Biology subjects

Letisse, F.

Publications and source records attributed to Letisse, F..

11 recordsLinked to original sources

bioRxiv2026

SpoT-mediated reduction of (p)ppGpp levels promotes Ralstonia pseudosolanacearum adaptation to both plant xylem and legume nodules

During evolution, bacteria have developed the ability to interact intimately with eukaryotic hosts. These interactions span a dynamic continuum ranging from pathogenicity to mutualism, along which bacteria can rapidly evolve and shift their lifestyles. However, the molecular mechanisms that enable bacteria to adapt to new hosts and to transition between distinct interaction modes remain poorly understood. Here, using a unique combination of two independent evolution experiments, we identified and characterized parallel adaptive mutations in spoT, which encodes the bifunctional (p)ppGpp synthetase-hydrolase. These mutations promote the adaptation of the plant pathogen Ralstonia pseudosolanacearum to two distinct plant-associated environments and two distinct lifestyles, the xylem of both susceptible and tolerant host plants as a pathogen and the root nodules of a legume as a symbiont, without compromising virulence on susceptible hosts. These mutations enhance the utilization of multiple carbon and nitrogen sources, including substrates known to be abundant in xylem sap, and increase bacterial exponential growth rate in minimal medium, suggesting reduced basal (p)ppGpp levels. Assessment of a strain deficient in SpoT synthetase activity confirmed that lowering basal (p)ppGpp levels is adaptive in both plant environments. Together, our findings reveal that fine-tuning intracellular (p)ppGpp concentrations represents an efficient strategy for optimizing bacterial adaptation to complex host-associated environments.

microbiology↗

bioRxiv2026

Metabolic engineering of Escherichia coli strains for the in vivo synthesis of GP-mediated oligosaccharides

Enzymatic synthesis of rare disaccharides by reverse-phosphorolysis is a potentially sustainable route to produce high-value glycosides for human health and nutrition. We report the metabolic engineering of Escherichia coli for in vivo production of {beta}-mannobiose with different osidic linkages from hexose sugars. We demonstrate production of {beta}-1,2-mannobiose with this approach as proof of concept. Phosphotransferase system (PTS) inactivation enables import of non-phosphorylated mannose via heterologous permease GalP, restoring growth on mannose in a PTS- background and allowing mannose into the reverse biosynthetic pathway. Deletion of pfkA, which promotes intracellular accumulation of key sugar phosphates (G1P, M1P), establishes a favorable metabolic chassis for oligosaccharide production using glycoside-phosphorylases. Using this chassis, we expressed two {beta}-mannoside phosphorylases to enable the direct production of {beta}-1,2- and {beta}-1,4-mannobiose from mannose. The same chassis was also employed for laminaribiose production through the expression of a laminaribiose-phosphorylase. pfkA deletion significantly increased product titer (> 0.6 g{middle dot}L-1) and yield (up to 9% g/g mannose), highlighting a favorable redistribution of carbon fluxes toward disaccharide formation. Moreover, a combination of mixed-substrate cultures using glycerol as carbon and energy source and further metabolic engineering enabled partial growth-production decoupling, redirecting mannose utilization primarily toward product synthesis, with a yield of 60%. These results demonstrate the modularity and efficiency of the proposed platform for fermentative production of non-conventional oligosaccharides and expand the scope of metabolic engineering strategies for glycoside biosynthesis.

synthetic biology↗

bioRxiv2025

Cross-regulation of (p)ppGpp and c-di-GMP pathways controls a cell-cycle transition

