Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint.

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of 'long'-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis.

Spontaneous mutations in a regulatory gene induce phenotypic heterogeneity and adaptation of Ralstonia solanacearum to changing environments.

An evolution experiment with the bacterial plant pathogen Ralstonia solanacearum revealed that several adaptive mutations conferring enhanced fitness in plants arose in the efpR gene encoding a regulator of virulence and metabolic functions. In this study, we found that an efpR mutant systematically displays colonies with two morphotypes: the type S ('smooth', similar to the wild type) and the type EV ('efpR variant'). We demonstrated that the efpH gene, a homologue of efpR, plays a key role in the control of phenotypic heterogeneity, the ΔefpR-ΔefpH double mutant being stably locked into the EV type. Using mixed infection assays, we demonstrated that the type EV is metabolically more proficient than the type S and displays fitness gain in specific environments, whereas the type S has a better fitness into the plant environment. We provide evidence that this efpR-dependent phenotypic heterogeneity is a general feature of strains of the R. solanacearum species complex and could occur in natural conditions. This study highlights the potential role of phenotypic heterogeneity in this plant pathogen as an adaptive trait to changing environments.

Comparative transcriptomic studies identify specific expression patterns of virulence factors under the control of the master regulator PhcA in the Ralstonia solanacearum species complex.

The global regulator PhcA controls numerous traits associated to virulence and bacterial proliferation in strains of the plant pathogen Ralstonia solanacearum species complex. Here, we conducted a genome-wide RNA sequencing study of the GMI1000 wild-type strain and a derived phcA mutant grown in complete medium. The PhcA regulon we identified is the largest regulon described to date in the R. solanacearum species complex with 1581 regulated genes, representing about 30% of the bacterial genome. Among these genes, 166 transcription regulators were identified including known regulators controlling major cellular functions such as the Type 3 secretion system and 27 novel regulators that were not identified in previous transcriptomic studies. This study highlights that PhcA controls other functions beside pathogenicity stricto sensu which participate to the global cell homeostasis (metabolism, energy storage). We then compared the PhcA regulon identified in complete medium to the recently published PhcA regulon obtained in planta. This comparison of the set of GMI1000 genes subjected to PhcA regulation in both conditions revealed 383 common genes. Among them, 326 (85%) had a similar PhcA dependent regulation pattern in complete medium and in planta, and 57 (15%) displayed an opposite regulation pattern. A large majority of the genes repressed by PhcA in complete medium but activated in planta belong to the HrpG-HrpB regulon, which represents a set of key genes required for R. solanacearum pathogenesis. This latter class of genes appears to be specifically induced by PhcA in the plant environment whereas PhcA represses their expression in complete medium. The large set of direct and indirect targets identified in this study will contribute to enrich our knowledge of the intricate regulatory network coordinating the expression of virulence and metabolic functions in the model plant pathogen R. solanacearum.

Introduction of Genetic Material in Ralstonia solanacearum Through Natural Transformation and Conjugation.

Ralstonia solanacearum is a soil-borne plant pathogen, responsible of the bacterial wilt disease. Its unusual wide host range (more than 250 plant species), aggressiveness, and broad geographic distribution have made of this bacterium the main plant pathogenic model in the beta-Proteobacteria class. Many R. solanacearum strains have the ability to internalize exogenous DNA through natural transformation. This property is widely used in reverse genetics studies to create mutants or reporter gene constructs, in the aim to study the molecular bases of pathogenesis of this bacterium. In this chapter, we describe three in vitro methods (natural transformation, electrotransformation, and conjugation) commonly used to produce recombinant R. solanacearum cells after introduction of exogenous DNA.

Recruitment of a Lineage-Specific Virulence Regulatory Pathway Promotes Intracellular Infection by a Plant Pathogen Experimentally Evolved into a Legume Symbiont.

Ecological transitions between different lifestyles, such as pathogenicity, mutualism and saprophytism, have been very frequent in the course of microbial evolution, and often driven by horizontal gene transfer. Yet, how genomes achieve the ecological transition initiated by the transfer of complex biological traits remains poorly known. Here, we used experimental evolution, genomics, transcriptomics and high-resolution phenotyping to analyze the evolution of the plant pathogen Ralstonia solanacearum into legume symbionts, following the transfer of a natural plasmid encoding the essential mutualistic genes. We show that a regulatory pathway of the recipient R. solanacearum genome involved in extracellular infection of natural hosts was reused to improve intracellular symbiosis with the Mimosa pudica legume. Optimization of intracellular infection capacity was gained through mutations affecting two components of a new regulatory pathway, the transcriptional regulator efpR and a region upstream from the RSc0965-0967 genes of unknown functions. Adaptive mutations caused the downregulation of efpR and the over-expression of a downstream regulatory module, the three unknown genes RSc3146-3148, two of which encoding proteins likely associated to the membrane. This over-expression led to important metabolic and transcriptomic changes and a drastic qualitative and quantitative improvement of nodule intracellular infection. In addition, these adaptive mutations decreased the virulence of the original pathogen. The complete efpR/RSc3146-3148 pathway could only be identified in the genomes of the pathogenic R. solanacearum species complex. Our findings illustrate how the rewiring of a genetic network regulating virulence allows a radically different type of symbiotic interaction and contributes to ecological transitions and trade-offs.

