Search

Horizontal gene transfer (HGT) is a major process by which genes are transferred between microbes in the rhizosphere. However, examining HGT remains challenging due to the complexity of mimicking conditions within the rhizosphere. Fabricated ecosystems (EcoFABs) have been used to investigate several complex processes in plant-associated environments. Here we show that EcoFABs are efficient tools to examine and measure HGT frequency in the rhizosphere. We provide the first demonstration of gene transfer via a triparental conjugation system in the Brachypodium distachyon rhizosphere in an EcoFAB using Pseudomonas putida KT2440 as both donor and recipient bacterial strain with the donor containing a mobilizable and non-self-transmissible plasmid. We observed that the frequency of plasmid transfer in the rhizosphere is potentially dependent on the plant developmental stage and the composition and amount of root exudates. The frequency of plasmid transfer also increased with higher numbers of donor cells. We demonstrate the transfer of plasmid from P. putida to another B. distachyon root colonizer, Burkholderia sp. OAS925, showing HGT within a rhizosphere microbial community. Environmental stresses also influenced the rate and efficiency of HGT in the rhizosphere between different species and genera. This study provides a robust workflow to evaluate transfer of engineered plasmids in the rhizosphere when such plasmids are potentially introduced in a field or other plant-associated environments.IMPORTANCEWe report the use of EcoFABs to investigate the HGT process in a rhizosphere environment. It highlights the potential of EcoFABs in recapitulating the dynamic rhizosphere conditions as well as their versatility in studying plant-microbe interactions. This study also emphasizes the importance of studying the parameters impacting the HGT frequency. Several factors such as plant developmental stages, nutrient conditions, number of donor cells, and environmental stresses influence gene transfer within the rhizosphere microbial community. This study paves the way for future investigations into understanding the fate and movement of engineered plasmids in a field environment.

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 TiO2 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.

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.

Following their entrance into natural water reservoirs, the use of pesticides may adversely affect human health and the environment. Biobeds facilitate decontamination of agricultural wastewaters. Bioaugmentation could enhance biodegradation of pesticides and addition of an adsorbent, such as natural zeolite, may increase biobed efficacy. In this study, the bioaugmentation utility of Pseudomonas putida (biomixture A) and the impact of zeolites supplement (biomixture C) in biomixtures contaminated with the fungicides fluopyram (20 mg/kg), myclobutanil (10 mg/kg), and triticonazole (30 mg/kg) were examined. A biomixture devoid of zeolites and inoculant was used as control (biomixture B). Bioaugmentation enhanced the dissipation rate of all fungicides (DT50 values of 9.5, 7.7, and 10.5 days for myclobutanil, fluopyram, and triticonazole, respectively), contrary to zeolite-treated samples in which the dissipation was prolonged. According to α-diveristy results, bioaugmentation treatment displayed higher microbial diversity than control samples, while the highest diversity was recorded in the case of natural zeolites. The results of β-diversity showed that bioaugmentation and natural zeolites affected microbial diversity during the first days of the experiment via the support of gammaproteobacteria (Proteobacteria) growth. [ABSTRACT FROM AUTHOR]

Petroleum hydrocarbons (PHCs) including phenolic compounds cause major environmental impacts just the once released into soils or groundwater. Among the core technologies, permeable reactive barriers (PRBs) have been so far exploited to contain and control such contaminants within the subsurface layers of the soil. Against this background, the present study investigated flow-biofilm interactions in a laboratory-scale permeable reactive bio-barrier (PRBB). To this end, an experimental setup was firstly built by embedding pressure measurement ports, at 10 cm intervals, onto a cylindrical column with a diameter of 57 mm and a height of 50 cm, and then filled with a porous medium, made up of sand with an average diameter of 1.78 mm. The bacterium, Pseudomonas putida (P. putida), was also utilized to generate the biofilm. Afterward, phenol-containing water was passed through the column at a rate of 2 L/h under a hydrodynamic laminar flow regime. Experimental evidence showed that the biofilm formed by bacterial growth could shrink the bio-barrier (BB) porosity from 0.35 to 0.07, instigating a drop by 590 and 840 times in the hydraulic pressure across the column at phenol concentrations, 200 and 400 mg/L, respectively. The desired biofilm additionally managed to remove 40 and 30% of phenol at concentrations of 200 and 400 mg/L, in that order. Exploring the variations in hydraulic conductivity in different layers plus the microscopic images further demonstrated that the biofilm created at phenol concentration of 200 mg/L seemed to be much stronger and even more stable, compared to the one at 400 mg/L. This was traceable to the better adaptation of P. putida to lower concentrations of phenol as the carbon source. Furthermore, the study results established that the given PRBB could help decompose only a small portion of phenol, but outperformed in terms of containing and controlling this contaminant through reducing hydraulic conductivity. [ABSTRACT FROM AUTHOR]

