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Nature Communications Jan 2024Efforts to produce aromatic monomers through catalytic lignin depolymerization have historically focused on aryl-ether bond cleavage. A large fraction of aromatic...
Efforts to produce aromatic monomers through catalytic lignin depolymerization have historically focused on aryl-ether bond cleavage. A large fraction of aromatic monomers in lignin, however, are linked by various carbon-carbon (C-C) bonds that are more challenging to cleave and limit the yields of aromatic monomers from lignin depolymerization. Here, we report a catalytic autoxidation method to cleave C-C bonds in lignin-derived dimers and oligomers from pine and poplar. The method uses manganese and zirconium salts as catalysts in acetic acid and produces aromatic carboxylic acids as primary products. The mixtures of the oxygenated monomers are efficiently converted to cis,cis-muconic acid in an engineered strain of Pseudomonas putida KT2440 that conducts aromatic O-demethylation reactions at the 4-position. This work demonstrates that autoxidation of lignin with Mn and Zr offers a catalytic strategy to increase the yield of valuable aromatic monomers from lignin.
PubMed: 38286984
DOI: 10.1038/s41467-024-45038-z -
Applied Microbiology and Biotechnology Jan 2024The resistance of biofilms to antibiotics is a key factor that makes bacterial infections unsusceptible to antimicrobial therapy. The results of classical tests of cell...
The resistance of biofilms to antibiotics is a key factor that makes bacterial infections unsusceptible to antimicrobial therapy. The results of classical tests of cell sensitivity to antibiotics cannot be used to predict therapeutic success in infections associated with biofilm formation. We describe a simple and rapid method for the real-time evaluation of bacterial biofilm sensitivity to antibiotics, with Pseudomonas putida and ampicillin as examples. The method uses an electric biosensor to detect the difference between changes in the biofilm electric polarizability, thereby evaluating antibiotic sensitivity. The electric signals showed that P. putida biofilms were susceptible to ampicillin and that at high antibiotic concentrations, the biofilms differed markedly in their susceptibility (dose-dependent effect). The sensor also detected differences between biofilms before and after ampicillin treatment. The electric-signal changes enabled us to describe the physical picture of the processes occurring in bacterial biofilms in the presence of ampicillin. The approach used in this study is promising for evaluating the activity of various compounds against biofilms, because it permits a conclusion about the antibiotic sensitivity of biofilm bacteria to be made in real time and in a short period (analysis time, not longer than 20 min). An added strong point is that analysis can be done directly in liquid, without preliminary sample preparation. KEY POINTS: • Sensor system to analyze biofilm antimicrobial susceptibility is described. • The signal change depended on the ampicillin concentration (dose-dependent effect). • The sensor allows real-time determination of the antibiofilm effect of ampicillin.
Topics: Ampicillin; Anti-Bacterial Agents; Biofilms; Electricity; Pseudomonas putida
PubMed: 38265501
DOI: 10.1007/s00253-023-12831-7 -
Frontiers in Microbiology 2023Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of...
Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of and in an artificial bacterial consortium comprising , , , and . Moreover, the growth inhibition of promoted the growth of and inhibited the growth of . These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.
PubMed: 38260881
DOI: 10.3389/fmicb.2023.1321428 -
The Journal of Biological Chemistry Feb 2024Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second...
Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle. The activity of these enzymes can be modulated by stimuli-sensing domains such as Per-ARNT-Sim (PAS). In Pseudomonas aeruginosa, more than 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual proteins possessing both canonical DGC and PDE motifs, that is, GGDEF and EAL, respectively. It was reported that deletion of the EAL/GGDEF dual enzyme PA0285, one of five c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms. PA0285 is anchored in the membrane and carries two PAS domains. Here, we confirm that its role is conserved in various P. aeruginosa strains and in Pseudomonas putida. Deletion of PA0285 impacts the early stage of colonization, and RNA-seq analysis suggests that expression of cupA fimbrial genes is involved. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds GTP and c-di-GMP, respectively, but only exhibits PDE activity in vitro. However, both GGDEF and EAL domains are important for PA0285 PDE activity in vivo. Complementation of the PA0285 mutant strain with a copy of the gene encoding the C-terminal GGDEF/EAL portion in trans was not as effective as complementation with the full-length gene. This suggests the N-terminal transmembrane and PAS domains influence the PDE activity in vivo, through modulating the protein conformation.
Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pseudomonas
PubMed: 38237678
DOI: 10.1016/j.jbc.2024.105659 -
BMC Genomics Jan 2024Pseudomonas putida S12 is a gram-negative bacterium renowned for its high tolerance to organic solvents and metabolic versatility, making it attractive for various...