Bacteria must adapt their physiology to changing environments, a process often orchestrated by nucleotide messengers such as (p)ppGpp and cyclic di-GMP (c-di-GMP). While (p)ppGpp promotes survival under nutrient stress by suppressing growth, c-di-GMP supports sessile lifestyles and cell differentiation. In the -proteobacterium Caulobacter crescentus, these messengers have opposite effects on the cell cycle, suggesting potential cross-regulations. Here, we uncover direct molecular cross-talk between (p)ppGpp and c-di-GMP signaling pathways. Using biochemical, structural and genetic approaches, we show that (p)ppGpp binds to the diguanylate cyclase PleD and inhibits c-di-GMP synthesis by competing with GTP at the active site. This interaction prevents c-di-GMP accumulation under nutrient-limiting conditions, linking metabolic stress to inhibition of developmental progression. Our findings reveal a new paradigm for second messenger interplay, in which one messenger directly suppresses synthesis of another. This work provides insight into how bacteria integrate metabolic and developmental cues through small-molecule cross-talk to guide adaptive behavior.

microbiology↗

bioRxiv2024

Inhibition of glycolysis in tuberculosis-mediated metabolic rewiring reduces HIV-1 spread across macrophages

Tuberculosis (TB) is a significant aggravating factor in individuals living with human immunodeficiency virus type 1 (HIV-1), the causative agent for acquired immunodeficiency syndrome (AIDS). Both Mycobacterium tuberculosis (Mtb), the bacterium responsible for TB, and HIV-1 target macrophages. Understanding how Mtb subverts these cells may facilitate the identification of new druggable targets. Here, we explored how TB can induce macrophages to form tunneling nanotubes (TNT), promoting HIV-1 spread. We found that TB triggers metabolic rewiring of macrophages, increasing their glycolytic ATP production. Using pharmacological inhibitors and glucose deprivation, we discovered that disrupting aerobic glycolysis significantly reduces HIV-1 exacerbation in these macrophages. Glycolysis is essential for tunneling nanotubes (TNT) formation, which facilitates viral transfer and cell-to-cell fusion and induces the expression of the sialoadhesin Siglec-1, enhancing both HIV-1 binding and TNT stabilization. Glycolysis did not exacerbate HIV-1 infection when TNT formation was pharmacologically prevented, indicating that higher metabolic activity is not sufficient per se to make macrophages more susceptible to HIV-1. Overall, these data might facilitate the development of targeted therapies aimed at inhibiting glycolytic activity in TB-induced immunomodulatory macrophages to ultimately halt HIV-1 dissemination in co-infected patients.

immunology↗

bioRxiv2023

Phosphogluconolactonase as the linchpin of an efficient pentose phosphate pathway

The metabolic networks of microorganisms are remarkably robust to genetic and environmental perturbations. This robustness stems from redundancies such as gene duplications, isoenzymes, alternative metabolic pathways, and also from non-enzymatic reactions. In the oxidative branch of the pentose-phosphate pathway (oxPPP), 6-phosphogluconolactone hydrolysis into 6-phosphogluconate is catalysed by 6-phosphogluconolactonase (Pgl) but in the absence of the latter, the oxPPP flux is thought to be maintained by spontaneous hydrolysis. However, in {Delta}pgl Escherichia coli, an extracellular pathway can also contribute to pentose-phosphate synthesis. This raises question as to whether the intracellular non-enzymatic reaction can compensate for the absence of 6-phosphogluconolactonase and, ultimately, on the role of 6-phosphogluconolactonase in central metabolism. Our results validate that the bypass pathway is active in the absence of Pgl, specifically involving the extracellular spontaneous hydrolysis of gluconolactones to gluconate. Under these conditions, metabolic flux analysis reveals that this bypass pathway accounts for the entire flux into the oxPPP. This alternative metabolic route - partially extracellular - sustains the flux through the oxPPP necessary for cell growth, albeit at a reduced rate in the absence of Pgl. Importantly, these findings imply that intracellular non-enzymatic hydrolysis of 6-phosphogluconolactone does not compensate for the absence of Pgl. This underscores the crucial role of Pgl in ensuring the efficient functioning of the oxPPP.

