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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 -
ACS Central Science Dec 2023Selective lignin depolymerization is a key step in lignin valorization to value-added products, and there are multiple catalytic methods to cleave labile aryl-ether...
Selective lignin depolymerization is a key step in lignin valorization to value-added products, and there are multiple catalytic methods to cleave labile aryl-ether bonds in lignin. However, the overall aromatic monomer yield is inherently limited by refractory carbon-carbon linkages, which are abundant in lignin and remain intact during most selective lignin deconstruction processes. In this work, we demonstrate that a Co/Mn/Br-based catalytic autoxidation method promotes carbon-carbon bond cleavage in acetylated lignin oligomers produced from reductive catalytic fractionation. The oxidation products include acetyl vanillic acid and acetyl vanillin, which are ideal substrates for bioconversion. Using an engineered strain of , we demonstrate the conversion of these aromatic monomers to ,-muconic acid. Overall, this study demonstrates that autoxidation enables higher yields of bioavailable aromatic monomers, exceeding the limits set by ether-bond cleavage alone.
PubMed: 38161372
DOI: 10.1021/acscentsci.3c00813 -
Metabolic Engineering Jan 2024Due to its tolerance properties, Pseudomonas has gained particular interest as host for oxidative upgrading of the toxic aldehyde 5-hydroxymethylfurfural (HMF) into...
Due to its tolerance properties, Pseudomonas has gained particular interest as host for oxidative upgrading of the toxic aldehyde 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA), a promising biobased alternative to terephthalate in polyesters. However, until now, the native enzymes responsible for aldehyde oxidation are unknown. Here, we report the identification of the primary HMF-converting enzymes of P. taiwanensis VLB120 and P. putida KT2440 by extended gene deletions. The key players in HMF oxidation are a molybdenum-dependent periplasmic oxidoreductase and a cytoplasmic dehydrogenase. Deletion of the corresponding genes almost completely abolished HMF oxidation, leading instead to aldehyde reduction. In this context, two HMF-reducing dehydrogenases were also revealed. These discoveries enabled enhancement of Pseudomonas' furanic aldehyde oxidation machinery by genomic overexpression of the respective genes. The resulting BOX strains (Boosted OXidation) represent superior hosts for biotechnological synthesis of FDCA from HMF. The increased oxidation rates provide greatly elevated HMF tolerance, thus tackling one of the major drawbacks of whole-cell catalysis with this aldehyde. Furthermore, the ROX (Reduced OXidation) and ROAR (Reduced Oxidation And Reduction) deletion mutants offer a solid foundation for future development of Pseudomonads as biotechnological chassis notably for scenarios where rapid HMF conversion is undesirable.
Topics: Pseudomonas; Furaldehyde; Furans; Dicarboxylic Acids
PubMed: 38154655
DOI: 10.1016/j.ymben.2023.12.010 -
Journal of Global Antimicrobial... Mar 2024Wild birds are vectors of antimicrobial resistance. Birds living in close contact with humans or other animals, like feral pigeons (Columba livia), might be especially...
OBJECTIVES
Wild birds are vectors of antimicrobial resistance. Birds living in close contact with humans or other animals, like feral pigeons (Columba livia), might be especially prone to acquire resistance genes such as those encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases.
METHODS
Cloacal samples (n = 206) of free-living feral pigeons (C. livia) were collected in Sousse and Monastir, Tunisia. Antimicrobial susceptibility profiles were determined by disc-diffusion, and resistant isolates were short- and long-read whole-genome sequenced. Sequence analysis was performed using tools of the Centre for Genomic Epidemiology, and Phylogenetic analysis was performed based on the core-genome MLST.
RESULTS
Fourteen (14/206, 6.8%) pigeons harboured Enterobacterales resistant to last-generations cephalosporins, of which 10 were CTX-M-15- or CTX-M-27-producers, while two (1.0%) carried a VIM-2-producing Pseudomonas putida. Positive pigeons lived on four different livestock farms. Three STs (ST206, ST5584, ST8149) were identified among E. coli, of which ST5584 and ST8149 were found in two different farms. Genetic diversity was also observed in Enterobacter cloacae and P. putida isolates. The bla genes were chromosomally encoded, while the bla genes were carried on highly similar IncF/F-:A-:B53 plasmids. The bla gene was located on a class 1 integron co-harbouring several resistance genes.