The use of species within the Eucalyptus genus has been widespread in traditional medicine practices globally. However, concerns regarding safety and toxicity data have been prominent due to the extensive use of its essential oil, which is widely regarded for its medicinal properties and various applications. This research aimed to analyze the chemical makeup of Eucalyptus globulus essential oil (EGEO) and explore its antibacterial and insecticidal properties both in laboratory settings and real-life scenarios. Gas chromatography/mass spectrometry (GC/MS) was employed to identify the chemical constituents of EGEO, revealing 1,8-cineole (86.1 %), ο-Cymene (4.9 %), α-Pinene (3.8 %), γ-Terpinene (2.2 %), and α-Terpinene (1.6 %) as the primary components. The disc diffusion method demonstrated EGEOʼs strongest antimicrobial activity against Micrococcus luteus (p < 0.05), while minimal inhibition concentration tests indicated notable effectiveness against Streptococcus pneumoniae (MIC 50 2.25 mg/mL and MIC 90 2.79 mg/mL). In situ antibacterial assessments using white radish and pear food models showed heightened activity compared to contact application, particularly with the highest concentration (500 µg/L), yielding optimal results against Pseudomonas putida (p < 0.05). Additionally, the study investigated EGEO's antibiofilm properties against Salmonella enterica using mass spectrometry and the crystal violet method. In terms of insecticidal action, EGEO at 100 % concentration resulted in the mortality of 90 % of Harmonia axyridis individuals. This comprehensive investigation contributes to a deeper understanding of both the chemical composition and biological activities of Eucalyptus globulus essential oil, highlighting its potential as a source of natural compounds with promising therapeutic properties. [Display omitted] • Eucalyptus globulus essential oil (EGEO) was characterized by GC/MS technique. • 1,8-Cineole (86.1 %) was the major compound. • EGEO showed strongest antimicrobial activity against Micrococcus luteus. • In situ antibacterial essays were performed using white radish and pear food models. • EGEOʼs antibiofilm properties against Salmonella enterica were evaluated. [ABSTRACT FROM AUTHOR]

Recently, there has been significant focus on microplastics in the environment, especially regarding their role in sorption–desorption processes of emerging contaminants, impacting pollutant migration between aquatic and terrestrial ecosystems. Notably, the newest pollutants in such environments are the herbicide formulations known as ionic liquids (ILs), which integrate the structure of classic herbicidal anion with surface-active cations acting as an adjuvant. In our study, we synthesized herbicidal ILs by combining 2,4-D anion with cetyltrimethylammonium [CTA] and didecyldimethylammonium [DDA] cations. We investigated whether ILs and the mixture of salts, when exposed to polyethylene (PE) microplastics, differ in properties. We analyzed their sorption on defined PE particles, evaluated toxicity on Pseudomonas putida KT2440 using trans/cis ratio of unsaturated fatty acids, and assessed biodegradability with OECD 301F standard test. Results indicate IL cations and anions behave as distinct entities, questioning IL synthesis feasibility. Hydrophobic adjuvants were found to adsorb onto PE microplastic surfaces (5–60% [CTA] > [DDA]), posing potential threats of surface-active xenobiotic accumulation. This highlights the need to explore microplastics' role as sorbents of hazardous adjuvants in agriculture, potentially competing with humic acids and affecting xenobiotic bioavailability. Consequently, xenobiotics may persist longer in the environment, facilitated by microplastic mobility between aquatic and terrestrial ecosystems. Key points: • Microplastics act as sorbents, accumulating xenobiotics and limiting biodegradation. • Sorption of surfactant cations on microplastics reduces soil bacteria toxicity. • Research confirms independent action of ions from ionic liquids in the environment. [ABSTRACT FROM AUTHOR]

