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Polyhydroxyalkanoates (PHA) represent an environmentally friendly alternative to petroleum based plastics for a broad range of applications from packaging to biomedical devices. In the prospect of an industrial PHA production, it is highly valuable to accurately control the incorporation of different repeating units into the polymer, to produce a polyester with specific material characteristics. In this study, we develop macroscopic dynamic models predicting the polymer production and composition when mixtures containing up to four volatile fatty acids (VFA) are used as substrates. These models successfully reproduce the sequential (and preferential) substrate consumption and polymer production/reconsumption patterns, experimentally observed during biomass growth, thanks to simple kinetic structures based on Monod and inhibition factors. These models can serve as a basis for numerical simulation and process analysis, as well as process intensification through model-based optimization and control.

The use of purple bacteria, which can assimilate volatile fatty acids, for biotechnological applications has increased, since they reduce the production costs of added-value compounds such as PHA. P(HB- co -HHx) and P(HB- co -HV- co -HHx) have demonstrated interesting properties, notably for biomedical applications.

Epidemiological studies have shown that obese subjects have an increased risk of developing triple‐negative breast cancer (TNBC) and an overall reduced survival. However, the relation between obesity and TNBC remains difficult to understand. We hypothesize that apelin, an adipokine whose levels are increased in obesity, could be a major factor contributing to both tumour growth and metastatization in TNBC obese patients. We observed that development of obesity under high‐fat diet in TNBC tumour‐bearing mice significantly increased tumour growth. By showing no effect of high‐fat diet in obesity‐resistant mice, we demonstrated the necessity to develop obesity‐related disorders to increase tumour growth. Apelin mRNA expression was also increased in the subcutaneous adipose tissue and tumours of obese mice. We further highlighted that the reproduction of obesity‐related levels of apelin in lean mice led to an increased TNBC growth and brain metastases formation. Finally, injections of the apelinergic antagonist F13A to obese mice significantly reduced TNBC growth, suggesting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.

Purple non-sulfur bacteria (PNSB) show great potential for environmental and industrial biotechnology, producing microbial protein, biohydrogen, polyhydroxyalkanoates (PHAs), pigments, etc. When grown photoheterotrophically, the carbon source is typically more reduced than the PNSB biomass, which leads to a redox imbalance. To mitigate the excess of electrons, PNSB can exhibit several ‘electron sinking’ strategies, such as CO2 fixation, N2 fixation, and H2 and PHA production. The lack of a comprehensive (over)view of these redox strategies is hindering the implementation of PNSB for biotechnology applications. This review aims to present the state of the art of redox homeostasis in phototrophically grown PNSB, presenting known and theoretically expected strategies, and discussing them from stoichiometric, thermodynamic, metabolic, and economic points of view.

Purple non-sulfur bacteria (PNSB) are known for their metabolic versatility and thrive as anoxygenic photoheterotrophs. In environmental engineering and resource recovery, cells would grow on mixtures of volatile fatty acids (VFA) generated by anaerobic fermentation of waste streams. In this study, we aim to better understand the behavior of Rhodospirillum rubrum, a model PNSB species, grown using multiple VFA as carbon sources. We highlighted that assimilation of individual VFA follows a sequential pattern. Based on observations in other PNSB, this seems to be specific to isocitrate lyase-lacking organisms. We hypothesized that the inhibition phenomenon could be due to the regulation of the metabolic fluxes in the substrate cycle between acetoacetyl-CoA and crotonyl-CoA. Developed macroscopic dynamic models showed a good predictive capability for substrate competition for every VFA mixture containing acetate, propionate, and/or butyrate. These novel insights provide valuable input for better design and operation of PNSB-based waste treatment solutions.