Enhanced in planta Fitness through Adaptive Mutations in EfpR, a Dual Regulator of Virulence and Metabolic Functions in the Plant Pathogen Ralstonia solanacearum.

Experimental evolution of the plant pathogen Ralstonia solanacearum, where bacteria were maintained on plant lineages for more than 300 generations, revealed that several independent single mutations in the efpR gene from populations propagated on beans were associated with fitness gain on bean. In the present work, novel allelic efpR variants were isolated from populations propagated on other plant species, thus suggesting that mutations in efpR were not solely associated to a fitness gain on bean, but also on additional hosts. A transcriptomic profiling and phenotypic characterization of the efpR deleted mutant showed that EfpR acts as a global catabolic repressor, directly or indirectly down-regulating the expression of multiple metabolic pathways. EfpR also controls virulence traits such as exopolysaccharide production, swimming and twitching motilities and deletion of efpR leads to reduced virulence on tomato plants after soil drenching inoculation. We studied the impact of the single mutations that occurred in efpR during experimental evolution and found that these allelic mutants displayed phenotypic characteristics similar to the deletion mutant, although not behaving as complete loss-of-function mutants. These adaptive mutations therefore strongly affected the function of efpR, leading to an expanded metabolic versatility that should benefit to the evolved clones. Altogether, these results indicated that EfpR is a novel central player of the R. solanacearum virulence regulatory network. Independent mutations therefore appeared during experimental evolution in the evolved clones, on a crucial node of this network, to favor adaptation to host vascular tissues through regulatory and metabolic rewiring.

Extracellular matrix produced by osteoblasts cultured under low-magnitude, high-frequency stimulation is favourable to osteogenic differentiation of mesenchymal stem cells.

The effects of low-magnitude, high-frequency (LMHF) mechanical stimulation on osteoblastic cells are poorly understood. We have developed a system that generates very small (15-40 µÎµ), high-frequency (400 Hz, sine) deformations on osteoblast cultures (MC3T3-E1). We investigated the effects of these LMHF stimulations mainly on extracellular matrix (ECM) synthesis. The functional properties of this ECM after decellularization were evaluated on C3H10T1/2 mesenchymal stem cells (MSCs). LMHF stimulations were applied 20 min once daily for 1, 3, or 7 days in MC3T3-E1 culture (1, 3, or 7 dLMHF). Cell number and viability were not affected after 3 or 7 dLMHF. Osteoblast response to LMHF was assessed by an increase in nitric oxide secretion, alteration of the cytoskeleton, and focal contacts. mRNA expression for fibronectin, osteopontin, bone sialoprotein, and type I collagen in LMHF cultures were 1.8-, 1.6-, 1.5-, and 1.7-fold higher than controls, respectively (P < 0.05). In terms of protein, osteopontin levels were increased after 3 dLMHF and ECM organization was altered as shown by fibronectin topology after 7 dLMHF. After decellularization, 7 dLMHF-ECM or control ECM was reseeded with MSCs. Seven dLMHF-ECM improved early events such as cell attachment (2 h) and focal contact adhesion (6 h) and, later (16 h), modified MSC morphological parameters. After 5 days in multipotential medium, gene-expression changes indicated that 7 dLMHF-ECM promoted the expression of osteoblast markers at the expense of adipogenic marker. LMHF stimulations of osteoblasts are therefore efficient and sufficient to generate osteogenic matrix.

The effect of dual frequency cyclic compression on matrix deposition by osteoblast-like cells grown in 3D scaffolds and on modulation of VEGF variant expression.

As a strategy to optimise osteointegration of biomaterials by inducing proper extracellular matrix synthesis, and specifically angiogenic growth factor production and storage, we tested the effects of cyclic mechanical compression on 3D cultures of human osteoblast-like cells. MG-63 cells were seeded into 3D porous hydroxyapatite ceramics under vacuum to enable a homogenous cellular distribution. A four-day culture period allowed cell proliferation throughout the scaffolds. Low amplitude cyclic compressions were then applied to the scaffolds for 15 min with different regimens generated by the ZetOS system. A 3 Hz sinusoidal (sine) signal increased slightly collagen and fibronectin expression. When 50 Hz or 100 Hz vibrations were superimposed to the 3 Hz signal, matrix protein expression was down-regulated. In contrast, adding a 25 Hz vibration up-regulated significantly collagen and fibronectin. Moreover, expression of a matrix-bound variant of vascular endothelial growth factor-A (VEGF-A) was specifically stimulated compared to control or 3 Hz sine, and non-soluble VEGF protein was increased. Our study enabled us to identify low-amplitude, high-frequency strain regimen able to increase major matrix proteins of bone tissue and to regulate the expression of VEGF variants, showing that an appropriate combined loading has the potential to functionalise cellularized bone-like constructs.