Background: The successful production of industrially relevant natural products hinges on two key factors: the cultivation of robust microbial chassis capable of synthesizing the desired compounds, and the availability of reliable genetic tools for expressing target genes. The development of versatile and portable genetic tools offers a streamlined pathway to efficiently produce a variety of compounds in well-established chassis organisms. The σ70lac and tet expression systems – adaptations of the widely used lac and tet regulatory systems developed in our laboratory – have shown effective regulation and robust expression of recombinant proteins in various Gram-negative bacteria. Understanding the strengths and limitations of these regulatory systems in controlling recombinant protein production is essential for progress in this area. Results: To assess their capacity for combinatorial control, both the σ70lac and tet expression systems were combined into a single plasmid and assessed for their performance in producing fluorescent reporters as well as the terpenoids lycopene and β-carotene. We thoroughly characterized the induction range, potential for synergistic effects, and metabolic costs of our dual σ70lac and tet expression system in the well-established microorganisms Escherichia coli, Pseudomonas putida, and Vibrio natriegens using combinations of fluorescent reporters. The dynamic range and basal transcriptional control of the σ70 expression systems were further improved through the incorporation of translational control mechanisms via toehold switches. This improvement was assessed using the highly sensitive luciferase reporter system. The improvement in control afforded by the integration of the toehold switches enabled the accumulation of a biosynthetic intermediate (lycopene) in the β-carotene synthesis pathway. Conclusion: This study presents the development and remaining challenges of a set of versatile genetic tools that are portable across well-established gammaproteobacterial chassis and capable of controlling the expression of multigene biosynthetic pathways. The enhanced σ70 expression systems, combined with toehold switches, facilitate the biosynthesis and study of enzymes, recombinant proteins, and natural products, thus providing a valuable resource for producing a variety of compounds in microbial cell factories. [ABSTRACT FROM AUTHOR]

Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of Pseudomonas putida KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (pcaIJ) gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the lva operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain P. putida HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, P. putida HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by P. putida. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications. • A strategy to produce levulinic acid from glucose was developed in P. putida KT2440. • Introducing aroG , asbF , and adc genes enabled levulinic acid production from glucose. • Deleting the pcaIJ and lvaE genes increased levulinic acid production in P. putida. • A levulinic acid yield of 36.3 mM (4.2 g/L) was achieved in fed-batch fermentation. • This is the first report of microbial levulinic acid production from glucose. [ABSTRACT FROM AUTHOR]

The complexities encountered in microbial metabolic engineering continue to elude prediction and design. Unravelling these complexities requires strategies that go beyond conventional genetics. Using multiplex mutagenesis with double stranded (ds) DNA, we extend the multiplex repertoire previously pioneered using single strand (ss) oligonucleotides. We present ReaL-MGE (Recombineering and Linear CRISPR/Cas9 assisted Multiplex Genome Engineering). ReaL-MGE enables precise manipulation of numerous large DNA sequences as demonstrated by the simultaneous insertion of multiple kilobase-scale sequences into E. coli, Schlegelella brevitalea and Pseudomonas putida genomes without any off-target errors. ReaL-MGE applications to enhance intracellular malonyl-CoA levels in these three genomes achieved 26-, 20-, and 13.5-fold elevations respectively, thereby promoting target polyketide yields by more than an order of magnitude. In a further round of ReaL-MGE, we adapt S. brevitalea to malonyl-CoA elevation utilizing a restricted carbon source (lignocellulose from straw) to realize production of the anti-cancer secondary metabolite, epothilone from lignocellulose. Multiplex mutagenesis with dsDNA enables the incorporation of lengthy segments that can fully encode additional functions. Additionally, the utilization of PCR to generate the dsDNAs brings flexible design advantages. ReaL-MGE presents strategic options in microbial metabolic engineering. ReaL-MGE (Recombineering and Linear CRISPR/Cas9 assisted Multiplex Genome Engineering) enables precise insertion of large DNA sequences in multiple species. Here, authors demonstrate its utility in enhancing malonyl-CoA levels and producing valuable compounds like epothilone from lignocellulose. [ABSTRACT FROM AUTHOR]

