Protein structure, amino acid composition and sequence determine proteome vulnerability to oxidation-induced damage.

Oxidative stress alters cell viability, from microorganism irradiation sensitivity to human aging and neurodegeneration. Deleterious effects of protein carbonylation by reactive oxygen species (ROS) make understanding molecular properties determining ROS susceptibility essential. The radiation-resistant bacterium Deinococcus radiodurans accumulates less carbonylation than sensitive organisms, making it a key model for deciphering properties governing oxidative stress resistance. We integrated shotgun redox proteomics, structural systems biology, and machine learning to resolve properties determining protein damage by γ-irradiation in Escherichia coli and D. radiodurans at multiple scales. Local accessibility, charge, and lysine enrichment accurately predict ROS susceptibility. Lysine, methionine, and cysteine usage also contribute to ROS resistance of the D. radiodurans proteome. Our model predicts proteome maintenance machinery, and proteins protecting against ROS are more resistant in D. radiodurans. Our findings substantiate that protein-intrinsic protection impacts oxidative stress resistance, identifying causal molecular properties.

Soil Metaproteomics for Microbial Community Profiling: Methodologies and Challenges.

Soil represents a complex and dynamic ecosystem, hosting a myriad of microorganisms that coexist and play vital roles in nutrient cycling and organic matter transformation. Among these microorganisms, bacteria and fungi are key members of the microbial community, profoundly influencing the fate of nitrogen, sulfur, and carbon in terrestrial environments. Understanding the intricacies of soil ecosystems and the biological processes orchestrated by microbial communities necessitates a deep dive into their composition and metabolic activities. The advent of next-generation sequencing and 'omics' techniques, such as metagenomics and metaproteomics, has revolutionized our understanding of microbial ecology and the functional dynamics of soil microbial communities. Metagenomics enables the identification of microbial community composition in soil, while metaproteomics sheds light on the current biological functions performed by these communities. However, metaproteomics presents several challenges, both technical and computational. Factors such as the presence of humic acids and variations in extraction methods can influence protein yield, while the absence of high-resolution mass spectrometry and comprehensive protein databases limits the depth of protein identification. Notwithstanding these limitations, metaproteomics remains a potent tool for unraveling the intricate biological processes and functions of soil microbial communities. In this review, we delve into the methodologies and challenges of metaproteomics in soil research, covering aspects such as protein extraction, identification, and bioinformatics analysis. Furthermore, we explore the applications of metaproteomics in soil bioremediation, highlighting its potential in addressing environmental challenges.

Diel Cycle Proteomics: Illuminating Molecular Dynamics in Purple Bacteria for Optimized Biotechnological Applications.

Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily variations. While ubiquitous in Eukaryotes, circadian clocks remain exclusively characterized in Cyanobacteria among Prokaryotes. These rhythms are regulated by a core oscillator, which is controlled by a cluster of three genes: kaiA, kaiB, and kaiC. Interestingly, recent studies revealed rhythmic activities, potentially tied to a circadian clock, in other Prokaryotes, including purple bacteria such as Rhodospirillum rubrum, known for its applications in fuel and plastic bioproduction. However, the pivotal question of how light and dark cycles influence protein dynamics and the expression of putative circadian clock genes remains unexplored in purple non-sulfur bacteria. Unraveling the regulation of these molecular clocks holds the key to unlocking optimal conditions for harnessing the biotechnological potential of R. rubrum. Understanding how its proteome responds to different light regimes-whether under continuous light or alternating light and dark cycles-could pave the way for precisely fine-tuning bioproduction processes. Here, we report for the first time the expressed proteome of R. rubrum grown under continuous light versus light and dark cycle conditions using a shotgun proteomic analysis. In addition, we measured the impact of light regimes on the expression of four putative circadian clock genes (kaiB1, kaiB2, kaiC1, kaiC2) at the transcriptional and translational levels using RT-qPCR and targeted proteomic (MRM-MS), respectively. The data revealed significant effects of light conditions on the overall differential regulation of the proteome, particularly during the early growth stages. Notably, several proteins were found to be differentially regulated during the light or dark period, thus impacting crucial biological processes such as energy conversion pathways and the general stress response. Furthermore, our study unveiled distinct regulation of the four kai genes at both the mRNA and protein levels in response to varying light conditions. Deciphering the impact of the diel cycle on purple bacteria not only enhances our understanding of their ecology but also holds promise for optimizing their applications in biotechnology, providing valuable insights into the origin and evolution of prokaryotic clock mechanisms.

