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Nucleic Acids Research Dec 2023The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad...
The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications. .
Topics: DNA, Single-Stranded; Gene Editing; Metabolic Engineering; Pseudomonas; Pseudomonas putida
PubMed: 37941137
DOI: 10.1093/nar/gkad1024 -
Scientific Reports Nov 2023Wastewater malodour is the proverbial 'elephant in the room' notwithstanding its severe implications on sanitation, health, and hygiene. The predominant malodorous...
Wastewater malodour is the proverbial 'elephant in the room' notwithstanding its severe implications on sanitation, health, and hygiene. The predominant malodorous compounds associated with wastewater treatment plants and toilets are volatile organic compounds, such as hydrogen sulphide, ammonia, methanethiol, and organic acids. Among them, methanethiol warrants more attention owing to its relatively low olfactory threshold and associated cytotoxicity. This requires an efficient odour-abatement method since conventional techniques are either cost-prohibitive or leave recalcitrant byproducts. Bacteriophage-based methodology holds promise, and the described work explores the potential. In this study, a non-lysogenous Pseudomonas putida strain is used as a model organism that produces methanethiol in the presence of methionine. Two double-stranded DNA phages of genome sizes > 10 Kb were isolated from sewage. ɸPh_PP01 and ɸPh_PP02 were stable at suboptimal pH, temperature, and at 10% chloroform. Moreover, they showed adsorption efficiencies of 53% and 89% in 12 min and burst sizes of 507 ± 187 and 105 ± 7 virions per cell, respectively. In augmented synthetic wastewater, ɸPh_PP01 and ɸPh_PP02 reduced methanethiol production by 52% and 47%, respectively, with the concomitant reduction in P. putida by 3 logs in 6 h. On extension of the study in P. putida spiked-sewage sample, maximum reduction in methanethiol production was achieved in 3 h, with 49% and 48% for ɸPh_PP01 and ɸPh_PP02, respectively. But at 6 h, efficiency reduced to 36% with both the phages. The study clearly demonstrates the potential of phages as biocontrol agents in the reduction of malodour in wastewater.
Topics: Bacteriophages; Pseudomonas putida; Wastewater; Sewage; Sulfhydryl Compounds
PubMed: 37945592
DOI: 10.1038/s41598-023-46938-8 -
Environmental Microbiology Dec 2023In sediments, the bioavailability and toxicity of Ni are strongly influenced by its sorption to manganese (Mn) oxides, which largely originate from the redox metabolism...
In sediments, the bioavailability and toxicity of Ni are strongly influenced by its sorption to manganese (Mn) oxides, which largely originate from the redox metabolism of microbes. However, microbes are concurrently susceptible to the toxic effects of Ni, which establishes complex interactions between toxicity and redox processes. This study measured the effect of Ni on growth, pellicle biofilm formation and oxidation of the Mn-oxidizing bacteria Pseudomonas putida GB-1. In liquid media, Ni exposure decreased the intrinsic growth rate but allowed growth to the stationary phase in all intermediate treatments. Manganese oxidation was 67% less than control for bacteria exposed to 5 μM Ni and completely ceased in all treatments above 50 μM. Pellicle biofilm development decreased exponentially with Ni concentration (maximum 92% reduction) and was replaced by planktonic growth in higher Ni treatments. In solid media assays, growth was unaffected by Ni exposure, but Mn oxidation completely ceased in treatments above 10 μM of Ni. Our results show that sublethal Ni concentrations substantially alter Mn oxidation rates and pellicle biofilm development in P. putida GB-1, which has implications for toxic metal bioavailability to the entire benthic community and the environmental consequences of metal contamination.
Topics: Manganese; Pseudomonas putida; Nickel; Oxidation-Reduction
PubMed: 37875338
DOI: 10.1111/1462-2920.16529 -
ACS Synthetic Biology Jul 2023Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link...
Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria ( and , respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium . In , we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; Transposases; DNA Transposable Elements; Proteobacteria; Mutagenesis; Gene Editing; Bacteria; RNA; CRISPR-Cas Systems
PubMed: 37368499
DOI: 10.1021/acssynbio.3c00065 -
The Science of the Total Environment Dec 2023Currently, discharge regulations for wastewater treatment plants (WWTPs) are based on conventional parameters, but more is needed to ensure safe water reuse. In...
Currently, discharge regulations for wastewater treatment plants (WWTPs) are based on conventional parameters, but more is needed to ensure safe water reuse. In particular, emerging pollutants, as antimicrobials and antibiotic resistance genes (ARGs), are not considered. This research focuses on the fate of emerging biological contaminants during wastewater treatment in Mexico City. intI1 and the ARGs cphA-02, OXA-10 and sul1 were analyzed by qPCR; pathogenic bacteria species were characterized by high throughput sequencing of complete 16S rRNA gene, and fragments of SARS-CoV-2 were quantified by RT-qPCR. Conventional parameters (chemical oxygen demand and coliform bacteria) were also determined. Two sampling campaigns (rainy and dry seasons) were carried out in four municipal WWTPs in Mexico City, representing five biological treatment processes: conventional activated sludge, extended aeration activated sludge, membrane bioreactor, direct anaerobic digestion, and constructed wetland, followed by ultraviolet light or chlorine disinfection. In most cases, gene fragments of SARS-CoV-2 were eliminated below the detection limit of RT-qPCR. The abundance of intI1 positively correlated with the sul1, OXA-10, and cphA-02 abundances; intI1 and the ARGs here studied were partially removed in the WWTPs, and in most cases, the number of copies per second discarded in the sludge were higher those in the effluent. The treatment processes decreased the abundance of dominant bacterial groups in the raw wastewater, while enriching bacterial groups in the effluent and the biological sludge, with possible pollutant removal capabilities. Bacterial communities in the raw wastewater showed the predominance of the genus Arcobacter (from 62.4 to 86.0 %) containing potentially pathogenic species. Additionally, DNA of some species persisted after the treatment processes: A. johnsonii, A. junii, A. caviae, A. hydrophila, A. veronii, A. butzleri, A. cryaerophilus, Chryseobacterium indologenes, Hafnia paralvei, M. osloensis, Pseudomonas putida and Vibrio cholerae, which deserves special attention in future regulation for safe water reuse.
