Search

UNLABELLED: There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules (functional modules), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida. Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE: This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids.

Here we report on light-triggered generation of local flow utilizing a bio-compatible non-ionic photo-active surfactant. The mechanism is based on diffusioosmotic phenomenon, where the gradient of relative concentration with respect to different chemical species near a surface leads to an osmotic pressure gradient driving liquid flow along the surface. The application of a photo-responsive surfactant allows for easy and reversible changes in concentration gradient by positioning a light source at the desired place. Along with the so-inscribed concentration gradient one can change reversible the direction and strength of the flow even in a closed system. The phenomenology of light-driven diffusioosmotic flow (LDDO) can be used in a rather flexible way: colloids can be gathered or dispersed and bio-compatibility extends the range of colloid types also to living microorganisms such as soil bacterium Pseudomonas putida. We show that DO flow can be considered a versatile method to set hydrodynamic conditions along the sample for investigating the motility of living cells. Further advantages of employing LDDO are the flexibility of flow generation in a reversible way and with spatiotemporal control, without the need to either change the channel geometry by loading a different device, or the periphery of pumps and connectors. [ABSTRACT FROM AUTHOR]

Bacteria indigenous to oil-contaminated water exhibited diverse metabolic capabilities in degrading various aromatic and monoaromatic hydrocarbons. Out of the 28 bacterial strains isolated from the wastewater, each was cultivated with at least one hydrocarbon, including kerosene, naphthalene, toluene, diesel, or aniline. Among these strains, Pseudomonas putida AD-128 emerged as one of the most effective polyaromatic hydrocarbon (PAH) degraders. Following a 6-day treatment period, strain P. putida AD-128 demonstrated proficiency in degrading various PAHs, including naphthalene, phenanthrene, and fluorine. After 6 days of incubation at 20 °C, the degradation of Naphthalene (NAP) notably increased. Gas Chromatography Mass Spectrometry analysis identified the degraded compounds, including pyruvic acid, salicylaldehyde, D-gluconic acid, and catechol. Optimal NAP degradation was observed at 20 °C and pH 6.0, with increased agitation speed correlating with enhanced bacterial growth and heightened degradation, particularly evident after 6 days at 20 °C. Peptone emerged as the most effective among the four nitrogen supplements (ammonium sulfate, potassium nitrate, beef extract, and peptone), significantly reducing residual naphthalene in the medium. The isolated indigenous bacterium, P. putida AD-128, exhibits robust capabilities in degrading PAHs under optimized conditions, making it a valuable asset for environmental management initiatives. [ABSTRACT FROM AUTHOR]

This study aimed to elucidate the potential of Pseudomonas species to degrade furfuryl alcohol (FA), a widely used furanic compound. FA-based resins and biopolymers have applications in various sectors, leading to the gradual discharge of FA into the environment through industrial wastewater. The objective was to find an eco-friendly solution for removing FA residues from industrial effluents, thereby eliminating the hazardous effects of FA. Pseudomonas species have emerged as potential bacterial genera for biodegradation of aromatic compounds; therefore, this study examined the effectiveness of Pseudomonas putida MTCC 1194 and Pseudomonas aeruginosa MTCC 1034 for FA degradation. Significant shifts in the fingerprint regions were observed using Fourier-transform infrared (FTIR) spectroscopy, providing evidence of successful FA degradation. A more than 50% reduction in FA content was observed by both strains, even at high concentrations (500 mgl− 1), as determined by spectrophotometric and high-performance liquid chromatography (HPLC) analysis. Liquid chromatography-mass spectrometry (LC-MS) identified the metabolite as 2-furoic acid (113 m/z). Both bacterial cultures followed substrate inhibition kinetics, and the specific growth rate best fit the Aiba and Haldane models for P. putida and the Haldane and Edwards models for P. aeruginosa. Furthermore, immobilized bacterial cells maintained approximately 50% FA degradation (300 mgl− 1) over five degradation cycles, highlighting their stability and reusability. The phytotoxicity bioassay demonstrated the non-hazardous nature of the metabolites formed after FA biodegradation. These findings underscore the potential of these strains for effective FA bioremediation, paving the way for their use in industrial effluent treatment. [ABSTRACT FROM AUTHOR]

In this study, the biosynthesis of polyhydroxyalkanoates (PHAs) was carried out using Pseudomonas putida and Pseudomonas aeruginosa. These PHAs were produced using reagent-grade glycerol and crude glycerol as the carbon sources. The objective was to compare the production of PHAs and to functionalize these polymers with silver nanoparticles to provide antibacterial properties for potential biomedical applications. The findings from the physical and chemical analyses confirmed the successful synthesis and extraction of PHAs, achieving comparable yields using both crude glycerol and reagent-grade glycerol as carbon sources across both strains. Approximately 16% higher PHAs production was obtained using Pseudomonas putida compared to Pseudomonas aeruginosa, and no significant difference was observed in the production rate of PHAs between the two carbon sources used, which means that crude glycerol could be utilized even though it has more impurities. Notably, PHAs functionalized with silver nanoparticles showed improved antibacterial effectiveness, especially those derived from reagent-grade glycerol and the Pseudomonas aeruginosa strain. [ABSTRACT FROM AUTHOR]

Aims This study aimed to evaluate the potential of endophytic plant growth-promoting bacterium (PGPB), Pseudomonas putida A32, to mitigate drought stress in two bell pepper genotypes, Amfora 19 and Amfora 26, and to assess the genotype-specific responses to bacterial treatment. Methods and results The isolate P. putida A32 was selected for its remarkable beneficial properties, exhibiting 13 out of 14 traits tested. Under drought conditions, Amfora 26 showed increased relative water content and decreased H2O2 and malondialdehyde following bacterial treatment, while Amfora 19 exhibited enhanced growth parameters but responded less to bacterial treatment regarding drought parameters. However, Amfora 19 displayed inherent drought tolerance mechanisms, as indicated by lower stress parameters compared to Amfora 26. Conclusions The study emphasizes the importance of genotype-specific responses to PGPB treatment and the mechanisms of drought tolerance in peppers. Pseudomonas putida A32 effectively mitigated drought stress in both genotypes, with differential responses influenced by plant genotype. Our study confirmed our initial hypothesis that Amfora 19, as a genotype tolerant to biotic stress, is also more tolerant to abiotic stress. Understanding these interactions is crucial for the development of customized strategies to improve plant productivity and tolerance to drought. [ABSTRACT FROM AUTHOR]