BACKGROUND
Pseudomonas putida S12 is a gram-negative bacterium renowned for its high tolerance to organic solvents and metabolic versatility, making it attractive for various applications, including bioremediation and the production of aromatic compounds, bioplastics, biofuels, and value-added compounds. However, a metabolic model of S12 has yet to be developed.
RESULTS
In this study, we present a comprehensive and highly curated genome-scale metabolic network model of S12 (iSH1474), containing 1,474 genes, 1,436 unique metabolites, and 2,938 metabolic reactions. The model was constructed by leveraging existing metabolic models and conducting comparative analyses of genomes and phenomes. Approximately 2,000 different phenotypes were measured for S12 and its closely related KT2440 strain under various nutritional and environmental conditions. These phenotypic data, combined with the reported experimental data, were used to refine and validate the reconstruction. Model predictions quantitatively agreed well with in vivo flux measurements and the batch cultivation of S12, which demonstrated that iSH1474 accurately represents the metabolic capabilities of S12. Furthermore, the model was simulated to investigate the maximum theoretical metabolic capacity of S12 growing on toxic organic solvents.
CONCLUSIONS
iSH1474 represents a significant advancement in our understanding of the cellular metabolism of P. putida S12. The combined results of metabolic simulation and comparative genome and phenome analyses identified the genetic and metabolic determinants of the characteristic phenotypes of S12. This study could accelerate the development of this versatile organism as an efficient cell factory for various biotechnological applications.
Topics: Solvents; Pseudomonas putida; Genome, Bacterial; Genomics; Metabolic Networks and Pathways
PubMed: 38229031
DOI: 10.1186/s12864-023-09940-y -
EFSA Journal. European Food Safety... Jan 2024The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. The...
Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 19: Suitability of taxonomic units notified to EFSA until September 2023.
The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. The QPS approach is based on an assessment of published data for each taxonomic unit (TU), with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a TU are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 71 microorganisms notified to EFSA between April and September 2023 (30 as feed additives, 22 as food enzymes or additives, 7 as novel foods and 12 from plant protection products [PPP]), 61 were not evaluated because: 26 were filamentous fungi, 1 was , 5 were , 1 was a bacteriophage (all excluded from the QPS evaluation) and 28 were TUs that already have a QPS status. The other 10 notifications belonged to 9 TUs which were evaluated for a possible QPS status: and did not get the QPS recommendation due to the limited body of knowledge about their occurrence in the food and/or feed chains and also due to its ability to generate biologically active compounds with antimicrobial activity; and due to safety concerns. is excluded from future QPS evaluations. is recommended for QPS status with the qualification 'for production purposes only'; is recommended for QPS status with the qualification 'absence of genetic determinants for toxigenic activity'; has been added as a synonym of . The Panel clarifies the extension of the QPS status for genetically modified strains.
PubMed: 38213415
DOI: 10.2903/j.efsa.2024.8517 -
Scientific Reports Jan 2024There has recently been an explosion of studies implicating the human microbiome in playing a critical role in many disease and wellness states. The etiology of abnormal... (Observational Study)
Observational Study
There has recently been an explosion of studies implicating the human microbiome in playing a critical role in many disease and wellness states. The etiology of abnormal semen analysis (SA) parameters is not identified in 30% of cases; investigations involving the semen microbiome may bridge this gap. Here, we explore the relationship between the semen microbiome and alterations of sperm parameters. We recruited men presenting for fertility evaluation or vasectomy consultation with proven biological paternity. SA and next generation sequencing was performed. Differential abundance testing using Analysis of composition of Microbiota with Bias Correction (ANCOM-BC) was performed along with canonical correlational analysis for microbial community profiling. Men with abnormal (N = 27) sperm motility showed a higher abundance of Lactobacillus iners compared to those with normal (N = 46) sperm motility (mean proportion 9.4% versus 2.6%, p = 0.046). This relationship persisted on canonical correlational analysis (r = 0.392, p = 0.011). Men with abnormal sperm concentration (N = 20) showed a higher abundance of Pseudomonas stutzeri (2.1% versus 1.0%, p = 0.024) and Pseudomonas fluorescens (0.9% versus 0.7%, p = 0.010), but a lower abundance of Pseudomonas putida (0.5% versus 0.8%, p = 0.020), compared to those with normal sperm concentration (N = 53). Major limitations are related to study design (cross-sectional, observational). Our results suggest that a small group of microorganisms may play a critical role in observed perturbations of SA parameters. Some of these microbes, most notably Lactobacillus iners, have been described extensively within other, fertility-related, contexts, whereas for others, this is the first report where they have potentially been implicated. Advances in our understanding of the semen microbiome may contribute to potentially new therapeutic avenues for correcting impairments in sperm parameters and improving male fertility.