CONCLUSION
Pigeons living on livestock farms carried clinically important resistance genes encoding ESBLs and carbapenemases. Our results evidenced that both clonal (ST8149 and ST5584) and plasmidic (IncF/F-:A-:B53) transfers played a role in the spread of resistance genes among pigeons. Further studies are needed to identify factors favouring the transfer and persistence of resistance genes within the pigeon communities.
Topics: Animals; Humans; Columbidae; Escherichia coli; Pseudomonas putida; Multilocus Sequence Typing; Tunisia; Phylogeny; beta-Lactamases; Anti-Infective Agents
PubMed: 38145799
DOI: 10.1016/j.jgar.2023.12.013 -
International Journal of Molecular... Dec 2023The rhizosphere represents a center of complex and dynamic interactions between plants and microbes, resulting in various positive effects on plant growth and...
The rhizosphere represents a center of complex and dynamic interactions between plants and microbes, resulting in various positive effects on plant growth and development. However, less is known about the effects of indole-3-acetic acid (IAA) on aquatic plants. In this study, we report the characterization of four strains isolated from the rhizosphere of the common duckweed () with IAA-degradation and -utilization ability. Our results confirm previous reports on the negative effect of IAA on aquatic plants, contrary to the effect on terrestrial plants. A3-104/5 demonstrated particularly beneficial traits, as it exhibited not only IAA-degrading and -producing activity but also a positive effect on the doubling time of duckweeds in the presence of IAA, positive chemotaxis in the presence of IAA, increased tolerance to oxidative stress in the presence of IAA and increased biofilm formation related to IAA. Similarly, C31-106/3 significantly shortened the doubling time of duckweeds in the presence of IAA, while having a neutral effect in the absence of IAA. These traits are important in the context of plant-bacteria interactions and highlight the role of IAA as a common metabolite in these interactions, especially in aquatic environments where plants are facing unique challenges compared to their terrestrial counterparts. We conclude that IAA-degrading and -producing strains presented in this study might regulate IAA effects on aquatic plants and confer evolutionary benefits under adverse conditions (e.g., under oxidative stress, excess of IAA or nutrient scarcity).
Topics: Pseudomonas; Indoleacetic Acids; Araceae
PubMed: 38139036
DOI: 10.3390/ijms242417207 -
STAR Protocols Mar 2024Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of...
Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy. We include procedures for image analysis, plotting, and statistics. For complete details on the use and execution of this protocol, please refer to Arellano-Caicedo et al. (2023)..
Topics: Microfluidics; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Nutrients
PubMed: 38103191
DOI: 10.1016/j.xpro.2023.102784 -
Microbiology Resource Announcements Jan 2024This study presents a comprehensive analysis of the genomic sequence of isolate L2890hy, having a 6,733,472 bp chromosome size. This particular strain was obtained from...
This study presents a comprehensive analysis of the genomic sequence of isolate L2890hy, having a 6,733,472 bp chromosome size. This particular strain was obtained from a fecal sample of a patient suffering from acute diarrhea in Hangzhou, China, in 2020.
PubMed: 38099686
DOI: 10.1128/mra.00885-23 -
Applied and Environmental Microbiology Jan 2024Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic...
Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated mutants of the wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, and . Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.
Topics: Plasmids; Xanthomonas; Biofilms; Drug Resistance, Microbial; Gene Transfer, Horizontal; Conjugation, Genetic; Pseudomonas putida; Anti-Bacterial Agents
PubMed: 38095456
DOI: 10.1128/aem.01510-23 -
Scientific Reports Dec 2023Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is...
Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is the first known phage infecting P. putida S12, a strain increasingly used as a synthetic biology chassis. MiCath was isolated from garden soil under a tomato plant using P. putida S12 as a host and was also found to infect four other P. putida strains. MiCath has a ~ 61 kbp double-stranded DNA genome which encodes 97 predicted open reading frames (ORFs); functions could only be predicted for 48 ORFs using comparative genomics. Functions include structural phage proteins, other common phage proteins (e.g., terminase), a queuosine gene cassette, a cas4 exonuclease, and an endosialidase. Restriction digestion analysis suggests the queuosine gene cassette encodes a pathway capable of modification of guanine residues. When compared to other phage genomes, MiCath shares at most 74% nucleotide identity over 2% of the genome with any sequenced phage. Overall, MiCath is a novel phage with no close relatives, encoding many unique gene products.
Topics: Bacteriophages; Genome, Viral; Pseudomonas putida; DNA, Viral; Nucleoside Q; Sequence Analysis, DNA; Soil; Open Reading Frames; Phylogeny
PubMed: 38071193
DOI: 10.1038/s41598-023-48634-z