Towards the discovery of novel molecular clocks in Prokaryotes.

Diel cycle is of enormous biological importance as it imposes daily oscillation in environmental conditions, which temporally structures most ecosystems. Organisms developed biological time-keeping mechanisms - circadian clocks - that provide a significant fitness advantage over competitors by optimising the synchronisation of their biological activities. While circadian clocks are ubiquitous in Eukaryotes, they are so far only characterised in Cyanobacteria within Prokaryotes. However, growing evidence suggests that circadian clocks are widespread in the bacterial and archaeal domains. As Prokaryotes are at the heart of crucial environmental processes and are essential to human health, unravelling their time-keeping systems provides numerous applications in medical research, environmental sciences, and biotechnology. In this review, we elaborate on how novel circadian clocks in Prokaryotes offer research and development perspectives. We compare and contrast the different circadian systems in Cyanobacteria and discuss about their evolution and taxonomic distribution. We necessarily provide an updated phylogenetic analysis of bacterial and archaeal species that harbour homologs of the main cyanobacterial clock components. Finally, we elaborate on new potential clock-controlled microorganisms that represent opportunities of ecological and industrial relevance in prokaryotic groups such as anoxygenic photosynthetic bacteria, methanogenic archaea, methanotrophs or sulphate-reducing bacteria.

Cataglyphis desert ants use distinct behavioral and physiological adaptations to cope with extreme thermal conditions.

Some ant species live in hot and arid environments, such as deserts and savannas. Worker polymorphism-variation in worker size and/or morphology within colonies-is adaptive in such ecosystems because it enhances resistance to heat stress and increases the efficiency of resource exploitation. However, species with small, monomorphic workers are also frequently found in these environments. How species with distinct worker size and degrees of polymorphism deal with such stressful environments remains poorly studied. We investigated the behavioral, physiological, and molecular adaptations that may enhance heat and desiccation tolerance in two sympatric species of Cataglyphis desert ants that differ dramatically in worker size and polymorphism: C. viatica is polymorphic, while C. cubica is small and monomorphic. We found that worker size, water content, water loss, and protein regulation play a key role in thermal resistance. (i) Large C. viatica workers better tolerated heat and desiccation stress than did small C. viatica or C. cubica workers. The former had greater water content and lost proportionally less water to evaporation under thermal stress. (ii) Despite their similar size distribution, workers of C. cubica are more heat tolerant than small C. viatica. This higher degree of tolerance likely stemmed from C. cubica workers having greater relative water content. (iii) Under thermal stress, small C. viatica workers metabolized larger quantities of fat and differentially expressed proteins involved in cellular homeostasis. In contrast, C. cubica downregulated the expression of numerous proteins involved in mitochondrial respiration likely reducing ROS accumulation. (iv) Consistent with these results, large C. viatica workers remained active throughout the day; C. cubica workers displayed a bimodal activity pattern, and small C. viatica remained poorly active outside the nest. Our study shows that ecologically similar ant species with different degrees of worker size polymorphism evolved distinct strategies for coping with extreme heat conditions.

Multiple Technology Approach Based on Stable Isotope Ratio Analysis, Fourier Transform Infrared Spectrometry and Thermogravimetric Analysis to Ensure the Fungal Origin of the Chitosan.

Chitosan is a natural polysaccharide which has been authorized for oenological practices for the treatment of musts and wines. This authorization is limited to chitosan of fungal origin while that of crustacean origin is prohibited. To guarantee its origin, a method based on the measurement of the stable isotope ratios (SIR) of carbon δ13C, nitrogen δ15N, oxygen δ18O and hydrogen δ2H of chitosan has been recently proposed without indicating the threshold authenticity limits of these parameters which, for the first time, were estimated in this paper. In addition, on part of the samples analysed through SIR, Fourier transform infrared spectrometry (FTIR) and thermogravimetric analysis (TGA) were performed as simple and rapid discrimination methods due to limited technological resources. Samples having δ13C values above -14.2‱ and below -125.1‱ can be considered as authentic fungal chitosan without needing to analyse other parameters. If the δ13C value falls between -25.1‱ and -24.9‱, it is necessary to proceed further with the evaluation of the parameter δ15N, which must be above +2.7‱. Samples having δ18O values lower than +25.3‱ can be considered as authentic fungal chitosan. The combination of maximum degradation temperatures (obtained using TGA) and peak areas of Amide I and NH2/Amide II (obtained using FTIR) also allows the discrimination between the two origins of the polysaccharide. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) based on TGA, FTIR and SIR data successfully distributed the tested samples into informative clusters. Therefore, we present the technologies described as part of a robust analytical strategy for the correct identification of chitosan samples from crustaceans or fungi.