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as Escherichia coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208, and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus, Nocardia, and Gordonia. This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux toward the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species. [ABSTRACT FROM AUTHOR]

The present study was proposed with the idea to screen and isolate efficient low-density polyethylene (LDPE) degrading novel bacterial strains from the plastic-contaminated dumping site. The identification of the bacterial isolate was performed with the help of microbiological and molecular characterization approaches. The screening of the best isolate was performed based on its growth in Bushnell-Hass broth supplemented with LDPE sheets as the sole carbon source. The molecular characterization revealed that the isolate WD4 showed a similarity with the Pseudomonas aeruginosa species. A comparative analysis of Pseudomonas aeruginosa WD4 identified in the current study with Pseudomonas putida MTCC 2445 strain was performed. The present study demonstrated that the bacterial isolate showed 9.2% degradation of LDPE films while Pseudomonas putida revealed a 6.5% weight reduction after 100 days of incubation at 37 °C. The end products of the LDPE degradation were analysed using Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC–MS). The LDPE degradation products eluted include fatty acids such as octadecanoic, hexadecanoic acid, dodecanal, and octyl palmitoleate, alkanes, and some of the unknown compounds after 100 days of microbial treatment with the isolated strain. The detailed analysis of the by-products generated in the current study indicates their contribution to the biochemical pathway of LDPE degradation. The profound scope lies in the scalability of these bacterial strains at the industrial level to combat the LDPE waste and similar plastic garbage problems, globally. [ABSTRACT FROM AUTHOR]

Surfactants are amphiphilic molecules that are capable of mixing water and oil. Biosurfactants are eco-friendly, low-toxicity, and stable to a variety of environmental factors. Optimizing conditions for microorganisms to produce biosurfactants can lead to improved production suitable for scaling up. In this study, we compared heterologous expression levels of the luminescence system luxCDABE operon controlled by regulatable promoters araC-PBAD and its strong version araC-PBAD-SD in Escherichia coli K12, Pseudomonas aeruginosa PAO1, and P. putida KT2440. Real-time monitoring of luminescence levels in the three strains indicated that luxCDABE controlled by araC-PBAD-SD promoter with 0.2% arabinose supplementation in P. putida produced the highest level of luminescence. By using the araC-PBAD-SD promoter-controlled rhlAB expression in P. putida, we were able to produce mono-rhamnolipid at a level of 1.5 g L−1 when 0.02% arabinose was supplemented. With the same system to express olsB, lyso-ornithine lipid was produced at a level of 10 mg L−1 when 0.2% arabinose was supplemented. To our knowledge, this is the first report about optimizing conditions for lyso-ornithine lipid production at a level up to 10 mg L−1. Taken together, our results demonstrate that regulatable araC-PBAD-SD promoter in P. putida KT2440 is a useful system for heterologous production of biosurfactants. [ABSTRACT FROM AUTHOR]

In nature, methylmercury (MeHg) is primarily generated through microbial metabolism, and the ability of bacteria to methylate Hg(II) depends on both bacterial properties and environmental factors. It is widely known that, as a metabolic analog, molybdate can inhibit the sulfate reduction process and affect the growth and methylation of sulfate-reducing bacteria (SRB). However, after it enters the cell, molybdate can be involved in various intracellular metabolic pathways as a molybdenum cofactor; whether fluctuations in its concentration affect the growth and methylation of aerobic mercury methylating strains remains unknown. To address this gap, aerobic γ-Proteobacteria strains Raoultella terrigena TGRB3 (B3) and Pseudomonas putida TGRB4 (B4), as well as an obligate anaerobic δ-Proteobacteria strain of the SRB Desulfomicrobium escambiense CGMCC 1.3481 (DE), were used as experimental strains. The growth and methylation ability of each strain were analyzed under conditions of 500 ng⋅L-1 Hg(II), 0 and 21% of oxygen, and 0, 0.25, 0.50, and 1 mM of MoO4 2-. In addition, in order to explore the metabolic specificity of aerobic strains, transcriptomic data of the facultative mercury-methylated strain B3 were further analyzed in an aerobic mercuric environment. The results indicated that: (a) molybdate significantly inhibited the growth of DE, while B3 and B4 exhibited normal growth. (b) Under anaerobic conditions, in DE, the MeHg content decreased significantly with increasing molybdate concentration, while in B3, MeHg production was unaffected. Furthermore, under aerobic conditions, the MeHg productions of B3 and B4 were not influenced by the molybdate concentration. (c) The transcriptomic analysis showed several genes that were annotated as members of the molybdenum oxidoreductase family of B3 and that exhibited significant differential expression. These findings suggest that the differential expression of molybdenum-binding proteins might be related to their involvement in energy metabolism pathways that utilize nitrate and dimethyl sulfoxide as electron acceptors. Aerobic bacteria, such as B3 and B4, might possess distinct Hg(II) biotransformation pathways from anaerobic SRB, rendering their growth and biomethylation abilities unaffected by molybdate. [ABSTRACT FROM AUTHOR]