biochemistry↗

bioRxiv2023

Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control

For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism of Pseudomonas putida at different levels of metabolic load. To this end, we constructed a P. putida KT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature of P. putidas central metabolic network is solicited to sustain energy production. HighlightsHeterologous protein production in P. putida reshuffles the periplasmic metabolism. Increased protein production progressively decouples catabolism from anabolism. Protein production exerts a stronger control on energy than on carbon fluxes. Glucose is directed towards ATP production to meet the elevated energy demands.

bioengineering↗

bioRxiv2021

Functional analysis of deoxyhexose sugar utilization in Escherichia coli reveals fermentative metabolism under aerobic conditions

L-rhamnose and L-fucose are the two main 6-deoxyhexoses Escherichia coli can use as carbon and energy sources. Deoxyhexose metabolism leads to the formation of lactaldehyde whose fate depends on oxygen availability. Under anaerobic conditions, lactaldehyde is reduced to 1,2-propanediol whereas under aerobic condition, it should be oxidised into lactate and then channelled into the central metabolism. However, although this all-or-nothing view is accepted in the literature, it seems overly simplistic since propanediol is also reported to be present in the culture medium during aerobic growth on L-fucose. To clarify the functioning of 6-deoxyhexose sugar metabolism, a quantitative metabolic analysis was performed to determine extra- and intracellular fluxes in E. coli K-12 MG1655 (a laboratory strain) and in E. coli Nissle 1917 (a human commensal strain) during anaerobic and aerobic growth on L-rhamnose and L-fucose. As expected, lactaldehyde is fully reduced to 1,2-propanediol in anoxic conditions allowing complete reoxidation of the NADH produced by glyceraldehyde-3-phosphate-dehydrogenase. We also found that net ATP synthesis is ensured by acetate production. More surprisingly, lactaldehyde is also primarily reduced into 1,2-propanediol under aerobic conditions. For growth on L-fucose, 13C-metabolic flux analysis revealed a large excess of available energy, highlighting the need to better characterize ATP utilization processes. The probiotic E. coli Nissle 1917 strain exhibits similar metabolic traits, indicating that they are not the result of the K-12 strains prolonged laboratory use. IMPORTANCEE. colis ability to survive, grow and colonize the gastrointestinal tract stems from its use of partially digested food and hydrolysed glycosylated proteins (mucins) from the intestinal mucus layer as substrates. These include L-fucose and L-rhamnose, two 6-deoxyhexose sugars, whose catabolic pathways have been established by genetic and biochemical studies. However, the functioning of these pathways has only partially been elucidated. Our quantitative metabolic analysis provides a comprehensive picture of 6-deoxyhexose sugar metabolism in E. coli under anaerobic and aerobic conditions. We found that 1,2-propanediol is a major by-product under both conditions, revealing the key role of fermentative pathways in 6-deoxyhexose sugar metabolism. This metabolic trait is shared by both E. coli strains studied here, a laboratory strain and a probiotic strain. Our findings add to our understanding of E. colis metabolism and of its functioning in the bacteriums natural environment.

physiology↗

bioRxiv2021

IsoSolve: an integrative framework to improve isotopic coverage and consolidate isotopic measurements by MS and/or NMR

Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS)and nuclear magnetic resonance (NMR)spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased, and the coverage of isotopic space improved, by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic dataset by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to i) clarify and improve the coverage of isotopic space, ii) evaluate the complementarity and redundancy of different techniques, iii) consolidate isotopic datasets, iv) design experiments, and v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSysLISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open source software that can be readily integrated into data analysis pipelines.

systems biology↗

bioRxiv2020

Engineering precursor pools for increasing production of odd-chain fatty acids in Yarrowia lipolytica