The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of this pathway results in colony morphology alterations and an increased ability to form biofilm. Such characteristics resemble the behavior of the Wsp pathway of Pseudomonas. In this work, we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. The purine derivatives guanine and hypoxanthine were identified as ligands of the recombinantly produced Htc10 ligand‐binding domain, with dissociation constants in the micromolar range, and its structure was solved by X‐ray protein crystallography. The sensor domain of Htc10 adopts a double Cache folding, with ligands bound to the membrane‐distal pocket. A high‐resolution structure of the occupied guanine‐binding pocket allowed the identification of residues involved in ligand recognition. Such residues were validated by site‐directed mutagenesis and isothermal titration calorimetry analyses of the protein variants. Moreover, heterologous expression of Htc10 in a Pseudomonas putida mutant lacking the native Wsp chemoreceptor promoted biofilm formation, a phenotype that was further enhanced by Htc10‐specific ligands. To our knowledge, this is the first description of binding specificity of a chemoreceptor that controls the activity of an associated diguanylate cyclase, opening the way for dynamic studies of the signaling behavior of this kind of sensory complex. [ABSTRACT FROM AUTHOR]

Pseudomonas plecoglossicida is a causative agent of visceral granulomas in large yellow croaker (Larimichthys crocea). Quorum sensing (QS) is widely involved in imparting virulence to pathogenic bacteria; however, it has not been studied in P. plecoglossicida. In this study, we annotated a LuxR family transcriptional regulator in P. plecoglossicida NB2011 and designated as PplR. We aligned the protein sequence by BlastP and Clustal X2, monitored the N-acyl-homoserine lactone (AHL) signal production through cross-feeding bioassay and HC-MS/MS; investigated exogenous AHL signal binding by recombinant expression and thin layer chromatography; constructed a deletion mutant of the target gene by method of double homologous recombination; sequenced the transcript RNA and analyzed the data; additionally, characterized phenotypes of wild type and mutant strain. The LuxR homolog PplR was found to share high similarity with PpoR—the LuxR solo of Pseudomonas putida —without a cognate LuxI. The wild-type strain did not produce any AHL signals and the recombinant LuxR protein was found to bind C6-L-homoserine lactone (C6-HSL), C8-HSL, 3-oxo-C10-HSL, and 3-oxo-C12-HSL. RNA-seq analysis indicated 84 differentially expressed genes—5 upregulated and 79 downregulated—mainly enriched in gene ontology terms, such as flagella-dependent motility, integral component of membrane, DNA binding and transcription, and metal ion binding, suggesting that PplR is a master transcription regulator. The mutant strain showed attenuated biofilm-forming ability and stress resistance, and the data support a role for PplR in the regulation of these traits in P. plecoglossicida NB2011 independent of the presence of AHL signals. This is the first study to provide QS-related information on P. plecoglossicida. [ABSTRACT FROM AUTHOR]

In Pseudomonas putida KT2440, a prime chassis for biotechnology, the clustered distribution of glucose catabolism genes and four related transcription factors (TFs) may facilitate the tight regulation of glucose catabolism. However, the genes under the direct control of these TFs remain unidentified, leaving their regulatory roles elusive. Furthermore, the carbon source gluconate was metabolised similarly to glucose in KT2440, but the responses of these catabolic and TF genes to gluconate were unclear. Here, these mysteries were unravelled through multi‐omics analysis integrated with physiological studies. First, we found that the expression of these catabolic and TF genes were significantly induced by both glucose and gluconate in KT2440. The independent responses of these genes to glucose and gluconate were differentiated in the gcd deletion mutant. We then defined the regulon of GnuR, one of the four related TFs, and discovered that GnuR directly repressed the expression of catabolic genes involved in the Entner–Doudoroff and the peripheral glucose and gluconate metabolism pathways. These results were further confirmed by physiological studies. Finally, a regulatory mode of an incoherent feedforward loop involving GnuR is proposed. [ABSTRACT FROM AUTHOR]