Long-duration human space missions require considerable amounts of water, oxygen, and nutritious biomass. Additionally, the space vehicles must be well equipped to deal with metabolic human waste. It is therefore important to develop life-support systems which make these missions self-sufficient in terms of water, food, and oxygen production as well as waste management. One such solution is the employment of regenerative life-support systems that use biological and chemical/physical processes to recycle crew waste, revitalize air, and produce water and food. Photosynthetic cyanobacteria Limnospira could play a significant role in meeting these objectives. Limnospira can metabolize CO2 and nitrogen-rich human waste to produce oxygen and edible biomass. So far, life-support system studies have mainly focused on using chemical/physical methods to recycle water, degrade human waste, and recycle CO2 into oxygen. Nowadays, additional microbial processes are considered, such as nitrification of urea–ammonium–rich human waste and then using the nitrate for cyanobacterial cultivation and air vitalization. This cascade of multiple processes tends to increase the complexity of the life-support systems. The possibility of using non-nitrified urine for Limnospira cultivation can partially solve these issues. Our previous studies have shown that it is possible to cultivate Limnospira with urea and ammonium, the prominent nitrogen forms present in non-nitrified urine. In this study, we investigated the possibility of cultivating Limnospira with the different nitrogen forms present in non-nitrified urine and also evaluated their effect on the oxygen production capacity of Limnospira. For this 35-day-long study, we worked on a simplified version of the European Space Agency’s MELiSSA. During this ground demonstration study, we monitored the effect of urea and ammonium (vs. nitrate) on the oxygen production capacity of Limnospira. A deterministic control law, developed and validated on the basis of a stochastic light-transfer model, modulated (increase/decrease) the incident light on the photobioreactor (with Limnospira) to control oxygen levels in the closed loop. The CO2 from the mouse compartment was recycled as a carbon source for Limnospira. We observed that while the system could meet the desired oxygen levels of 20.3% under the nitrate and urea regime, it could only reach a maximum O2 level of 19.5% under the ammonium regime.

Salvia miltiorrhiza is a medicinal plant highly appreciated by its content of tanshinones and salvianolic acids. Tanshinones are of particular relevance for their anti-oxidant, anti-tumoral and anti-inflammatory properties. Abiotic and biotic agents as silver nitrate and yeast extract have shown efficiently to stimulate tanshinone accumulation, but the underlying molecular mechanism remains essentially unknown. By using hairy roots as experimental material and the elicitors mentioned, were obtained up to 22 mg of tanshinones per gram of dry weight. Differential label-free quantitative proteomic analysis was applied to study the proteins involved in tanshinone biosynthesis. A total of 2650 proteins were identified in roots extracts, of which 893 showed statistically (p

Plasmids carrying metal resistance genes (MRGs) have been suggested to be key ecological players in the adaptation of metal-impacted microbial communities, making them promising drivers of bio-remediation processes. However, the impact of metals on plasmid-mediated spread of MRGs through selection, plasmid loss, and transfer is far from being fully understood. In the present study, we used two-member bacterial communities to test the impact of lead on the dispersal of the IncP plasmid pKJK5 from a Pseudomonas putida KT2440 plasmid donor and two distinct recipients, Variovorax paradoxus B4 or Delftia acidovorans SPH-1 after 4 and 10 days of mating. Two versions of the plasmid were used, carrying or not carrying the lead resistance pbrTRABCD operon, to assess the importance of fitness benefit and conjugative potential for the dispersal of the plasmid. The spread dynamics of metal resistance conveyed by the conjugative plasmid were dependent on the recipient and the lead concentration: For V. paradoxus, the pbr operon did not facilitate neither lead resistance nor variation in plasmid spread. The growth gain brought by the pbr operon to D. acidovorans SPH-1 and P. putida KT2440 at 1 mM Pb enhanced the spread of the plasmid. At 1.5 mM Pb after 4 days, the proteomics results revealed an oxidative stress response and an increased abundance of pKJK5-encoded conjugation and partitioning proteins, which most likely increased the transfer of the control plasmid to D. acidovorans SPH-1 and ensured plasmid maintenance. As a consequence, we observed an increased spread of pKJK5-gfp. Conversely, the pbr operon reduced the oxidative stress response and impeded the rise of conjugation- and partitioning-associated proteins, which slowed down the spread of the pbr carrying plasmid. Ultimately, when a fitness gain was recorded in the recipient strain, the spread of MRG-carrying plasmids was facilitated through positive selection at an intermediate metal concentration, while a high lead concentration induced oxidative stress with positive impacts on proteins encoding plasmid conjugation and partitioning.