The contamination of food by spoilage microorganisms and/or pathogens is a concern in the food industry. Pseudomonas species, Gram-negative, aerobic, and psychrotrophic bacteria, can develop in food products stored at low temperatures. The species causing spoilage in raw meat include Pseudomonas fragi, Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas lundensis. Psychrotrophic pseudomonads cause bad odors, off-tastes, discoloration, and mucus production in raw meat stored under refrigeration and aerobic conditions. Some Pseudomonas species that develop in meats stored at low temperatures are strong biofilm producers. The spoilage activities of pseudomonads are linked to a cell-to-cell communication system known as quorum sensing (QS). The QS system is responsible for encoding genes related to pathogenicity by sensing population density through molecules produced under adverse conditions. Understanding the spoilage mechanisms of pseudomonads in meat is important for inhibiting their development. Research on the QS system of meat pseudomonads is limited. This review discusses the biodiversity, spoilage properties, biofilm formation abilities, and QS effects of Pseudomonas species on meat spoilage. [ABSTRACT FROM AUTHOR]

Pseudomonas putida , a Gram-negative aerobe widely used in biotechnological applications, has attracted attention as a novel exoelectrogen in BESs. BESs exploit the EET capability of exoelectrogens in microbial fuel cells, microbial electrolysis cells, microbial electrosynthesis, and microbial desalination cells. EET enables anaerobic respiration with various external redox-active surfaces such as electrodes and metal oxides, allowing energy conversion and electron transfer both inside and outside the cell. The performance of BESs with P. putida as the exoelectrogen is affected by key factors such as substrates, electrode materials, and EET pathways. BESs with P. putida as the exoelectrogen have been used for removal of organics, the biosynthesis of valuable chemicals, and environmental toxicity testing. Bioelectrochemical systems (BESs), a group of promising integrated systems that combine the advantages of biotechnology and electrochemical techniques, offer new opportunities to address environmental and energy challenges. Exoelectrogens capable of extracellular electron transfer (EET) are the critical factor enabling electrocatalytic activity in BESs. Pseudomonas putida , an aerobe widely used in environmental bioremediation, the biosynthesis of valuable chemicals, and energy bioproduction, has attracted much attention due to its unique application potential in BESs. This review provides a comprehensive understanding of the working principles, key factors, and applications of BESs using P. putida as the exoelectrogen. The challenges and perspectives for the development of BESs with P. putida as the exoelectrogen are also proposed and discussed. [ABSTRACT FROM AUTHOR]

Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever‐improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one‐plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I‐SceI expressing plasmid pSW‐2 as a counterselection marker to accelerated plasmid curing; (2) double‐strand break introducing gene I‐sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG‐RIS. The single plasmid of pEMG‐RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I‐SceI encoding gene. Here, the usability of the pEMG‐RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform. [ABSTRACT FROM AUTHOR]

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O -demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O -demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O -demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida , and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O- demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA , involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms , yielding a platform strain for efficient O -demethylation of lignin-related aromatic compounds to value-added products. • Depolymerization of lignin, followed by bioconversion, is a promising strategy to convert lignin to valuable products. • Methoxylated monomers, like vanillate, must undergo O -demethylation to enter catabolic pathways in aerobic bacteria. • Vanillate O-demethylation pathways were compared in Pseudomonas putida KT2440, focusing on the LigM and VanAB systems. • ALE optimized each pathway, but the optimized VanAB pathway demonstrated faster vanillate utilization. • Mutations were reverse-engineered into P. putida to generate an improved vanillate biocatalyst for valorization of lignin. [ABSTRACT FROM AUTHOR]

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO 2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production. • Meta-Analysis of 13C-MFA and GEMs show P. putida to be an ideal host for shinorine production. • CRISPRi-mediated gene downregulation of PP_1444 improves shinorine production. • Shinorine was produced at high titers and amino acid supplementation improved the titer. • Shinorine accumulates exclusively in the supernatant in P. putida cultures. [ABSTRACT FROM AUTHOR]

Background The aac(6 ′)-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. Objectives To identify sources for the aac(6 ′)-Im fragment found in A. baumannii. Methods MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6 ′)-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. Results Among >60 000 non- Acinetobacter draft genomes in the MRSN collection, the aac(6 ′)-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6 ′)-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6 ′)-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6 ′)-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6 ′)-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6 ′)-Im cassette was different. Conclusions The aac(6 ′)-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype. [ABSTRACT FROM AUTHOR]

The metagenome-assembled genome (MAG) sequences of Pseudomonas putida PP14A and PP20A were obtained by metagenomic sequencing from the gut microbiomes of a female and a male patient both 24 years old from the same household presenting to a health outreach laboratory with complaint of headache, and occasional diarrhoea in Mararaba, Nasarawa State, Nigeria. The phylogenetic relationship observed between the two PP MAGs with other Pseudomonas spp MAGs from human, points to the global spread of Pseudomonas putida through human activity and migration. [ABSTRACT FROM AUTHOR]

To achieve future food security, reliable crop protection will be critically important. Development of biological control for soil borne disease is accepted as a safe, durable and ecofriendly alternative for chemical management strategy. Pseudomonads possess many traits like production antibiotics, lytic enzymes, siderophores, hydrogen cyanide (HCN) that make them well suited as biocontrol and growth-promoting agents. An attempt was taken to isolate and identify different fluorescent Pseudomonads and to evaluate their antagonistic ability. Twelve fluorescent Pseudomonads isolates were collected from different crop rhizosphere from different locations of northern parts of West Bengal. Isolates characterized morphologically and biochemically. All the isolates were gram negative rods with smooth and glossy colony on kings B and nutrient agar medium with convex elevation but variation was found in respect to pigment production. A huge variation was found in respect to biochemical characterization. From the result of biochemical analysis, the isolated sp. can be identified as Pseudomonas fluorescens or Pseudomonas putida or Pseudomonas aeruginosa. Some of the isolate produce green, some isolates produce yellowish green and rest of them produce red/pink pigments. Isolates showed a huge variation in respect to pathogenicity against soil borne pathogen Slerotium rolfsii. Percent inhibition varied from 31 to 74 %. Isolate 3 and 8 can be efficient biocontrol agent as highest growth inhibition of the pathogen Sclerotium rolfsii was obtained by the isolate 8 (74.37%) followed by isolate 3 (63.15%). In respect to growth parameter isolate 8, 9 and 10 were found better among the isolates where as in respect to disease reduction isolate 3, 8, 9 and 11 were promising under in vivo study. [ABSTRACT FROM AUTHOR]