Study of the Production of Poly(Hydroxybutyrate-co-Hydroxyhexanoate) and Poly(Hydroxybutyrate-co-Hydroxyvalerate-co-Hydroxyhexanoate) in Rhodospirillum rubrum.

Poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] and poly(hydroxybutyrate-co-hydroxyvalerate-co-hydroxyhexanoate) [P(HB-co-HV-co-HHx)] demonstrate interesting mechanical and thermal properties as well as excellent biocompatibility, making them suitable for multiple applications and notably biomedical purposes. The production of such polymers was described in Rhodospirillum rubrum, a purple nonsulfur bacteria in a nutrient-lacking environment where the HHx synthesis is triggered by the presence of hexanoate in the medium. However, the production of P(HB-co-HHx) under nutrient-balanced growth conditions in R. rubrum has not been described so far, and the assimilation of hexanoate is poorly documented. In this study, we used proteomic analysis and a mutant fitness assay to demonstrate that hexanoate assimilation involve ß-oxidation and the ethylmalonyl-coenzyme A (CoA) (EMC) and methylbutanoyl-CoA (MBC) pathways, both being anaplerotic pathways already described in R. rubrum. Polyhydroxyalkanoate (PHA) production is likely to involve the de novo fatty acid synthesis pathway. Concerning the polymer composition, HB is the main component of the polymer, probably as acetyl-CoA and butyryl-CoA are intermediates of hexanoate assimilation pathways. When no essential nutrient is lacking in the medium, the synthesis of PHA seems to help maintain the redox balance of the cell. In this framework, we showed that the fixation of CO2 is required to sustain the growth. An increase in the proportion of HHx in the polymer was observed when redox stress was engendered in the cell under bicarbonate-limiting growth conditions. The addition of isoleucine or valerate in the medium also increased the HHx content of the polymer and allowed the production of a terpolymer of P(HB-co-HV-co-HHx). IMPORTANCE 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. In a nutrient-lacking environment, R. rubrum is known to synthesize such polymers when hexanoate is used as the carbon source. However, their production in R. rubrum in non-nutrient-lacking growth conditions has not been described so far, and the assimilation of hexanoate is poorly documented. As the carbon source and its assimilation directly impact the polymer composition, we studied under non-nutrient-lacking growth conditions the assimilation pathway of hexanoate and PHA production in R. rubrum. Proteomic analysis and mutant fitness assays allowed us to explain PHA production and composition. An increase in HHx content of the polymer and production of P(HB-co-HV-co-HHx) was possible using the knowledge gained on metabolism under hexanoate growth conditions.

Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters.

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.

Growth and biofilm formation of Cupriavidus metallidurans CH34 on different metallic and polymeric materials used in spaceflight applications.

Bacteria biofilm formation and its complications are of special concern in isolated structures, such as offshore stations, manned submarines and space habitats, as maintenance and technical support are poorly accessible due to costs and/or logistical challenges. In addition, considering that future exploration missions are planned to adventure farther and longer in space, unlocking biofilm formation mechanisms and developing new antifouling solutions are key goals in order to ensure spacecraft's efficiency, crew's safety and mission success. In this work, we explored the interactions between Cupriavidus metallidurans, a prevalently identified contaminant onboard the International Space Station, and aerospace grade materials such as the titanium alloy TiAl6V4, the stainless steel AISI 316 (SS316) and Polytetrafluoroethylene (PTFE) or Teflon. Borosilicate glass was used as a control and all surfaces were investigated at two different pH values (5.0 and 7.0). Biofilms were almost absent on stainless steel and the titanium alloy contrary to Teflon and glass that were covered by an extensive biofilm formed via monolayers of scattered matrix-free cells and complex multilayered clusters or communities. Filamentous extracellular DNA structures were observed specifically in the complex multilayered clusters adherent to Teflon, indicating that the employed attachment machinery might depend on the physicochemical characteristics of the surface.

Targeted Analysis of HSP70 Isoforms in Human Spermatozoa in the Context of Capacitation and Motility.