Purple nonsulfur bacteria are increasingly recognized for industrial applications in bioplastics, pigment, and biomass production. In order to optimize the yield of future biotechnological processes, the assimilation of different carbon sources by Rhodospirillum rubrum has to be understood. As they are released from several fermentation processes, volatile fatty acids (VFAs) represent a promising carbon source in the development of circular industrial applications. To obtain an exhaustive characterization of the photoheterotrophic metabolism of R. rubrum in the presence of valerate, we combined phenotypic, proteomic, and genomic approaches. We obtained evidence that valerate is cleaved into acetyl coenzyme A (acetyl-CoA) and propionyl-CoA and depends on the presence of bicarbonate ions. Genomic and enzyme inhibition data showed that a functional methylmalonyl-CoA pathway is essential. Our proteomic data showed that the photoheterotrophic assimilation of valerate induces an intracellular redox stress which is accompanied by an increased abundance of phasins (the main proteins present in polyhydroxyalkanoate [PHA] granules). Finally, we observed a significant increase in the production of the copolymer P(HB-co-HV), accounting for a very high (>80%) percentage of HV monomer. Moreover, an increase in the PHA content was obtained when bicarbonate ions were progressively added to the medium. The experimental conditions used in this study suggest that the redox imbalance is responsible for PHA production. These findings also reinforce the idea that purple nonsulfur bacteria are suitable for PHA production through a strategy other than the well-known feast-and-famine process.IMPORTANCE The use and the littering of plastics represent major issues that humanity has to face. Polyhydroxyalkanoates (PHAs) are good candidates for the replacement of oil-based plastics, as they exhibit comparable physicochemical properties but are biobased and biodegradable. However, the current industrial production of PHAs is curbed by the production costs, which are mainly linked to the carbon source. Volatile fatty acids issued from the fermentation processes constitute interesting carbon sources, since they are inexpensive and readily available. Among them, valerate is gaining interest regarding the ability of many bacteria to produce a copolymer of PHAs. Here, we describe the photoheterotrophic assimilation of valerate by Rhodospirillum rubrum, a purple nonsulfur bacterium mainly known for its metabolic versatility. Using a knowledge-based optimization process, we present a new strategy for the improvement of PHA production, paving the way for the use of R. rubrum in industrial processes.

Background The great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. Rhodospirillum rubrum S1H is an α-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied. Results In this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate. Conclusion Photoheterotrophic growth of Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.

Purple nonsulfur bacteria are increasingly recognized for industrial applications in bioplastics, pigment, and biomass production. In order to optimize the yield of future biotechnological processes, the assimilation of different carbon sources by Rhodospirillum rubrum has to be understood. As they are released from several fermentation processes, volatile fatty acids (VFAs) represent a promising carbon source in the development of circular industrial applications. To obtain an exhaustive characterization of the photoheterotrophic metabolism of R. rubrum in the presence of valerate, we combined phenotypic, proteomic, and genomic approaches. We obtained evidence that valerate is cleaved into acetyl coenzyme A (acetyl-CoA) and propionyl-CoA and depends on the presence of bicarbonate ions. Genomic and enzyme inhibition data showed that a functional methylmalonyl-CoA pathway is essential. Our proteomic data showed that the photoheterotrophic assimilation of valerate induces an intracellular redox stress which is accompanied by an increased abundance of phasins (the main proteins present in polyhydroxyalkanoate [PHA] granules). Finally, we observed a significant increase in the production of the copolymer P(HB-co-HV), accounting for a very high (>80%) percentage of HV monomer. Moreover, an increase in the PHA content was obtained when bicarbonate ions were progressively added to the medium. The experimental conditions used in this study suggest that the redox imbalance is responsible for PHA production. These findings also reinforce the idea that purple nonsulfur bacteria are suitable for PHA production through a strategy other than the well-known feast-and-famine process. IMPORTANCE The use and the littering of plastics represent major issues that humanity has to face. Polyhydroxyalkanoates (PHAs) are good candidates for the replacement of oil-based plastics, as they exhibit comparable physicochemical properties but are biobased and biodegradable. However, the current industrial production of PHAs is curbed by the production costs, which are mainly linked to the carbon source. Volatile fatty acids issued from the fermentation processes constitute interesting carbon sources, since they are inexpensive and readily available. Among them, valerate is gaining interest regarding the ability of many bacteria to produce a copolymer of PHAs. Here, we describe the photoheterotrophic assimilation of valerate by Rhodospirillum rubrum, a purple nonsulfur bacterium mainly known for its metabolic versatility. Using a knowledge-based optimization process, we present a new strategy for the improvement of PHA production, paving the way for the use of R. rubrum in industrial processes.

