A widespread family of phage-inducible chromosomal islands only steals bacteriophage tails to spread in nature.

Phage satellites are genetic elements that couple their life cycle to that of helper phages they parasitize, interfering with phage packaging through the production of small capsids, where only satellites are packaged. So far, in all analyzed systems, the satellite-sized capsids are composed of phage proteins. Here, we report that a family of phage-inducible chromosomal islands (PICIs), a type of satellites, encodes all the proteins required for both the production of small-sized capsids and the exclusive packaging of the PICIs into these capsids. Therefore, this new family, named capsid-forming PICIs (cf-PICIs), only requires phage tails to generate PICI particles. Remarkably, the representative cf-PICIs are produced with no cost from their helper phages, suggesting that the relationship between these elements is not parasitic. Finally, our phylogenomic studies indicate that cf-PICIs are present both in gram-positive and gram-negative bacteria and have evolved at least three times independently to spread in nature.

Non-canonical Staphylococcus aureus pathogenicity island repression.

Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.

Beyond the CRISPR-Cas safeguard: PICI-encoded innate immune systems protect bacteria from bacteriophage predation.

Phage satellites are genetic elements that depend on helper phages for induction, packaging and transfer. To promote their lifestyles, they have evolved elegant and sophisticated strategies to inhibit phage reproduction, which will be reviewed here. We will principally focus on the convergent interference mechanisms used by phage-inducible chromosomal islands (PICIs), which are a family of satellite phages present in both Gram-positive and Gram-negative bacteria. While some PICI elements have been extensively studied for their roles in virulence and antibiotic resistance, recent studies have highlighted their relevance in controlling phage ecology and diversity. In many cases, these interference mechanisms are complemented by additional strategies that promote the preferential PICI packaging and dissemination of these elements in nature. Since the PICI-encoded mechanisms target conserved phage mechanisms, we propose here that the PICIs form part of the initial innate immune system that phages must overcome to infect their bacterial host.

The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines.

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

Author Correction: Revisiting the pH-gated conformational switch on the activities of HisKA-family histidine kinases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Revisiting the pH-gated conformational switch on the activities of HisKA-family histidine kinases.

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.

Structural and Functional Characterization of Autophosphorylation in Bacterial Histidine Kinases.

Autophosphorylation of histidine kinases (HK) is the first step for signal transduction in bacterial two-component signalling systems. As HKs dimerize, the His residue is phosphorylated in cis or trans depending on whether the ATP molecule used in the reaction is bound to the same or the neighboring subunit, respectively. The cis or trans autophosphorylation results from an alternative directionality in the connection between helices α1 and α2 in the HK DHp domain, in such a way that α2 could be oriented almost 90° counterclockwise or clockwise with respect to α1. Sequence and length variability of this connection appears to lie behind the different directionality and is implicated in partner recognition with the response regulator (RR), highlighting its importance in signal transduction. Despite this mechanistic difference, HK autophosphorylation appears to be universal, involving conserved residues neighboring the phosphoacceptor His residue. Herein, we describe a simple protocol to determine both autophosphorylation directionality of HKs and the roles of the catalytic residues in these protein kinases.

Conformational dynamism for DNA interaction in the Salmonella RcsB response regulator.

The RcsCDB phosphorelay system controls an extremely large regulon in Enterobacteriaceae that involves processes such as biofilm formation, flagella production, synthesis of extracellular capsules and cell division. Therefore, fine-tuning of this system is essential for virulence in pathogenic microorganisms of this group. The final master effector of the RcsCDB system is the response regulator (RR) RcsB, which activates or represses multiple genes by binding to different promoter regions. This regulatory activity of RcsB can be done alone or in combination with additional transcriptional factors in phosphorylated or dephosphorylated states. The capacity of RcsB to interact with multiple promoters and partners, either dephosphorylated or phosphorylated, suggests an extremely conformational dynamism for this RR. To shed light on the activation mechanism of RcsB and its implication on promoter recognition, we solved the crystal structure of full-length RcsB from Salmonella enterica serovar Typhimurium in the presence and absence of a phosphomimetic molecule BeF3-. These two novel structures have guided an extensive site-directed mutagenesis study at the structural and functional level that confirms RcsB conformational plasticity and dynamism. Our data allowed us to propose a ß5-T switch mechanism where phosphorylation is coupled to alternative DNA binding ways and which highlights the conformational dynamism of RcsB to be so pleiotropic.

Broadening the antibacterial spectrum of histidine kinase autophosphorylation inhibitors via the use of ε-poly-L-lysine capped mesoporous silica-based nanoparticles.

Two-component systems (TCS) regulate diverse processes such as virulence, stress responses, metabolism and antibiotic resistance in bacteria but are absent in humans, making them promising targets for novel antibacterials. By incorporating recently described TCS histidine kinase autophosphorylation inhibitors (HKAIs) into ε-poly-L-lysine capped nanoparticles (NPs) we could overcome the Gram negative (Gr-) permeability barrier for the HKAIs. The observed bactericidal activity against Gr- bacteria was shown to be due to the enhanced delivery and internalization of the HKAIs and not an inhibitory or synergistic effect of the NPs. The NPs had no adverse effects on mammalian cell viability or the immune function of macrophages in vitro and showed no signs of toxicity to zebrafish larvae in vivo. These results show that HKAIs are promising antibacterials for both Gr- and Gr+pathogens and that NPs are a safe drug delivery technology that can enhance the selectivity and efficacy of HKAIs against bacteria.

The closed conformation of the LDL receptor is destabilized by the low Ca(++) concentration but favored by the high Mg(++) concentration in the endosome.

The LDL receptor (LDLR) internalizes LDL and VLDL particles. In the endosomes, it adopts a closed conformation important for recycling, by interaction of two modules of the ligand binding domain (LR4-5) and a ß-propeller motif. Here, we investigate by SPR the interactions between those two modules and the ß-propeller. Our results indicate that the two modules cooperate to bind the ß-propeller. The binding is favored by low pH and by high [Ca(++)]. Our data show that Mg(++), at high concentration in the endosome, favors the formation of the closed conformation by replacing the structuring effect of Ca(++) in LR5. We propose a sequential model of LDL release where formation of the close conformation follows LDL release.

Visualizing autophosphorylation in histidine kinases.

Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis- and trans-phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality.

Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96.

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high light stress require activation by the phosphorylation-independent response regulator NblR. Structural modelling of its receiver domain suggested a role for Cys69 and Cys96 on activation of NblR. Here, we investigate this hypothesis by engineering Cys to Ala substitutions. In vivo and in vitro analyses indicated that mutations Cys69Ala and/or Cys96Ala have a minor impact on NblR function, structure, size, or oligomerization state of the protein, and that Cys69 and Cys96 do not seem to form disulphide bridges. Our results argue against the predicted involvement of Cys69 and Cys96 on NblR activation by redox sensing.

Structural and functional insights into endoglin ligand recognition and binding.

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-ß receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-ß signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.