The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O2), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O2. The encoded mutations cluster around a putative O2 tunnel, increasing flexibility and accessibility to O2 in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

The strict requirement of cytochrome P450cam for its native ferredoxin redox partner, putidaredoxin (Pdx), is not exhibited by any other known cytochrome P450 (CYP) system and the molecular details of redox partner selectivity are still not completely understood. We therefore examined the selectivity of a related Pseudomonas cytochrome P450, P450lin, by testing its activity with non-native redox partners. We found that P450lin could utilize Arx, the native redox partner of CYP101D1, to enable turnover of its substrate, linalool, while Pdx showed limited activity. Arx exhibited a higher sequence similarity to P450lins native redox partner, linredoxin (Ldx) than Pdx, including several residues that are believed to be at the interface of the two proteins, based on the P450cam-Pdx complex structure. We therefore mutated Pdx to resemble Ldx and Arx and found that a double mutant, D38L/∆106, displayed higher activity than Arx. In addition, Pdx D38L/∆106 does not induce a low-spin shift in linalool bound P450lin but does destabilize the P450lin-oxycomplex. Together our results suggest that P450lin and its redox partners may form a similar interface to P450cam-Pdx, but the interactions that allow for productive turnover are different.

Anthracnose disease caused by Colletotrichum species has appeared in many crops, particularly chilies which have been heavily affected. In Viet Nam, Lam Dong Province is one of the most chili planting areas then it is enormously impacted on chili production. Currently, biological methods have been increasingly used in plant disease prevention due to their effectiveness and safety. Specifically, Pseudomonas species have been widely studied and become the great potential candidates owing to their antagonistic ability against the anthracnose causing fungi. For this reason, the research of isolation and selection of the Pseudomonas species strain possessing the highest inhibitory activity against the fungus Colletotrichum scovillei, giving rise to Chili anthracnose disease in Lam Dong Province, was conducted. From 12 soil samples, 19 isolates were initially categorized as Pseudomonas species. Out of all candidates, the LD3.1 strain exhibited the strongest inhibitory effectiveness on the development of Colletotrichum scovillei (60.78% after 15 days). The molecular identification result based on 16S ribosomal DNA revealed that LD3.1 is identical to Pseudomonas putida (100%).

Abstract The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL− 1, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD50 equal to 4.67 × 107 colony forming unit (CFU) fish− 1. After intraperitoneal (IP) challenge, fish treated with 10 mg kg− 1 of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg− 1. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL− 1 at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg− 1, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg− 1) effectively protected the challenged fish.

Abstract Background Petrochemicals contribute to environmental issues, with concerns ranging from energy consumption and carbon emission to pollution. In contrast, microbial biorefineries offer eco-friendly alternatives. The solvent-tolerant Pseudomonas putida DOT-T1E serves as a suitable host for producing aromatic compounds, specifically l-phenylalanine and its derivative, 2-phenylethanol (2-PE), which find widespread applications in various industries. Results This study focuses on enhancing 2-PE production in two l-phenylalanine overproducing strains of DOT-T1E, namely CM12-5 and CM12-5Δgcd (xylABE), which grow with glucose and glucose-xylose, respectively. To synthesize 2-PE from l-phenylalanine, these strains were transformed with plasmid pPE-1, bearing the Ehrlich pathway genes, and it was found higher 2-PE production with glucose (about 50–60 ppm) than with xylose (

Abstract Background Pseudomonas putida is a pathogenic bacterium that induces great losses in fishes, including Nile tilapia (Oreochromis niloticus). Currently, the application of nanomaterials in aquaculture practices has gained more success as it endows promising results in therapies compared to traditional protocols. Objective Therefore, the current perspective is considered the first report to assess the anti-bacterial efficacy of titanium dioxide nanogel (TDNG) against Pseudomonas putida (P. putida) in Nile tilapia. Methods The fish (n = 200; average body weight: 47.50±1.32 g) were allocated into four random groups (control, TDNG, P. putida, and TDNG + P. putida), where 0.9 mg/L of TDNG was applied as bath treatment for ten days. Results Outcomes revealed that P. putida infection caused ethological alterations (surfacing, abnormal movement, and aggression) and depression of immune-antioxidant variables (complement 3, lysozyme activity, total antioxidant capacity, superoxide dismutase, and reduced glutathione content). Additionally, a substantial elevation in hepatorenal biomarkers (aspartate and alanine aminotransferases and creatinine) with clear histopathological changes and immuno-histochemical alterations (very weak BCL-2 and potent caspase-3 immuno-expressions) were seen. Surprisingly, treating P. putida-infected fish with TDNG improved these variables and obvious restoration of the tissue architectures. Conclusion Overall, this report encompasses the key role of TDNG as an anti-bacterial agent for controlling P. putida infection and improving the health status of Nile tilapia.

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis. [ABSTRACT FROM AUTHOR]

The aim of this research was to estimate the immunopotentiation effect of brown algae Padina boergesenii water extract on Nile tilapia, Oreochromis niloticus through resistance to Pseudomonas putida infection. Gas Chromatography Mass Spectrometry was utilized to characterize the seaweed phytoconstituents. One hundred and twenty-six fish were divided in triplicates into two equal groups corresponding to two diet variants that used to feed Nile tilapia for 20 successive days: a basal (control), and P. boergesenii water extract supplemented group. Fish samples were collected at 10-days intervals throughout the experiment. Serum biochemical constituents, total antioxidant capacity (TAC), and some immune related genes expression of the spleen and intestinal tissues of experimental fish were studied, as well as histological examination of fish immune tissues. Moreover, following 20 days of feeding, the susceptibility of Nile tilapia to P. putida infection was evaluated to assess the protective effect of the used extract. The findings indicated that the studied parameters were significantly increased, and the best immune response profiles were observed in fish fed P. boergesenii water extract for 20 successive days. A bacterial challenge experiment using P. putida resulted in higher survival within the supplemented fish group than the control. Thus, the lowered post-challenge mortality of the fish may be related to the protection provided by the stimulation of the innate immune system, reduced oxidative stress by higher activity of TAC, and elevated levels of expression of iterleukin-1beta (IL-1β), beta-defensin (β-defensin), and natural killer-lysin (NKl). Moreover, the constituents of the extract used showed potential protective activity for histological features of the supplemented fish group when compared to the control. Collectively, this study presents a great insight on the protective role of P. boergesenii water extract as an additive in Nile tilapia feed which suggests its potential for improving the immune response against P. putida infection. [ABSTRACT FROM AUTHOR]

Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics' impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species. ATP dynamics influence bioproduction yet are largely unexplored in this context. Here, authors unravel ATP dynamics across various conditions, identify carbon sources which boost ATP levels and bioproduction, and uncover metabolic bottlenecks, shedding light on how ATP dynamics can be used to enhance bioproduction. [ABSTRACT FROM AUTHOR]

Oil-degrading bacteria have the ability to degrade alkanes present in crude oil because of a special enzymatic system, the alkane hydroxylase complex (AlkH). The mechanism for the transport and degradation of alkanes present in crude oil remains unclear, especially related to the first step in hydrocarbons oxidation. In this work, we present a novel model of the crude oil biodegradation mechanism by considering the contact between the oil drop and the cell and calculating the mass transfer coefficients in three oleophilic bacteria (B. licheniformis, P. putida and P. glucanolyticus). The mass transfer coefficients are evaluated under critical time conditions, when the kinetics and mass transport are in balance, and the difference in the values obtained (kL α = 1.60 × 10−3, 5.25 × 10−4 and 6.19 × 10−4 m/d, respectively) shows the higher value of the mass transfer coefficient and higher biodegradation potential for B. licheniformis. Because the morphology of the cells has been analyzed by optical and electron microscopy, in the proposed model, the increase in the size of the cells in P. glucanolyticus compared to P. putida exhibits higher values of the mass transfer coefficients and this is attributed, as a novel statement, to a bigger window for alkanes transport (contact area) when the external area of the cell is bigger. [ABSTRACT FROM AUTHOR]

Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene terephthalate) (PET). Recently, the use of EG as a starting material for the production of bio-fuels or bio-chemicals is gaining attention as a sustainable process since EG can be derived from materials not competing with human food stocks including CO2, syngas, lignocellulolytic biomass, and PET waste. In order to design and construct microbial process for the conversion of EG to value-added chemicals, microbes capable of catabolizing EG such as Escherichia coli, Pseudomonas putida, Rhodococcus jostii, Ideonella sakaiensis, Paracoccus denitrificans, and Acetobacterium woodii are candidates of chassis for the construction of synthetic pathways. In this mini-review, we describe EG catabolic pathways and catabolic enzymes in these microbes, and further review recent advances in microbial conversion of EG to value-added chemicals by means of metabolic engineering. Key points: • Ethylene glycol is a potential next-generation feedstock for sustainable industry. • Microbial conversion of ethylene glycol to value-added chemicals is gaining attention. • Ethylene glycol-utilizing microbes are useful as chassis for synthetic pathways. [ABSTRACT FROM AUTHOR]

Cellular metabolic activity can be detected by tetrazolium-based colorimetric assays, which rely on dehydrogenase enzymes from living cells to reduce tetrazolium compounds into colored formazan products. Although these methods have been used in different fields of microbiology, their application to the detection of bacteria with plastic-degrading activity has not been well documented. Here, we report a microplate-adapted method for the detection of bacteria metabolically active on the commercial polyester polyurethane (PU) Impranil®DLN using the tetrazolium salt 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT). Bacterial cells that are active on PU reduce XTT to a water-soluble orange dye, which can be quantitatively measured using a microplate reader. We used the Pseudomonas putida KT2440 strain as a study model. Its metabolic activity on Impranil detected by our novel method was further verified by Fourier-transform infrared spectroscopy (FTIR) analyses. Measurements of the absorbance of reduced XTT at 470 nm in microplate wells were not affected by the colloidal properties of Impranil or cell density. In summary, we provide here an easy and high-throughput method for screening bacteria active on PU that can be adapted to other plastic substrates. [ABSTRACT FROM AUTHOR]

Simultaneous application of modified Fe3O4 with biological treatments in remediating multi-metal polluted soils, has rarely been investigated. Thus, a pioneering approach towards sustainable environmental remediation strategies is crucial. In this study, we aimed to improve the efficiency of Fe3O4 as adsorbents for heavy metals (HMs) by applying protective coatings. We synthesized core-shell magnetite nanoparticles coated with modified nanocellulose, nanohydrochar, and nanobiochar, and investigated their effectiveness in conjunction with bacteria (Pseudomonas putida and Bacillus megaterium) for remediating a multi-metal contamination soil. The results showed that the coatings significantly enhanced the immobilization of heavy metals in the soil, even at low doses (0.5%). The coating of nanocellulose had the highest efficiency in stabilizing metals due to the greater variety of surface functional groups and higher specific surface area (63.86 m2 g-1) than the other two coatings. Interestingly, uncoated Fe3O4 had lower performance (113.6 m2 g-1) due to their susceptibility to deformation and oxidation. The use of bacteria as a biological treatment led to an increase in the stabilization of metals in soil. In fact, Pseudomonas putida and Bacillus megaterium increased immobilization of HMs in soil successfully because of extracellular polymeric substances and intensive negative charges. Analysis of metal concentrations in plants revealed that Ni and Zn accumulated in the roots, while Pb and Cd were transferred from the roots to the shoots. Treatment Fe3O4 coated with modified nanocellulose at rates of 0.5 and 1% along with Pseudomonas putida showed the highest effect in stabilizing metals. Application of coated Fe3O4 for in-situ immobilization of HMs in contamination soils is recommendable due to their high metal stabilization efficiency and suitability to apply in large quantities. [ABSTRACT FROM AUTHOR]