Topics: Humans; Male; Cross-Sectional Studies; Fertility; Infertility, Male; Lactobacillus; Semen; Semen Analysis; Sperm Count; Sperm Motility; Spermatozoa
PubMed: 38212576
DOI: 10.1038/s41598-024-51686-4 -
Microbial Biotechnology Jan 2024Microbial synthetic consortia are a promising alternative to classical monoculture for biotechnological applications and fermentative processes. Their versatile use...
Establishment of synthetic microbial consortia with Corynebacterium glutamicum and Pseudomonas putida: Design, construction, and application to production of γ-glutamylisopropylamide and l-theanine.
Microbial synthetic consortia are a promising alternative to classical monoculture for biotechnological applications and fermentative processes. Their versatile use offers advantages in the degradation of complex substrates, the allocation of the metabolic burden between individual partners, or the division of labour in energy utilisation, substrate supply or product formation. Here, stable synthetic consortia between the two industrially relevant production hosts, Pseudomonas putida KT2440 and Corynebacterium glutamicum ATCC13032, were established for the first time. By applying arginine auxotrophy/overproduction and/or formamidase-based utilisation of the rare nitrogen source formamide, different types of interaction were realised, such as commensal relationships (+/0 and 0/+) and mutualistic cross-feeding (+/+). These consortia did not only show stable growth but could also be used for fermentative production of the γ-glutamylated amines theanine and γ-glutamyl-isopropylamide (GIPA). The consortia produced up to 2.8 g L of GIPA and up to 2.6 g L of theanine, a taste-enhancing constituent of green tea leaves. Thus, the advantageous approach of using synthetic microbial consortia for fermentative production of value-added compounds was successfully demonstrated.
Topics: Corynebacterium glutamicum; Pseudomonas putida; Microbial Consortia; Metabolic Engineering; Glutamates
PubMed: 38206115
DOI: 10.1111/1751-7915.14400 -
Bioelectrochemistry (Amsterdam,... Jun 2024Pseudomonas aeruginosa phenazines contribute to survival under microaerobic and anaerobic conditions by extracellular electron discharge to regulate cellular redox...
Pseudomonas aeruginosa phenazines contribute to survival under microaerobic and anaerobic conditions by extracellular electron discharge to regulate cellular redox balances. This electron discharge is also attractive to be used for bioelectrochemical applications. However, elements of the respiratory pathways that interact with phenazines are not well understood. Five terminal oxidases are involved in the aerobic electron transport chain (ETC) of Pseudomonas putida and P. aeruginosa. The latter bacterium also includes four reductases that allow for denitrification. Here, we explored if phenazine-1-carboxylic acid interacts with those elements to enhance anodic electron discharge and drive bacterial growth in oxygen-limited conditions. Bioelectrochemical evaluations of terminal oxidase-deficient mutants of both Pseudomonas strains and P. aeruginosa with stimulated denitrification pathways indicated no direct beneficial interaction of phenazines with ETC elements for extracellular electron discharge. However, the single usage of the Cbb3-2 oxidase increased phenazine production, electron discharge, and cell growth. Assays with purified periplasmic cytochromes NirM and NirS indicated that pyocyanin acts as their electron donor. We conclude that phenazines play an important role in electron transfer to, between, and from terminal oxidases under oxygen-limiting conditions and their modulation might enhance EET. However, the phenazine-anode interaction cannot replace oxygen respiration to deliver energy for biomass formation.
Topics: Electron Transport; Pseudomonas aeruginosa; Pseudomonas putida; Electrons; Phenazines; Oxygen
PubMed: 38181591
DOI: 10.1016/j.bioelechem.2023.108636 -
Nucleic Acids Research Feb 2024A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was...
A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) and an adenine base-editor (ABE) have been optimized for precise single-nucleotide modification of plasmid and genome targets. CBE comprises a cytidine deaminase conjugated to a Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting in C→T (or G→A) substitutions. Conversely, ABE consists of an adenine deaminase fused to SpnCas9 for A→G (or T→C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems and a novel protospacer adjacent motif (PAM)-relaxed SpnCas9 (SpRY) variant. Base-editing was validated in Pseudomonas putida and other Gram-negative bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins and metabolic (antibiotic-resistance) functions. The functional knockouts obtained by engineering STOP codons via CBE were reverted to the wild-type genotype using ABE. Additionally, a series of induction-responsive vectors was developed to facilitate the curing of the base-editing platform in a single cultivation step, simplifying complex strain engineering programs without relying on homologous recombination and yielding plasmid-free, modified bacterial cells.
Topics: Adenine; Cytidine; Gene Editing; Gram-Negative Bacteria; Nucleotides; Software
PubMed: 38180826
DOI: 10.1093/nar/gkad1236