Purple non-sulfur bacteria (PNSBs) are well known for their metabolic versatility. Among them, Rhodospirillum rubrum can assimilate a broad range of carbon sources, including volatile fatty acids (VFAs), such as acetate, propionate or butyrate. These carbon sources are gaining increasing interest in bioindustrial processes since they allow reduction of the production costs. Recently, our lab discovered that, after long term cultivation with acetate as unique carbon source, Rs. rubrum got acclimated to this carbon source which resulted in a drastic reduction of the lag phase. This acclimation was characterized by the amplification of the genomic region containing, among others, genes belonging to the ethylmalonyl-CoA (EMC) pathway, which has been demonstrated to be required for acetate assimilation in Rs. rubrum. In this paper, we combined bacterial growth analysis with proteomic (SWATH -Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra-processing) investigation to better understand the bacterial response to a sudden increase of the light intensity. We compared the impact of suddenly increasing light intensity on the WT strain to that on the newly described acetate-competent strain in the presence of acetate. Contrary to what was observed with the WT strain, we observed that the acetate-competent strain was tolerant to the light stress. Proteomic analysis revealed that increasing light intensity had a significant impact on the photosynthetic apparatus, especially in the wild-type strain cultivated in the presence of acetate and low concentration of HCO3–. This phenomenon was accompanied by a relatively higher abundance of certain stress related proteins. Our results suggested that the production of PHA, but also potentially of branched chain amino acids synthesis, could be part of the mechanism used by Rs. rubrum to adapt to the light stress and the redox imbalance it triggered.

The ability of cyanobacterium Arthrospira sp. to assimilate waste nitrogen sources (ammonium and urea) makes it an important candidate for wastewater management. The aim of this work was to evaluate a cultivation approach based on continuous-transitional-feeding regime (nitrate-ammonium-nitrate) in a photobioreactor to assess the effects of ammonium salts on Arthrospira sp. PCC 8005 metabolism. Using a comprehensive biochemical, proteomic and stoichiometric profiling of biomass, this study demonstrated that the proposed cultivation approach could increase the proteins and pigments yields by 20-30%, compared to the respective yields obtained from wild-type Arthrospira sp. strain A light-energy-transfer model was used to predict the biomass and oxygen productivities of Arthrospira sp. cultivated under transitional-feeding regime. 95 ± 2% match was observed between the experimental and simulated productivities. This study thus opened new avenues for use of ammonium rich wastewater for commercial production of high value pigments, biofuel and bioplastics using Arthrospira sp.

Rhodospirillum rubrum has a versatile metabolism, and as such can assimilate a broad range of carbon sources, including volatile fatty acids. These carbon sources are gaining increasing interest for biotechnological processes, since they reduce the production costs for numerous value-added compounds and contribute to the development of a more circular economy. Usually, studies characterizing carbon metabolism are performed by supplying a single carbon source, however, in both environmental and engineered conditions, cells would rather grow on mixtures of volatile fatty acids (VFAs) generated via anaerobic fermentation. In this study, we show that the use of a mixture of VFAs as carbon source appears to have a synergy effect on growth phenotype. In addition, while propionate and butyrate assimilation in Rs. rubrum is known to require an excess of bicarbonate in the culture medium, mixing them reduces the requirement for bicarbonate supplementation. The fixation of CO2 is one of the main electron sinks in purple bacteria, therefore, this observation suggests an adaptation of both metabolic pathways used for the assimilation of these VFAs and redox homeostasis mechanism. Based on proteomic data, modification of the propionate assimilation pathway seems to occur with a switch from a methylmalonyl-CoA intermediate to the methylcitrate cycle. Moreover, it seems that the presence of a mixture of VFAs switches electron sinking from CO2 fixation to H2 and isoleucine production.

The aim of the present study was to evaluate the effects of varying concentrations of different nitrogen sources (individually or in combination) on the biochemical, transcriptomic and proteomic profiles of Arthrospira sp. PCC 8005 under batch and continuous modes. In batch mode, while ammonium showed a repressive effect on nitrate-assimilation pathway of the cyanobacteria; better growth and nutrient uptake rate were observed in presence of urea than nitrate. The inhibitory effect of ammonium was further confirmed by the continuous photobioreactor study wherein the nutrient feed was transiently replaced from nitrate to ammonium (28mM turbiostat regime). The changes in lipid, exopolysaccharide, transcriptomic and proteomic profiles of cyanobacteria on transition from nitrate to ammonium indicated at an onset of nutrient stress.