PubMed: 37574072
DOI: 10.1016/j.scitotenv.2023.165984 -
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 2024Pseudomonas putida KT2440 is a robust, aromatic catabolic bacterium that has been widely engineered to convert bio-based and waste-based feedstocks to target products....
Pseudomonas putida KT2440 is a robust, aromatic catabolic bacterium that has been widely engineered to convert bio-based and waste-based feedstocks to target products. Towards industrial domestication of P. putida KT2440, rational genome reduction has been previously conducted, resulting in P. putida strain EM42, which exhibited characteristics that could be advantageous for production strains. Here, we compared P. putida KT2440- and EM42-derived strains for cis,cis-muconic acid production from an aromatic compound, p-coumarate, and in separate strains, from glucose. To our surprise, the EM42-derived strains did not outperform the KT2440-derived strains in muconate production from either substrate. In bioreactor cultivations, KT2440- and EM42-derived strains produced muconate from p-coumarate at titers of 45 g/L and 37 g/L, respectively, and from glucose at 20 g/L and 13 g/L, respectively. To provide additional insights about the differences in the parent strains, we analyzed growth profiles of KT2440 and EM42 on aromatic compounds as the sole carbon and energy sources. In general, the EM42 strain exhibited reduced growth rates but shorter growth lags than KT2440. We also observed that EM42-derived strains resulted in higher growth rates on glucose compared to KT2440-derived strains, but only at the lowest glucose concentrations tested. Transcriptomics revealed that genome reduction in EM42 had global effects on transcript levels and showed that the EM42-derived strains that produce muconate from glucose exhibit reduced modulation of gene expression in response to changes in glucose concentrations. Overall, our results highlight that additional studies are warranted to understand the effects of genome reduction on microbial metabolism and physiology, especially when intended for use in production strains.
Topics: Pseudomonas putida; Glucose; Bioreactors
PubMed: 38000549
DOI: 10.1016/j.ymben.2023.11.004 -
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 -
New Biotechnology Dec 2023Phenolic acids including hydroxybenzoic and hydroxycinnamic acids are secondary plant and fungal metabolites involved in many physiological processes offering health and...
Phenolic acids including hydroxybenzoic and hydroxycinnamic acids are secondary plant and fungal metabolites involved in many physiological processes offering health and dietary benefits. They are often utilised as precursors for production of value-added compounds. The limited availability of synthetic biology tools, such as whole-cell biosensors suitable for monitoring the dynamics of phenolic acids intracellularly and extracellularly, hinders the capabilities to develop high-throughput screens to study their metabolism and forward engineering. Here, by applying a multi-genome approach, we have identified phenolic acid-inducible gene expression systems composed of transcription factor-inducible promoter pairs responding to eleven different phenolic acids. Subsequently, they were used for the development of whole-cell biosensors based on model bacterial hosts, such as Escherichia coli, Cupriavidus necator and Pseudomonas putida. The dynamics and range of the biosensors were evaluated by establishing their response and sensitivity landscapes. The specificity and previously uncharacterised interactions between transcription factor and its effector(s) were identified by a screen of twenty major phenolic acids. To exemplify applicability, we utilise a protocatechuic acid-biosensor to identify enzymes with enhanced activity for conversion of p-hydroxybenzoate to protocatechuate. Transcription factor-based biosensors developed in this study will advance the analytics of phenolic acids and expedite research into their metabolism.
Topics: Transcription Factors; Bacteria; Promoter Regions, Genetic; Escherichia coli; Biosensing Techniques
PubMed: 37714511
DOI: 10.1016/j.nbt.2023.09.004 -
AMB Express Sep 2023The phenylurea herbicides are persistent in soil and water, necessitating the creation of methods for removing them from the environment. This study aimed to examine the...
The phenylurea herbicides are persistent in soil and water, necessitating the creation of methods for removing them from the environment. This study aimed to examine the soil microbial diversity, searching for local bacterial isolates able to efficiently degrade the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea (IPU). The best isolates able to effectively degrade IPU were selected, characterized, and identified as Pseudomonas putida and Acinetobacter johnsonii. The catechol 1, 2-dioxygenase enzyme's catA gene was amplified, cloned, and expressed in E. coli M15. The Expressed E. coli showed high degradation efficiency (44.80%) as analyzed by HPLC after 15 days of inoculation in comparison to P. putida (21.60%). The expression of the catA gene in P. putida and expressed E. coli was measured using quantitative polymerase chain reaction (qPCR). The results displayed a significant increase in the mRNA levels of the catA gene by increasing the incubation time with IPU. Hydrophilic interaction chromatography (HILIC) mass spectrometry analysis revealed that three intermediate metabolites, 1-(4-isopropylphenyl)-3-methylurea (MDIPU), 4-Isopropylaniline (4-IA) and 1-(4-isopropylphenyl) urea (DDIPU) were generated by both P. putida and expressed E. coli. In addition, IPU-induced catA activity was detected in both P. putida and expressed E. coli. The supernatant of both P. putida and expressed E. coli had a significant influence on weed growth. The study clearly exhibited that P. putida and expressed E. coli were capable of metabolizing IPU influentially and thus could be utilized for bioremediation and biodegradation technology development.
PubMed: 37751014
DOI: 10.1186/s13568-023-01609-9