Pseudomonas putida is widely used in industrial applications, including the recombinant proteins production, because of its natural advantageous properties. In this study, the gene encoding FleQ, the primary regulator of flagellar synthesis, was deleted to construct a new non‐motile P. putida KT2440‐derived strain (ΔfleQ). The non‐motile cells showed reduced biofilm formation and enhanced expression of a heterologous gene in nutrient‐rich media compared with the wild‐type (WT) strain, attributed to the reallocation of cellular resources from flagellar synthesis and cellular motility. Additionally, the ΔfleQ strain exhibited enhanced tolerance to chloramphenicol, indicating higher ribosome production, confirmed by a higher RNA/protein ratio relative to the WT. While the WT strain showed decreased growth and a three‐fold increase in reporter gene activity in minimal media, the ΔfleQ strain maintained consistent reporter gene expression and exhibited a relatively higher growth rate. This suggests that the FleQ is involved in modulating proteome allocation based on nutrient quality. The removal of FleQ allows for more flexible resource allocation, creating a chassis strain with nutrient quality‐independent gene expression capacity, which could be valuable in industrial applications where consistent output is essential. [ABSTRACT FROM AUTHOR]

Malonyl‐coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value‐added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl‐CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome‐reduced Pseudomonas putida strain to increase acetyl‐CoA carboxylation while limiting malonyl‐CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked‐out in specific combinations towards increasing the malonyl‐CoA pool. An enzyme‐coupled biosensor, based on the rppA gene, was employed to monitor malonyl‐CoA levels in vivo. RppA is a type III polyketide synthase that converts malonyl‐CoA into flaviolin, a red‐colored polyketide. We isolated strains displaying enhanced malonyl‐CoA availability via a colorimetric screening method based on the RppA‐dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl‐CoA levels. We further modified these strains by adding a non‐canonical pathway that uses malonyl‐CoA as precursor for poly(3‐hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl‐CoA–dependent pathways in P. putida. [ABSTRACT FROM AUTHOR]

The Pseudomonas putida F1 genome and those of many other pseudomonads contain two tandem genes encoding acyl-CoA ligases Pput_1340 (fadD1) and Pput_1339 (fadD2) with Pput_1339 (fadD2) being the upstream gene. The fadD designation was assigned when both genes were found to complement the growth of an Escherichia coli acyl-CoA synthetase fadD deletion strain with oleic acid as sole carbon source. Site-directed mutagenesis showed that residues of the ATP/AMP domain required for function of E. coli FadD were also essential for full function of FadD1 and FadD2. Growth of the constructed ∆fadD1, ∆fadD2, and ∆fadD1∆fadD2 strains was tested in minimal medium with different chain length fatty acids as sole carbon sources. Lack of FadD1 significantly retarded growth with different chain length fatty acids and lack of both FadD1 and FadD2 further retarded growth. Derivatives of the ∆fabA∆desA unsaturated fatty acid auxotrophic strain carrying a deletion of either ∆fadD1 or ∆fadD2 were constructed. Growth of the ∆fabA∆desA∆fadD1 strain was very weak, whereas the ∆fabA∆desA∆fadD2 strain grew as well as the ∆fabA∆desA parent strain. Overexpression of either fadD1 or fadD2 restored growth of the ∆fabA∆desA∆fadD1 strain with fadD2 overexpression having a greater effect than fadD1 overexpression. The ∆fadD1 or ∆fadD2 genes are cotranscribed although the expression level of fadD1 is much higher than that of fadD2. This is attributed to a fadD1 promoter located within the upstream FadD2 coding sequence. IMPORTANCE Pseudomonas bacteria demonstrate a great deal of metabolic diversity and consequently colonize a wide range of ecological niches. A characteristic of these bacteria is a pair of genes in tandem annotated as acyl-CoA ligases involved in fatty acid degradation. The Pseudomonas putida F1 genome is annotated as having at least nine genes encoding acyl-CoA ligases which are scattered around the chromosome excepting the tandem pair. Since similar tandem pairs are found in other pseudomonads, we have constructed and characterized deletion mutants of the tandem ligases. We report that the encoded proteins are authentic acyl-CoA ligases involved in fatty acid degradation. [ABSTRACT FROM AUTHOR]