Typically, nitrification is a two-stage microbial process and is key in wastewater treatment and nutrient recovery from waste streams. Changes in salinity represent a major stress factor that can trigger response mechanisms, impacting the activity and the physiology of bacteria. Despite its pivotal biotechnological role, little information is available on the specific response of nitrifying bacteria to varying levels of salinity. In this study, synthetic communities of ammonia-oxidizing bacteria (AOB Nitrosomonas europaea and/or Nitrosomonas ureae) and nitrite-oxidizing bacteria (NOB Nitrobacter winogradskyi and/or Nitrobacter vulgaris) were tested at 5, 10, and 30 mS cm-1 by adding sodium chloride to the mineral medium (0, 40, and 200 mM NaCl, respectively). Ammonia oxidation activity was less affected by salinity than nitrite oxidation. AOB, on their own or in combination with NOB, showed no significant difference in the ammonia oxidation rate among the three conditions. However, N. winogradskyi improved the absolute ammonia oxidation rate of both N. europaea and N. ureae. N. winogradskyis nitrite oxidation rate decreased to 42% residual activity upon exposure to 30 mS cm-1, also showing a similar behavior when tested with Nitrosomonas spp. The nitrite oxidation rate of N. vulgaris, as a single species, was not affected when adding sodium chloride up to 30 mS cm-1, however, its activity was completely inhibited when combined with Nitrosomonas spp. in the presence of ammonium/ammonia. The proteomic analysis of a co-culture of N. europaea and N. winogradskyi revealed the production of osmolytes, regulation of cell permeability and an oxidative stress response in N. europaea and an oxidative stress response in N. winogradskyi, as a result of increasing the salt concentration from 5 to 30 mS cm-1. A specific metabolic response observed in N. europaea suggests the role of carbon metabolism in the production of reducing power, possibly to meet the energy demands of the stress response mechanisms, induced by high salinity. For the first time, metabolic modifications and response mechanisms caused by the exposure to salinity were described, serving as a tool toward controllability and predictability of nitrifying systems exposed to salt fluctuations.

Aminobacter sp. MSH1 uses the groundwater micropollutant 2,6-dichlorobenzamide (BAM) as sole source of carbon and energy. In the first step, MSH1 converts BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) by means of the BbdA amidase encoded on the IncP-1β plasmid pBAM1. Information about the genes and degradation steps involved in 2,6-DCBA metabolism in MSH1 or any other organism is currently lacking. Here, we show that the genes for 2,6-DCBA degradation in strain MSH1 reside on a second catabolic plasmid in MSH1, designated as pBAM2. The complete sequence of pBAM2 was determined revealing that it is a 53.9 kb repABC family plasmid. The 2,6-DCBA catabolic genes on pBAM2 are organized in two main clusters bordered by IS elements and integrase genes and encode putative functions like Rieske mono-/dioxygenase, meta-cleavage dioxygenase, and reductive dehalogenases. The putative mono-oxygenase encoded by the bbdD gene was shown to convert 2,6-DCBA to 3-hydroxy-2,6-dichlorobenzoate (3-OH-2,6-DCBA). 3-OH-DCBA was degraded by wild-type MSH1 and not by a pBAM2-free MSH1 variant indicating that it is a likely intermediate in the pBAM2-encoded DCBA catabolic pathway. Based on the activity of BbdD and the putative functions of the other catabolic genes on pBAM2, a metabolic pathway for BAM/2,6-DCBA in strain MSH1 was suggested. ispartof: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY vol:102 issue:18 pages:7963-7979 ispartof: location:Germany status: published

This study was conducted with the aim of embedding circular economies (waste recycling) with photosynthetic biorefineries, for production of commercially viable by-products. Since nitrogen source constitute the major input costs for commercial Arthrospira sp. production, the use of nitrogen rich wastewater for Arthrospira sp. cultivation could significantly reduce their production costs. This study evaluated the effects of high concentrations (8.5–120 mM) of alternative nitrogen sources (urea, ammonium and nitrite) on the biochemical, pigment and proteomic profile of Arthrospira sp., under batch and continuous conditions. Arthrospira sp. cells fed with urea were quantified with modified biochemical and proteomic profile compared to the nitrate fed cells. No inhibitory effect of urea was observed on the biomass even at 120 mM. Nitrite fed cells exhibited comparable biochemical and proteomic profiles as nitrate fed cells. These results clearly indicated at the possibility of using urea rich wastewater streams for profitable cultivation of Arthrospira sp.