HSP70s constitute a family of chaperones, some isoforms of which appear to play a role in sperm function. Notably, global proteomic studies analyzing proteins deregulated in asthenozoospermia, a main cause of male infertility characterized by low sperm motility, showed the dysregulation of some HSP70 isoforms. However, to date, no clear trend has been established since the variations in the abundance of HSP70 isoforms differed between studies. The HSPA2 isoform has been reported to play a key role in fertilization, but its dysregulation and possible relocation during capacitation, a maturation process making the spermatozoon capable of fertilizing an oocyte, is debated in the literature. The aim of the present study was to investigate the fate of all sperm HSP70 isoforms during capacitation and in relation to sperm motility. Using Multiple-Reaction Monitoring (MRM) mass spectrometry, we showed that the relative abundance of all detected isoforms was stable between non-capacitated and capacitated spermatozoa. Immunofluorescence using two different antibodies also demonstrated the stability of HSP70 isoform localization during capacitation. We also investigated spermatozoa purified from 20 sperm samples displaying various levels of total and progressive sperm motility. We showed that the abundance of HSP70 isoforms is not correlated to sperm total or progressive motility.

PfHRP2 detection using plasmonic optrodes: performance analysis.

BACKGROUND: Early malaria diagnosis and its profiling require the development of new sensing platforms enabling rapid and early analysis of parasites in blood or saliva, aside the widespread rapid diagnostic tests (RDTs). METHODS: This study shows the performance of a cost-effective optical fiber-based solution to target the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Unclad multimode optical fiber probes are coated with a thin gold film to excite Surface Plasmon Resonance (SPR) yielding high sensitivity to bio-interactions between targets and bioreceptors grafted on the metal surface. RESULTS: Their performances are presented in laboratory conditions using PBS spiked with growing concentrations of purified target proteins and within in vitro cultures. Two probe configurations are studied through label-free detection and amplification using secondary antibodies to show the possibility to lower the intrisic limit of detection. CONCLUSIONS: As malaria hits millions of people worldwide, the improvement and multiplexing of this optical fiber technique can be of great interest, especially for a future purpose of using multiple receptors on the fiber surface or several coated-nanoparticles as amplifiers.

Photoheterotrophic Assimilation of Valerate and Associated Polyhydroxyalkanoate Production by Rhodospirillum 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.

New perspectives on butyrate assimilation in Rhodospirillum rubrum S1H under photoheterotrophic conditions.

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.

Selective detection of cadmium ions using plasmonic optical fiber gratings functionalized with bacteria.

Environmental monitoring and potable water control are key applications where optical fiber sensing solutions can outperform other technologies. In this work, we report a highly sensitive plasmonic fiber-optic probe that has been developed to determine the concentration of cadmium ions (Cd2+) in solution. This original sensor was fabricated by immobilizing the Acinetobacter sp. around gold-coated tilted fiber Bragg gratings (TFBGs). To this aim, the immobilization conditions of bacteria on the gold-coated optical fiber surface were first experimentally determined. Then, the coated sensors were tested in vitro. The relative intensity of the sensor response experienced a change of 1.1 dB for a Cd2+ concentration increase from 0.1 to 1000 ppb. According to our test procedure, we estimate the experimental limit of detection to be close to 1 ppb. Cadmium ions strongly bind to the sensing surface, so the sensor exhibits a much higher sensitivity to Cd2+ than to other heavy metal ions such as Pb2+, Zn2+ and CrO42- found in contaminated water, which ensures a good selectivity.

Multimodal plasmonic optical fiber grating aptasensor.

Tilted fiber Bragg gratings (TFBGs) are now a well-established technology in the scientific literature, bringing numerous advantages, especially for biodetection. Significant sensitivity improvements are achieved by exciting plasmon waves on their metal-coated surface. Nowadays, a large part of advances in this topic relies on new strategies aimed at providing sensitivity enhancements. In this work, TFBGs are produced in both single-mode and multimode telecommunication-grade optical fibers, and their relative performances are evaluated for refractometry and biosensing purposes. TFBGs are biofunctionalized with aptamers oriented against HER2 (Human Epidermal Growth Factor Receptor-2), a relevant protein biomarker for breast cancer diagnosis. In vitro assays confirm that the sensing performances of TFBGs in multimode fiber are higher or identical to those of their counterparts in single-mode fiber, respectively, when bulk refractometry or surface biosensing is considered. These observations are confirmed by numerical simulations. TFBGs in multimode fiber bring valuable practical assets, featuring a reduced spectral bandwidth for improved multiplexing possibilities enabling the detection of several biomarkers.

Isoniazid Bactericidal Activity Involves Electron Transport Chain Perturbation.