This study demonstrated the biodegradation of two different brands of cigarette butts (CBs), which are primarily composed of cellulose acetate, by four distinct microorganisms. These included the white rot fungus Pleurotus ostreatus, the brown rot fungus Lentinus lepideus, and the bacteria Bacillus cereus and Pseudomonas putida. After 31 days of treatment, weight loss measurements revealed a mass loss of 24–34%, where B. cereus exhibited the greatest efficacy in terms of mass loss for both brands of CBs. Fourier-Transform Infrared Spectroscopy (FTIR), confocal microscopy, and scanning electron microscopy (SEM) confirmed changes in the surface of the CBs, attributable to structural wear and material breakdown, indicating effective biodegradation by the evaluated microorganisms. Furthermore, the analyses confirmed changes in the surface of the CBs, attributable to structural wear and material breakdown, indicating effective biodegradation by the evaluated microorganisms. [ABSTRACT FROM AUTHOR]

Many species of the genus Pseudomonas are known to be highly tolerant to solvents and other environmental stressors. Based on phylogenomic and comparative genomic analyses, several Pseudomonas species were recently transferred to a new genus named Halopseudomonas. Because of their unique enzymatic machinery, these strains are being discussed as novel biocatalysts in biotechnology. In order to test their growth parameters and stress tolerance, five Halopseudomonas strains were assessed regarding their tolerance toward different n-alkanols (1-butanol, 1-hexanol, 1-octanol, 1-decanol), as well as to salt stress and elevated temperatures. The toxicity of the solvents was investigated by their effects on bacterial growth rates and presented as EC50 concentrations. Hereby, all Halopseudomonas strains showed EC50 values up to two-fold lower than those previously detected for Pseudomonas putida. In addition, the activity of the cis-trans isomerase of unsaturated fatty acids (Cti), which is an urgent stress response mechanism known to be present in all Pseudomonas species, was monitored in the five Halopseudomonas strains. Although several of the tested species were known to contain the cti gene, no significant phenotypic activity could be detected in the presence of the assayed stressors. A bioinformatic analysis of eight cti-carrying Halopseudomonas strains examining promotor binding sites, binding motifs and signal peptides showed that most of the cti genes have a lipoprotein signal peptide and promotor regions and binding motifs that do not coincide with those of Pseudomonas. These insights represent putative reasons for the absence of the expected Cti activity in Halopseudomonas, which in turn has always been observed in cti-carrying Pseudomonas. The lack of Cti activity under membrane stress conditions when the cti gene is present has never been documented, and this could represent potential negative implications on the utility of the genus Halopseudomonas for some biotechnological applications. [ABSTRACT FROM AUTHOR]

Background/Objectives: Vitamin B12 is very important for human health, as it is a cofactor for enzymatic activities and plays various roles in human physiology. It is highly valued in the pharmaceutical, food, and additive production industries. Some of the bacteria currently used for the vitamin production are difficult to modify with gene-editing tools and may have slow growth. We propose the use of the bacteria Pseudomonas putida KT2440 for the production of vitamin B12 because it has a robust chassis for genetic modifications. The present wok evaluates P. putida KT2440 as a host for vitamin B12 production and explore potential gene-editing optimization strategies. Methods: We curated and modified a genome-scale metabolic model of Pseudomonas putida KT2440 and evaluated different strategies to optimize vitamin B12 production using the knockin and OptGene algorithms from the COBRA Toolbox. Furthermore, we examined the presence of riboswitches as cis-regulatory elements and calculated theoretical biomass growth yields and vitamin B12 production using a flux balance analysis (FBA). Results: According to the flux balance analysis of P. putida KT2440 under culture conditions, the biomass production values could reach 1.802 gDW−1·h1·L−1, and vitamin B12 production could reach 0.359 µmol·gDW−1·h−1·L−1. The theoretical vitamin B12 synthesis rate calculated using P. putida KT2040 with two additional reactions was 14 times higher than that calculated using the control, Pseudomonas denitrificans, which has been used for the industrial production of this vitamin. Conclusions: We propose that, with the addition of aminopropanol linker genes and the modification of riboswitches, P. putida KT2440 may become a suitable host for the industrial production of vitamin B12. [ABSTRACT FROM AUTHOR]