Purple nonsulfur bacteria represent a promising resource for biotechnology because of their great metabolic versatility. Rhodospirillum rubrum has been widely studied regarding its metabolism of volatile fatty acid, mainly acetate. As the glyoxylate shunt is unavailable in Rs. rubrum , the citramalate cycle pathway and the ethylmalonyl-coenzyme A (CoA) pathway are proposed as alternative anaplerotic pathways for acetate assimilation. However, despite years of debate, neither has been confirmed to be essential. Here, using functional genomics, we demonstrate that the ethylmalonyl-CoA pathway is required for acetate photoassimilation. Moreover, an unexpected reversible long-term adaptation is observed, leading to a drastic decrease in the lag phase characterizing the growth of Rs. rubrum in the presence of acetate. Using proteomic and genomic analyses, we present evidence that the adaptation phenomenon is associated with reversible amplification and overexpression of a 60-kb genome fragment containing key enzymes of the ethylmalonyl-CoA pathway. Our observations suggest that a genome duplication and amplification phenomenon is not only involved in adaptation to acute stress but can also be important for basic carbon metabolism and the redox balance. IMPORTANCE Purple nonsulfur bacteria represent a major group of anoxygenic photosynthetic bacteria that emerged as a promising resource for biotechnology because of their great metabolic versatility and ability to grow under various conditions. Rhodospirillum rubrum S1H has notably been selected by the European Space Agency to colonize its life support system, called MELiSSA, due to its capacity to perform photoheterotrophic assimilation of volatile fatty acids (VFAs), mainly acetate. VFAs are valuable carbon sources for many applications, combining bioremediation of contaminated environments with the generation of added-value products. Acetate is one of the major volatile fatty acids generated as a by-product of fermentation processes. In Rs. rubrum , purple nonsulfur bacteria, the assimilation of acetate is still under debate since two different pathways have been proposed. Here, we clearly demonstrate that the ethylmalonyl-CoA pathway is the major anaplerotic pathway for acetate assimilation in this strain. Interestingly, we further observed that gene duplication and amplification, which represent a well-known phenomenon in antibiotic resistance, also play a regulatory function in carbon metabolism and redox homeostasis.

2,6-dichlorobenzamide (BAM) is a recalcitrant groundwater micropollutant that poses a major problem for drinking water production in European countries. Aminobacter sp. MSH1 and related strains have the unique ability to mineralize BAM at micropollutant concentrations but no information exists on the genetics of BAM biodegradation. An amidase-BbdA-converting BAM to 2,6-dichlorobenzoic acid (DCBA) was purified from Aminobacter sp. MSH1. Heterologous expression of the corresponding bbdA gene and its absence in MSH1 mutants defective in BAM degradation, confirmed its BAM degrading function. BbdA shows low amino acid sequence identity with reported amidases and is encoded by an IncP1-β plasmid (pBAM1, 40.6 kb) that lacks several genes for conjugation. BbdA has a remarkably low KM for BAM (0.71 μM) and also shows activity against benzamide and ortho-chlorobenzamide (OBAM). Differential proteomics and transcriptional reporter analysis suggest the constitutive expression of bbdA in MSH1. Also in other BAM mineralizing Aminobacter sp. strains, bbdA and pBAM1 appear to be involved in BAM degradation. BbdA's high affinity for BAM and its constitutive expression are of interest for using strain MSH1 in treatment of groundwater containing micropollutant concentrations of BAM for drinking water production.

