to the scarcity of local and longitudinal studies.

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontal disease, as well as other systemic diseases. The ability of the bacterium to bind collagen, the principal component of the extracellular matrix, is mediated by the extracellular matrix protein adhesin A (EmaA). EmaA forms antennae-like appendages on the bacterial cell surface that are comprised of three monomers. The functional domain, of approximately 30 nm in length, is located at the distal end of the adhesin and is subdivided into three subdomains (SI-SIII) [1]. Glycosylation of EmaA adhesins is critical for collagen binding, and it has been demonstrated that this post-translational modification exploits the same pathway as the O-polysaccharide (O-PS) of the lipopolysaccharide [2]. However, it is still unclear how glycosylation facilitates collagen binding. In this study we have analyzed the 3D structure of the functional domain of the EmaA adhesin from mutant strains with a disrupted glycosylation mechanism (rmlC and waaL mutants). The rmlC mutant strain lacks the rhamnose epimerase and the waaL mutant strain does not express the O-antigen ligase, WaaL, an essential component of the O-PS glycosylation pathway. Structural comparison of the glycosylated and non-glycosylated adhesins will help to determine if this modification promotes a structural conformation that is required for collagen binding.EmaA adhesins from both the rmlC and the waaL mutant strains were analyzed by electron tomography of whole-mount negatively-stained preparations of the bacteria as previously described for other strains [3]. Bacteria were adsorbed on carbon-coated grids pretreated with a colloidal gold solution and negatively stained with Nano W (2% methylamine tungstate). Tomographic single-axis tilt series were acquired over a ±64° angular range in 2° intervals with a calibrated 3.08 Å pixelsize at the specimen scale. Single-axis tilt series were processed using the IMOD processing software to generate tomograms. EmaA adhesins were selected from the tomograms by marking two points on their axis. For each selected adhesin, a tilt series of subprojections was extracted and subvolumes were calculated with the adhesin's axis approximately parallel to the Y-axis using algorithms in both Spider and EMIRA [4,5]. These subvolumes were visualized in Chimera [6] and further aligned to a reference subvolume of the wild-type EmaA [1]. Lower quality EmaA subvolumes were removed from further processing (<10% subvolumes removed). Probabilistic Principal Component Analysis (PPCAEM) implemented in EMIRA was used to assess differences between all the EmaA subvolumes and estimate the missing data [5,7]. The 3D reconstructions were divided into groups of similar structures. The subvolumes were further aligned to a reference selected from the volumes present in each group. Chimera [6] was used to generate an average subvolume for each group.The 3D electron microscopy analysis of the EmaA adhesins from the mutant strain suggests that glyco...

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