Simple Summary: Pseudomonas putida KT2440 is an attractive platform for bioremediation with versatile growth substrate and tolerance capabilities. The limited research on the salt tolerance of strain KT2440 hinders its application in bioremediation in high-salinity environments, where the composition of pollutants becomes increasingly complex and wastewater salinity often remains high. Therefore, the salt tolerance of strain KT2440 was explored in this work. By analyzing transcriptomic data from the high-salinity minimal salts medium, we identified key salt-tolerance genes and used them to improve the salt tolerance of strain KT2440 under 4% w/v NaCl. With co-expression of salt-tolerant genes, the maximum salt tolerance of strain KT2440 increased to 5% w/v NaCl. Further addition of compatible solutes increased the salt tolerance of the strain to 6% w/v NaCl. The engineered strain KT2440 could degrade common pollutants under 4% w/v NaCl in 48 h, and could be a promising platform for bioremediation in high-salinity environments. Pseudomonas putida KT2440 is a popular platform for bioremediation due to its robust tolerance to environmental stress and strong biodegradation capacity. Limited research on the salt tolerance of P. putida KT2440 has hindered its application. In this study, the strain KT2440 was tested to tolerate a maximum of 4% w/v NaCl cultured with minimal salts medium. Transcriptomic data in a high-salinity environment showed significant expression changes in genes in membrane components, redox processes, chemotaxis, and cellular catabolic processes. betB-encoding betaine-aldehyde dehydrogenase was identified from the transcriptome data to overexpress and enhance growth profile of the strain KT2440 in minimal salts medium containing 4% w/v NaCl. Meanwhile, screening for exogenous salt-tolerant genes revealed that the Na+/H+ antiporter EcnhaA from Escherichia coli significantly increased the growth of the strain KT2440 in 4% w/v NaCl. Then, co-expression of EcnhaA and betB (KT2440-EcnhaA-betB) increased the maximum salt tolerance of strain KT2440 to 5% w/v NaCl. Further addition of betaine and proline improved the salt tolerance of the engineered strain to 6% w/v NaCl. Finally, the engineered strain KT2440-EcnhaA-betB was able to degrade 56.70% of benzoic acid and 95.64% of protocatechuic acid in minimal salt medium containing 4% w/v NaCl in 48 h, while no biodegradation was observed in the normal strain KT2440 in the same conditions. However, the strain KT2440-EcnhaA-betB failed to degrade catechol in minimal salt medium containing 3% w/v NaCl. This study illustrated the improvement in the salt tolerance performance of Pseudomonas putida KT2440 and the feasibility of engineered strain KT2440 as a potential salt-tolerant bioremediation platform. [ABSTRACT FROM AUTHOR]

The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double‐stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin–antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications. [ABSTRACT FROM AUTHOR]

Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl‐CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl‐CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non‐preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl‐CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH‐null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild‐type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH‐null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds. [ABSTRACT FROM AUTHOR]

Many nonsporulating bacterial species survive prolonged resource exhaustion, by entering a state termed long-term stationary phase. Here, we performed long-term stationary phase evolutionary experiments on the bacterium Pseudomonas putida , followed by whole-genome sequencing of evolved clones. We show that P. putida is able to persist and adapt genetically under long-term stationary phase. We observed an accumulation of mutations within the evolving P. putida populations. Within each population, independently evolving lineages are established early on and persist throughout the 4-month-long experiment. Mutations accumulate in a highly convergent manner, with similar loci being mutated across independently evolving populations. Across populations, mutators emerge, that due to mutations within mismatch repair genes developed a much higher rate of mutation than other clones with which they coexisted within their respective populations. While these general dynamics of the adaptive process are quite similar to those we previously observed in the model bacterium Escherichia coli , the specific loci that are involved in adaptation only partially overlap between P. putida and E. coli. [ABSTRACT FROM AUTHOR]

Microbial biofilm formation represents a serious problem for both food industry and households. Natural biofilms are formed mostly by multiple species, and show resistance against most of the usual sanitizers. In this study, the effects of cinnamon (Cinnamomum zeylanicum), marjoram (Origanum majorana) and thyme (Thymus vulgaris) essential oils (EOs) and their main components (cinnamaldehyde, terpinene-4-ol, and thymol) were investigated on four-species biofilms of Escherichia coli, Listeria monocytogenes, Pseudomonas putida and Staphylococcus aureus. Minimum bactericide concentration (MBC) and killing time were determined by means of the microdilution method. MBC of the investigated EOs and components was between 0.5 mg/mL (cinnamaldehyde) to 25 mg/mL (terpinene-4-ol). Killing times for the four-species suspension were 5 or 10 min, time spans usable in the food industry. For eradication of the mixed-population biofilm from stainless steel (SS), polypropylene (PP), tile and wood surfaces, EO- or EO component-based disinfectant solutions were developed, and their effects were compared to a peracetic acid-based industrial sanitizer (HC-DPE). Total eradication of biofilms (99.9%) was achieved, with solutions containing cinnamon and thyme EO and EO components, from SS and PP, but not from tile or wood surfaces. Apparently, cinnamon EO, terpinene-4-ol and thymol have better disinfectant activity than HC-DPE. [ABSTRACT FROM AUTHOR]

The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250 mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30 °C using a shaking incubator for 24–48 h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69 kDa after purification. The purified enzyme was stable at 35 °C and a pH of 6.0. [ABSTRACT FROM AUTHOR]