For decades, cyanobacteria have been of great interest in research because their metabolic versatility and their capacity to adapt to a variety of environmental conditions have enabled cyanobacteria to colonize various habitats. Furthermore, the metabolic abilities of cyanobacteria also make them useful for human applications, and therefore, they have been extensively characterized. To understand more deeply the molecular basis for the underlying metabolic versatility, cyanobacteria have been subjected to various culture conditions (e.g., nutrient limitations, salt and high-light exposure), followed by characterization by molecular “omic” approaches. Here, we reviewed the proteomic approach and focused on its application in the characterization of the molecular ability through the phylum Cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes that play a crucial role in the Earth’s nitrogen and carbon cycles. Nitrogen availability is one of the most important factors in cyanobacterial growth. Interestingly, filamentous non-diazotrophic cyanobacteria, such asArthrospirasp. PCC 8005, have developed survival strategies that enable them to adapt to nitrogen deprivation. Metabolic studies recently demonstrated a substantial synthesis and accumulation of glycogen derived from amino acids during nitrogen starvation. Nevertheless, the regulatory mechanism of this adaptation is poorly understood. To the best of our knowledge, this study is the first proteomic and cellular analysis ofArthrospirasp. PCC 8005 under nitrogen depletion. Label-free differential proteomic analysis indicated the global carbon and nitrogen reprogramming of the cells during nitrogen depletion as characterized by an upregulation of glycogen synthesis and the use of endogenous nitrogen sources. The degradation of proteins and cyanophycin provided endogenous nitrogen when exogenous nitrogen was limited. Moreover, formamides, cyanates and urea were also potential endogenous nitrogen sources. The transporters of some amino acids and alternative nitrogen sources such as ammonium permease 1 were induced under nitrogen depletion. Intriguingly, althoughArthrospirais a non-diazotrophic cyanobacterium, we observed the upregulation of HetR and HglK proteins, which are involved in heterocyst differentiation. Moreover, after a long period without nitrate, only a few highly fluorescent cells in each trichome were observed, and they might be involved in the long-term survival mechanism of this non-diazotrophic cyanobacterium under nitrogen deprivation.

Glycoproteins form an interesting class of macromolecules involved in bacterial-host interactions, but they are not yet widely explored in Gram-positive and beneficial species. Here, an integrated and widely applicable approach was followed to identify putative bacterial glycoproteins, combining proteome fractionation with 2D protein and glycostained gels and lectin blots. This approach was validated for the microbiota isolate Lactobacillus rhamnosus GG. The approach resulted in a list of putative glycosylated proteins receiving a 'glycosylation score'. Ultimately, we could identify 41 unique glycosylated proteins in L. rhamnosus GG (6 top-confidence, 10 high-confidence and 25 putative hits; classification based on glycosylation score). Most glycoproteins are associated with the cell wall and membrane. Identified glycoproteins include proteins involved in transport, translation, and sugar metabolism processes. A robust screening resulted in a comprehensive mapping of glycoproteins in L. rhamnosus GG. Our results reflect the glycosylation of sugar metabolism enzymes, transporters, and other proteins crucial for cell physiology. We hypothesize that protein glycosylation can confer an extra level of regulation, for example by affecting enzyme functions. This is the first systematic study of the glycoproteome of a probiotic and beneficial gut isolate.

Herpesviruses are highly prevalent viruses associated with numerous pathologies both in animal and human populations. Until now, most of the strategies used to prevent or to cure these infections have been unsuccessful because these viruses have developed numerous immune evasion mechanisms. Therefore, a better understanding of their complex lifecycle is needed. In particular, while the genome of numerous herpesviruses has been sequenced, the exact composition of virions remains unknown for most of them. Mass spectrometry has recently emerged as a central method and has permitted fundamental discoveries in virology. Here, we review mass spectrometry-based approaches that have recently allowed a better understanding of the composition of the herpesvirus virion. In particular, we describe strategies commonly used for proper sample preparation and fractionation to allow protein localization inside the particle but also to avoid contamination by nonstructural proteins. A collection of other important data regarding post-translational modifications or the relative abundance of structural proteins is also described. This review also discusses the poorly studied importance of host proteins in herpesvirus structural proteins and the necessity to develop a quantitative workflow to better understand the dynamics of the structural proteome. In the future, we hope that this collaborative effort will assist in the development of new strategies to fight these infections.