Accumulating evidence suggests that the bactericidal activity of some antibiotics may not be directly initiated by target inhibition. The activity of isoniazid (INH), a key first-line bactericidal antituberculosis drug currently known to inhibit mycolic acid synthesis, becomes extremely poor under stress conditions, such as hypoxia and starvation. This suggests that the target inhibition may not fully explain the bactericidal activity of the drug. Here, we report that INH rapidly increased Mycobacterium bovis BCG cellular ATP levels and enhanced oxygen consumption. The INH-triggered ATP increase and bactericidal activity were strongly compromised by Q203 and bedaquiline, which inhibit mycobacterial cytochrome bc1 and FoF1 ATP synthase, respectively. Moreover, the antioxidant N-acetylcysteine (NAC) but not 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) abrogated the INH-triggered ATP increase and killing. These results reveal a link between the energetic (ATP) perturbation and INH's killing. Furthermore, the INH-induced energetic perturbation and killing were also abrogated by chemical inhibition of NADH dehydrogenases (NDHs) and succinate dehydrogenases (SDHs), linking INH's bactericidal activity further to the electron transport chain (ETC) perturbation. This notion was also supported by the observation that INH dissipated mycobacterial membrane potential. Importantly, inhibition of cytochrome bd oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome bd oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome bc1, and FoF1 ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome bd oxidase in protection against this drug under stress conditions.

Aminobacter sp. MSH1 Mineralizes the Groundwater Micropollutant 2,6-Dichlorobenzamide through a Unique Chlorobenzoate Catabolic Pathway.

2,6-Dichlorobenzamide (BAM) is a major groundwater micropollutant posing problems for drinking water treatment plants (DWTPs) that depend on groundwater intake. Aminobacter sp. MSH1 uses BAM as the sole source of carbon, nitrogen, and energy and is considered a prime biocatalyst for groundwater bioremediation in DWTPs. Its use in bioremediation requires knowledge of its BAM-catabolic pathway, which is currently restricted to the amidase BbdA converting BAM into 2,6-dichlorobenzoic acid (2,6-DCBA) and the monooxygenase BbdD transforming 2,6-DCBA into 2,6-dichloro-3-hydroxybenzoic acid. Here, we show that the 2,6-DCBA catabolic pathway is unique and differs substantially from catabolism of other chlorobenzoates. BbdD catalyzes a second hydroxylation, forming 2,6-dichloro-3,5-dihydroxybenzoic acid. Subsequently, glutathione-dependent dehalogenases (BbdI and BbdE) catalyze the thiolytic removal of the first chlorine. The remaining chlorine is then removed hydrolytically by a dehalogenase of the α/ß hydrolase superfamily (BbdC). BbdC is the first enzyme in that superfamily associated with dehalogenation of chlorinated aromatics and appears to represent a new subtype within the α/ß hydrolase dehalogenases. The activity of BbdC yields a unique trihydroxylated aromatic intermediate for ring cleavage that is performed by an extradiol dioxygenase (BbdF) producing 2,4,6-trioxoheptanedioic acid, which is likely converted to Krebs cycle intermediates by BbdG.

Optical Fiber Gratings Immunoassays.

Optical fibers are of growing interest for biosensing, especially for point-of-care and biomedical assays. Their intrinsic properties bestow them sought-after assets for the detection of low concentrations of analytes. Tilted fiber Bragg gratings (TFBGs) photo-inscribed in the core of telecommunication-grade optical fibers are known to be highly-sensitive refractometers. In this work, we present different strategies to use them for label-free immunoassays. Bare, gold-sputtered, gold-electroless-plated (ELP) and hybrid configurations are biofunctionalized with antibodies, aiming at the detection of cancer biomarkers. We discuss the relative performances of the tested configurations and show that each leads to singular key features, which therefore drives their selection as a function of the target application. The most sensitive configuration presents a limit of detection of 10-12 g/mL in laboratory settings and was successfully used ex vivo in freshly resected lung tissues.

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro, and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs.IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the major findings were: (i) the FL strain encodes 16 virion transmembrane proteins; (ii) eight of these proteins are essential for viral growth in vitro; (iii) seven of the nonessential proteins affect viral growth in vitro, and two affect virulence in vivo; and (iv) a mutant lacking ORF25 is highly attenuated but induces moderate immune protection. This study represents a major breakthrough in understanding the biology of CyHV-3 and will contribute to the development of prophylactic methods. It also provides a firm basis for the further research on alloherpesvirus virion transmembrane proteins.

Genetic Plasticity and Ethylmalonyl Coenzyme A Pathway during Acetate Assimilation in Rhodospirillum rubrum S1H under Photoheterotrophic Conditions.

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.