In natural microbiomes, microorganisms interact with each other and exhibit diverse functions. Microbiome engineering, which enables bacterial knockdown, is a promising method to elucidate the functions of targeted bacteria in microbiomes. However, few methods to selectively kill target microorganisms in the microbiome without affecting the growth of nontarget microorganisms are available. In this study, we focused on the host-specific lytic ability of virulent phages and validated their potency for precise microbiome engineering. In an artificial microbiome consisting of Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus plantarum, the addition of bacteriophages infecting their respective host strains specifically reduced the number of these bacteria more than 102 orders. Remarkably, the reduction in target bacteria did not affect the growth of nontarget bacteria, indicating that bacteriophages were effective tools for precise microbiome engineering. Moreover, a virulent derivative of the phage was synthesized from prophage DNA in the genome of lysogen by in vivo DNA assembly and phage-rebooting techniques, and E. coli-targeted microbiome engineering was achieved. These results propose a novel approach for precise microbiome engineering using bacteriophages, in which virulent phages are synthesized from prophage DNA in lysogenic strains without isolating phages from environmental samples. [ABSTRACT FROM AUTHOR]

Background: Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3 dihydroxyacetone (DHA). FLS lays the foundation for several artificial carbon fixation and valorization pathways, such as the artificial starch anabolic pathway. However, the application of FLS is limited by its low catalytic activity and product promiscuity. Findings: FLS, designed and engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected as a candidate for modification. To evaluate its catalytic activity, 25 residues located within an 8 Å distance from the active center were screened using single-point saturation mutagenesis. A screening approach based on the color reaction of the DHA product was applied to identify the desired FLS variants. After screening approximately 5,000 variants (approximately 200 transformants per site), several amino acid sites that were not identified by directed evolution were found to improve DHA formation. The serine-to-phenylalanine substitution at position 236 improved the activity towards DHA formation by 7.6-fold. Molecular dynamics simulations suggested that the mutation increased local hydrophobicity at the active site, predisposing the cofactor-C2 intermediate to nucleophilic attack by the third formaldehyde molecule for subsequent DHA generation. Conclusions: This study provides improved FLS variants and valuable information into the influence of residues adjacent to the active center affecting catalytic efficiency, which can guide the rational engineering or directed evolution of FLS to optimize its performance in artificial carbon fixation and valorization. [ABSTRACT FROM AUTHOR]

Purpose: This paper aims to study the application of high-tolerance and flexible indigenous bacteria and fungi, along with the co-metabolism in recycled paper and cardboard mill (RPCM) wastewater treatment (WWT). Design/methodology/approach: The molecular characterization of isolated indigenous bacteria and fungi was performed by 16S rRNA and 18S rRNA gene sequencing, respectively. Glucose was used as a cometabolic substrate to enhance the bioremediation process. Findings: The highest removal efficiency was achieved for both chemical oxygen demand (COD) and color [78% COD and 45% color removal by Pseudomonas aeruginosa RW-2 (MZ603673), as well as approximately 70% COD and 48% color removal by Geotrichum candidum RW-4 (ON024394)]. The corresponding percentages were higher in comparison with the efficiency obtained from the oxidation ditch unit in the full-scale RPCM WWT plant. Originality/value: Indigenous P. aeruginosa RW-2 and G. candidum RW-4 demonstrated effective capability in RPCM WWT despite the highly toxic and low biodegradable nature, especially with the assistance of glucose. [ABSTRACT FROM AUTHOR]

Objectives To characterize VIM-type metallo-β-lactamase (MBL)-encoding genomic islands (GIs) in Pseudomonas aeruginosa and P. putida group isolates from Polish hospitals from 2001–2015/16. Methods Twelve P. aeruginosa and 20 P. putida group isolates producing VIM-like MBLs were selected from a large collection of these based on epidemiological and typing data. The organisms represented all major epidemic genotypes of these species spread in Poland with chromosomally located bla VIM gene-carrying integrons. The previously determined short-read sequences were complemented by long-read sequencing in this study. The comparative structural analysis of the GIs used a variety of bioinformatic tools. Results Thirty different GIs with bla VIM integrons were identified in the 32 isolates, of which 24 GIs from 26 isolates were integrative and conjugative elements (ICEs) of the clc family. These in turn were dominated by 21 variants of the GI2/ICE 6441 subfamily with a total of 19 VIM integrons, each inserted in the same position within the ICE's Tn 21 -like transposon Tn 4380. The three other ICEs formed a novel ICE 6705 subfamily, lacking Tn 4380 and having different VIM integrons located in another site of the elements. The remaining six non-ICE GIs represented miscellaneous structures. The presence of various integrons in the same ICE sublineage, and of the same integron in different GIs, indicated circulation and recombination of the integron-carrying genetic platforms across Pseudomonas species/genotypes. Conclusions Despite the general diversity of the bla VIM-carrying GIs in Pseudomonas spp. in Poland, a clear predominance of broadly spread and rapidly evolving clc -type ICEs was documented, confirming their significant role in antimicrobial resistance epidemiology. [ABSTRACT FROM AUTHOR]

Climate change is a universal issue and plastic trash is the major cause of global warming. The present study was specifically designed to address the escalating plastic waste problem, the biodegradation of low-density polyethylene (LDPE) bags was investigated over an 8-week period using liquid culture approach. Bacterial strain (Pseudomonas putida) capable of degrading LDPE was isolated from common plastic garbage dumping sites. The strain was identified after sequencing analysis, as P. putida was evaluated for its effectiveness in degrading LDPE. Various analytical techniques, including weight loss, SEM, FTIR and GC-MS were employed to assess the degradation process. Results revealed that bacterial strain achieved 0.0776 % degradation. SEM and FTIR confirmed surface and chemical changes, while GC MS identified degradation by products in comparison to the control (where no bacteria was used). [ABSTRACT FROM AUTHOR]