Microbial production of lipids is one of the promising alternatives to fossil fuels with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and five-carbon chain ({beta}-ketovaleryl-CoA) for the production of OCFAs in Yarrowia lipolytica. We evaluated different propionate-activating enzymes and the overexpression of propionyl-CoA transferase gene from Ralstonia eutropha increased the accumulation of OCFAs by 3.8 times over control strain, indicating propionate activation is the limiting step of OCFAs synthesis. It was shown that acetate supplement was necessary to restore growth and to produce a higher OCFA contents in total lipids, suggesting the balance of the precursors between acetyl-CoA and propionyl-CoA is crucial for OCFA accumulation. To improve {beta}-ketovaleryl-CoA pools for further increase of OCFA production, we co-expressed the bktB encoding {beta}-ketothiolase in the producing strain, and the OCFA production was increased by 33 % compared to control. Combining strain engineering and the optimization of the C/N ratio promoted the OCFA production up to 1.87 g/L representing 62% of total lipids, the highest recombinant OCFAs titer reported in yeast, up to date. This study provides a strong basis for the microbial production of OCFAs and its derivatives having high potentials in a wide range of applications.

synthetic biology↗

bioRxiv2020

Crucial role of ppGpp in the resilience of Escherichia coli to growth disruption

Bacteria grow in constantly changing environments that can suddenly become completely deleted in essential nutrients. The stringent response, a rewiring of the cellular metabolism mediated by the alarmone (p)ppGpp, plays a crucial role in adjusting bacterial growth to the severity of the nutritional insult. The ability of (p)ppGpp to trigger a slowdown of cell growth or induce bacterial dormancy has been widely investigated. However, little is known about the role of (p)ppGpp in promoting growth recovery after severe growth inhibition. In this study, we performed a time-resolved analysis of (p)ppGpp metabolism in Escherichia coli as it recovered from a sudden slowdown in growth. Results show that E. coli recovers by itself from the growth disruption provoked by the addition of serine hydroxamate, the serine analogue that we used to induce the stringent response. Growth inhibition was accompanied by a severe disturbance of metabolic activity and more surprisingly, by a transient overflow of valine and alanine. Our data also show that ppGpp is crucial for growth recovery since in the absence of ppGpp, E. colis growth recovery was slower. In contrast, an increased concentration of pppGpp was found to have no significant effect on growth recovery. Interestingly, the observed decrease in intracellular ppGpp levels in the recovery phase correlated with bacterial growth and the main effect involved was identified as growth dilution rather than active degradative process. This report thus significantly expands our knowledge of (p)ppGpp metabolism in E. coli physiology. IMPORTANCEThe capacity of microbes to resist and overcome environmental insults, know as resilience, allows them to survive in changing environments but also to resist antibiotic and biocide treatments, immune system responses. Although the role of the stringent response in bacterial resilience to nutritional insults has been well studied, little is known about its importance in the ability of the bacteria to not just resist but also recover from these disturbances. To address this important question, we investigated growth disruption resilience in the model bacterium Escherichia coli and its dependency on the stringent response alarmone (p)ppGpp by quantifying ppGpp and pppGpp levels as growth was disrupted and then recovered. Our findings may thus contribute to understanding how ppGpp improves E. colis resilience to nutritional stress and other environmental insults.

systems biology↗

bioRxiv2020

Control and regulation of acetate overflow in Escherichia coli

Overflow metabolism refers to the production of seemingly wasteful by-products by cells during growth on glucose even when oxygen is abundant. Two theories have been proposed to explain acetate overflow in Escherichia coli - global control of the central metabolism and local control of the acetate pathway - but neither accounts for all observations. Here, we develop a kinetic model of E. coli metabolism that quantitatively accounts for observed behaviors and successfully predicts the response of E. coli to new perturbations. We reconcile these theories and clarify the origin, control and regulation of the acetate flux. We also find that, in turns, acetate regulates glucose metabolism by coordinating the expression of glycolytic and TCA genes. Acetate should not be considered a wasteful end-product since it is also a co-substrate and a global regulator of glucose metabolism in E. coli. This has broad implications for our understanding of overflow metabolism.

systems biology↗