Snow avalanche data in the French Alps and Pyrenees have been recorded for more than 100 years in several databases. The increasing amount of observed data required a more integrative and automated service. Here we report the comprehensive web-based Snow Avalanche Information System newly developed to this end for three important data sets: an avalanche chronicle (Enquête Permanente sur les Avalanches, EPA), an avalanche map (Carte de Localisation des Phénomènes d'Avalanche, CLPA) and a compilation of hazard and vulnerability data recorded on selected paths endangering human settlements (Sites Habités Sensibles aux Avalanches, SSA). These data sets are now integrated into a common database, enabling full interoperability between all different types of snow avalanche records: digitized geographic data, avalanche descriptive parameters, eyewitness reports, photographs, hazard and risk levels, etc. The new information system is implemented through modular components using Java-based web technologies with Spring and Hibernate frameworks. It automates the manual data entry and improves the process of information collection and sharing, enhancing user experience and data quality, and offering new outlooks to explore and exploit the huge amount of snow avalanche data available for fundamental research and more applied risk assessment.

A proteomic approach was used to explore the metabolism of the phenylurea herbicide linuron and 3,4-dichloroaniline (3,4-DCA) in Variovorax sp. WDL1. This bacterium grows on linuron as sole source of carbon, nitrogen and energy, while it transiently accumulates 3,4-DCA as a metabolite. Differential protein expression analysis of Variovorax sp. WDL1 grown in a heterotrophic medium in the presence and absence of linuron or 3,4-DCA was conducted using 2-D PAGE. Selected up- and downregulated proteins were identified with nanoLC-ESI-MS/MS. In the 3,4-DCA-supplemented culture, upregulation of several proteins showing high amino acid sequence similarity to different components of the multicomponent aniline dioxygenase in aniline-degrading Proteobacteria was observed. For one of the components, multiple variant proteins were detected, suggesting that strain WDL1 harbors several copies of the aniline dioxygenase (AD) gene cluster which are simultaneously expressed in the presence of 3,4-DCA. A number of unidentifiable proteins, which were upregulated in the linuron- and/or 3,4-DCA-supplemented cultures, might represent up to now uncharacterized proteins with a role in linuron and/or 3,4-DCA degradation in strain WDL1. In addition, several stress-related proteins were differentially expressed. ispartof: Research in Microbiology vol:161 issue:3 pages:208-218 ispartof: location:France status: published

In view of long-haul space exploration missions, the European Space Agency initiated the Micro-Ecological Life Support System Alternative (MELiSSA) project targeting the total recycling of organic waste produced by the astronauts into oxygen, water and food using a loop of bacterial and higher plant bioreactors. In that purpose, the alpha-proteobacterium, Rhodospirillum rubrum S1H, was sent twice to the International Space Station and was analyzed post-flight using a newly developed R. rubrum whole genome oligonucleotide microarray and high throughput gel-free proteomics with Isotope-Coded Protein Label technology. Moreover, in an effort to identify a specific response of R. rubrum S1H to space flight, simulation of microgravity and space-ionizing radiation were performed on Earth under identical culture set-up and growth conditions as encountered during the actual space journeys. Transcriptomic and proteomic data were integrated and permitted to put forward the importance of medium composition and culture set-up on the response of the bacterium to space flight-related environmental conditions. In addition, we showed for the first time that a low dose of ionizing radiation (2 mGy) can induce a significant response at the transcriptomic level, although no change in cell viability and only a few significant differentially expressed proteins were observed. From the MELiSSA perspective, we could argue the effect of microgravity to be minimized, whereas R. rubrum S1H could be more sensitive to ionizing radiation during long-term space exploration mission.

Paratuberculosis is a chronic enteritis caused in domestic and wild ruminant species by Mycobacterium avium subsp. paratuberculosis (MAP) that is responsible for major economic losses to the agricultural industry. To date, no satisfactory therapeutic, vaccine, or diagnostic tools are available, globally impairing all control programs. In this study, we have undertaken a large-scale postgenomic analysis of MAP proteins, to identify specific antigens that could potentially improve the diagnosis of paratuberculosis. Two complementary approaches were implemented, the first one consisting in the systematic proteomic identification of proteins present in MAP culture filtrates (CFs), followed by the selection of MAP-specific proteins by BLAST query on available mycobacterial genomes. The resulting database represents the first established secretome of MAP and a useful source of potentially specific antigens. The second approach consisted in the immunoproteomic analysis of both MAP extracts and CFs, using sera from MAP-infected and uninfected cattle. Combining results obtained with both approaches resulted in the identification of 25 candidate diagnostic antigens. Five of these were tested in an ELISA assay for their diagnostic potential, on a limited panel of field sera, and the combination of three of them competed in performance with available commercial assays, reaching a test sensitivity of 94.74% and specificity of 97.92%.