Introduction: Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The objectives of this research were to investigate changes in microbial diversity and community structure during the growth of Pholiota nameko and to analyze the adaptability of the dominant strains to their respective habitats through metabolic. Methods: Specifically, we conducted second-generation sequencing of the 16S rRNA gene (Illumina) on samples obtained from these stages. In addition, we isolated and characterized endophytes present in Pholiota nameko, focusing on examining the impact of dominant endophyte genera on autolysis. We also conducted a metabolic pathway analysis. Results and discussion: The results unveiled 578,414 valid sequences of Pholiota nameko endophytic fungi. At the phylum level, the dominant taxa were Basidiomycota, Ascomycota, Zoopagomycota, and Mucoromycota. At the genus level, the dominant taxa observed were Pholiota, Inocybe, Fusarium, and Hortiboletus. For endophytic bacteria, we obtained 458,475 valid sequences. The dominant phyla were Proteobacteria, TM6, Firmicutes, and Bacteroidetes, while the dominant genera were Edaphobacter, Xanthomonas, Burkholderia, and Pseudomonas. Moreover, we identified the isolated strains in Pholiota nameko using 16S rDNA, and most of them were found to belong to the genus Pseudomonas, with Pseudomonas putida being the most prevalent strain. The findings revealed that the Pseudomonas putida strain has the ability to slow down the breakdown of soluble proteins and partially suppress the metabolic processes that generate superoxide anion radicals in Pholiota nameko, thereby reducing autolysis. Additionally, our results demonstrated that molybdenum enzyme-mediated anaerobic oxidative phosphorylation reactions were the primary energy metabolism pathway in the Pseudomonas putida strain. This suggests that the molybdenum cofactor synthesis pathway might be the main mechanism through which Pholiota nameko adapts to its complex and diverse habitats. [ABSTRACT FROM AUTHOR]

The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL− 1, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD50 equal to 4.67 × 107 colony forming unit (CFU) fish− 1. After intraperitoneal (IP) challenge, fish treated with 10 mg kg− 1 of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg− 1. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL− 1 at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg− 1, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg− 1) effectively protected the challenged fish. [ABSTRACT FROM AUTHOR]

Modern agriculture practices heavily depend on the application of chemical fertilizers and continuous application disturbs the soil quality and health. Fluorescent Pseudomonas are the well characterized group of bacteria proficient in plant growth promotion and have excellent phosphate solubilizing abilities and solubilize insoluble phosphate to plants available form via secretion of organic acids. This study focused on the phosphate solubilization ability of Pseudomonas, arouse of Aloin-A molecule, gluconic acid production, as well as biometric parameters of key medicinal plant, the Aloe vera (Aloe barbadensis). Two strains (P. putida and Pseudomonas sp.) were found very effective PSBs and solubilized 236.73 and 153.73 mg L−1 phosphate, respectively. High-performance liquid chromatography analysis (HPLC) showed the production of gluconic acid (GA). P. putida and P. sp. produced 23970 ± 231 and 21538 ± 129 mg L−1 GA, respectively. Inoculation of the plants with consortium of P. putida and Pseudomonas sp. enhanced the plant growth, soil available P and phosphate uptake in plants than individual inoculation. An increase in aloin-A content (18.15 and 28.71 fold) was recorded in A. barbadensis treated with a PSB consortium in a soil amended without and with TCP (tricalcium phosphate), respectively. P-uptake ultimately enhances the aloin-A synthesis and biomass of plant. Therefore, agronomic attributes of Aloe vera plants can be improved for used as a medicinal plant through application of potent Fluorescent Pseudomonas. [ABSTRACT FROM AUTHOR]

The potato dry rot disease caused by Fusarium spp. seriously reduces potato yield and threatens human health. However, potential biocontrol agents cannot guarantee the stability and activity of biocontrol. Here, 18 synthetic microbial communities of different scales were constructed, and the synthetic microbial communities with the best biocontrol effect on potato dry rot disease were screened through in vitro and in vivo experiments. The results show that the synthetic community composed of Paenibacillus amylolyticus, Pseudomonas putida, Acinetobacter calcoaceticus, Serratia proteamaculans, Actinomycetia bacterium and Bacillus subtilis has the best biocontrol activity. Metabolomics results show that Serratia protoamaculans interacts with other member strains to produce caproic acid and reduce the disease index to 38.01%. Furthermore, the mycelial growth inhibition after treatment with caproic acid was 77.54%, and flow cytometry analysis showed that the living conidia rate after treatment with caproic acid was 11.2%. This study provides potential value for the application of synthetic microbial communities in potatoes, as well as the interaction mechanisms between member strains of synthetic microbial communities. [ABSTRACT FROM AUTHOR]

The use of constructed wetlands (CWs) is one of the best options to treat wastewater. In CWs, microorganisms play a major role in the degradation of organic pollutants but the concentration of nutrients, surfactant, and aeration (NSA) in oil-contaminated water is one of the factors that affect the persistence and metabolic functioning of hydrocarbon-degrading microorganisms. In the present investigation, the influence of the addition of NSA on the persistence of the augmented bacteria, copy of (alkane hydroxylase gene) alkB gene, and its expression level in the water, soil, and plants of CWs were evaluated. The CWs mesocosms were developed by the vegetation of Typha latifolia and Cyperus laevigatus and inoculated with the bacterial consortium (Pseudomonas putida TYRI39, Acinetobacter junii TYRH47, Acinetobacter sp. CYRH17, Pseudomonas sp. CYSI27, and Pseudomonas sp. TYRH42). The mesocosms were provided with nutrients (20 mg l− 1 N, 2.6 mg l− 1 P, and 16.4 mg l− 1 K) in liquid form, surfactant Tween-20 (0.2%, v/v) in liquid form, and aeration (≥ 7.0 ± 1 mg l− 1 DO) using aeration pump. The addition of NSA in CWs enhanced the persistence and metabolic functioning of the inoculated bacteria in the water, rhizospheric soil, and plants. The maximum hydrocarbon removal (97%) was observed in the water treated by CWs having C. laevigatus, bacteria, and NSA, and it is correlated with the copy numbers of alkB and its expression level. The application of NSA in CWs not only improved bacterial persistence and catabolic gene expression but also increased plant development and hydrocarbon removal. [ABSTRACT FROM AUTHOR]