Pneumococcal competence is a populational health sensor driving multilevel heterogeneity in response to antibiotics.

Competence for natural transformation is a central driver of genetic diversity in bacteria. In the human pathogen Streptococcus pneumoniae, competence exhibits a populational character mediated by the stress-induced ComABCDE quorum-sensing (QS) system. Here, we explore how this cell-to-cell communication mechanism proceeds and the functional properties acquired by competent cells grown under lethal stress. We show that populational competence development depends on self-induced cells stochastically emerging in response to stresses, including antibiotics. Competence then propagates through the population from a low threshold density of self-induced cells, defining a biphasic Self-Induction and Propagation (SI&P) QS mechanism. We also reveal that a competent population displays either increased sensitivity or improved tolerance to lethal doses of antibiotics, dependent in the latter case on the competence-induced ComM division inhibitor. Remarkably, these surviving competent cells also display an altered transformation potential. Thus, the unveiled SI&P QS mechanism shapes pneumococcal competence as a health sensor of the clonal population, promoting a bet-hedging strategy that both responds to and drives cells towards heterogeneity.

Mechanistic and functional aspects of the Ruminococcin C sactipeptide isoforms.

In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1-C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

Multiple detection of both attractants and repellents by the dCache-chemoreceptor SO_1056 of Shewanella oneidensis.

Chemoreceptors are usually transmembrane proteins dedicated to the detection of compound gradients or signals in the surroundings of a bacterium. After detection, they modulate the activation of CheA-CheY, the core of the chemotactic pathway, to allow cells to move upwards or downwards depending on whether the signal is an attractant or a repellent, respectively. Environmental bacteria such as Shewanella oneidensis harbour dozens of chemoreceptors or MCPs (methyl-accepting chemotaxis proteins). A recent study revealed that MCP SO_1056 of S. oneidensis binds chromate. Here, we show that this MCP also detects an additional attractant (l-malate) and two repellents (nickel and cobalt). The experiments were performed in vivo by the agarose-in-plug technique after overproducing MCP SO_1056 and in vitro, when possible, by submitting the purified ligand-binding domain (LBD) of SO_1056 to a thermal shift assay (TSA) coupled to isothermal titration calorimetry (ITC). ITC assays revealed a KD of 3.4 µm for l-malate and of 47.7 µm for nickel. We conclude that MCP SO_1056 binds attractants and repellents of unrelated composition. The LBD of SO_1056 belongs to the double Cache_1 family and is highly homologous to PctA, a chemoreceptor from Pseudomonas aeruginosa that detects several amino acids. Therefore, LBDs of the same family can bind diverse compounds, confirming that experimental approaches are required to define accurate LBD-binding molecules or signals.

Combining two optimized and affordable methods to assign chemoreceptors to a specific signal.

Chemotaxis allows bacteria to detect specific compounds and move accordingly. This pathway involves signal detection by chemoreceptors (MCPs). Attributing a chemoreceptor to a ligand is difficult because there is a lot of redundancy in the MCPs that recognize a single ligand. We propose a methodology to define which chemoreceptors bind a given ligand. First, an MCP is overproduced to increase sensitivity to the ligand(s) it recognizes, thus promoting accumulation of cells around an agarose plug containing a low attractant concentration. Second, the ligand-binding domain (LBD) of the chemoreceptor is fused to maltose-binding protein (MBP), which facilitates purification and provides a control for a thermal shift assay (TSA). An increase in the melting temperature of the LBD in the presence of the ligand indicates that the chemoreceptor directly binds it. We showed that overexpression of two Shewanella oneidensis chemoreceptors (SO_0987 and SO_1056) promoted swimming toward an agarose plug containing a low concentration of chromate. The LBD of each of the two chemoreceptors was fused to MBP. A TSA revealed that only the LBD from SO_1056 had its melting temperature increased by chromate. In conclusion, we describe an efficient approach to define chemoreceptor-ligand pairs before undertaking more-sophisticated biochemical and structural studies.

The phosphorylated regulator of chemotaxis is crucial throughout biofilm biogenesis in Shewanella oneidensis.

The core of the chemotaxis system of Shewanella oneidensis is made of the CheA3 kinase and the CheY3 regulator. When appropriated, CheA3 phosphorylates CheY3, which, in turn, binds to the rotor of the flagellum to modify the swimming direction. In this study, we showed that phosphorylated CheY3 (CheY3-P) also plays an essential role during biogenesis of the solid-surface-associated biofilm (SSA-biofilm). Indeed, in a ΔcheY3 strain, the formation of this biofilm is abolished. Using the phospho-mimetic CheY3D56E mutant, we showed that CheY-P is required throughout the biogenesis of the biofilm but CheY3 phosphorylation is independent of CheA3 during this process. We have recently found that CheY3 interacts with two diguanylate cyclases (DGCs) and with MxdA, the c-di-GMP effector, probably triggering exopolysaccharide synthesis by the Mxd machinery. Here, we discovered two additional DGCs involved in SSA-biofilm development and showed that one of them interacts with CheY3. We therefore propose that CheY3-P acts together with DGCs to control SSA-biofilm formation. Interestingly, two orthologous CheY regulators complement the biofilm defect of a ΔcheY3 strain, supporting the idea that biofilm formation could involve CheY regulators in other bacteria.

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c-di-GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis.

Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA-biofilm) or at the air-liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non-pellicle-forming cheY3-deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c-di-GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c-di-GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c-di-GMP binding protein MxdA.

ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction.

The chromate efflux pump encoding gene chrASO was identified on the chromosome of Shewanella oneidensis MR1. Although chrASO is expressed without chromate, its expression level increases when Cr(VI) is added. When deleted, the resulting mutant ΔchrASO exhibits a chromate sensitive phenotype compared to that of the wild-type strain. Interestingly, heterologous expression of chrASO in E. coli confers resistance to high chromate concentration. Moreover, expression of chrASO in S. oneidensis and E. coli significantly improves Cr(VI) reduction. This effect could result either from extracytoplasmic chromate reduction or from a better cell survival leading to enhanced Cr(VI) reduction.