Medium chain‐length polyhydroxyalkanoates (mcl‐PHAs) are efficiently synthesized from fatty acids. It was hypothesized that under non‐axenic conditions, the anti‐microbial properties of fatty acids would reduce growth of microbial competitors and allow Pseudomonas putida LS46 to dominate the population resulting in non‐sterile mcl‐PHA production. Non‐sterile batch and fed‐batch cultures produced similar final mcl‐PHA content, monomer composition, and productivity during 24–27 h cultivations when compared to sterile control cultures for initial octanoic acid concentrations ranging from 5 to 40 mM at an initial pH of 6.5. In the absence of a P. putida inoculum, growth was eventually observed in non‐sterile medium after a lag period of up to 120 h, the length of which was dependent on the octanoic acid concentration. The efficacy of prolonged non‐sterile cultivation was tested using a sequential batch reactor (SBR). After 28 days (672 h), 1.62 g L−1 of total biomass was observed, of which the mcl‐PHA content was 47%. This resulted in a PHA titer of 0.77 g L−1, and a volumetric PHA productivity of 32 mg L−1 h−1. The polymer composition remained stable throughout at 87.0 ± 2.7% C8 monomers, and short‐chain length (scl‐) monomers were not detected. This study is the first of its kind to report that purely mcl‐PHAs were produced after prolonged periods in a non‐sterile environment and demonstrate that medium chain‐length fatty acids exert a strong selective pressure toward organisms that synthesize mcl‐PHA. This suggests an opportunity for mcl‐PHA production in open continuous cultivation, which could reduce both fixed and operating costs. [ABSTRACT FROM AUTHOR]

Pearl millet (Pennisetum glaucum (L.) R. Br.), a vital C4 Panicoid millet crop, predominantly thrives in rainfed regions subject to various abiotic stresses, notably drought and cold stress, limiting its growth potential and yield. As climate change exacerbates water scarcity, understanding methods to mitigate drought's adverse effects becomes crucial. However, particular bacteria flourishing in the rhizosphere, demonstrating resilience to drought and skilled at nurturing plant health, are recognized for their ability to enhance growth under various abiotic stresses. The current study demonstrated the varying effects of Pseudomonas putida MTCC5279 (RA) on mitigating drought stress under low-temperature field conditions for the pearl millet genotypes PRLT2/89–33 (drought-tolerant) and H77/833–2 (drought-resistant). Plants of both genotypes are grown till panicle emergence and subjected to drought stress at the start of January where temperature also drops in field conditions. The compound effect of drought with low temperature severely affects the inflorescence of both the genotypes but RA-inoculated PRLT2/89–33 plants have better performance than their respective control and drought plants as well compared to H77/833–2 genotypes. Abiotic stresses markedly influenced growth metrics, osmolyte buildup, MDA levels, and the capability to scavenge reactive oxygen species (ROS), all of which saw positive modulation following the application of RA in PRLT2/89–33. To our knowledge, this study represents the first comprehensive examination of P. putida-mediated plant growth enhancement in pearl millet under the combined effects of abiotic stresses. [ABSTRACT FROM AUTHOR]

The continuous emergence of new illegal compounds, particularly psychoactive chemicals, poses significant challenges for current drug detection methods. Developing new protocols and kits for each new drug requires substantial time, effort, and dedicated manpower. Whole-cell bacterial bioreporters have been proven capable of detecting diverse hazardous compounds in both laboratory and field settings, identifying not only single compounds but also chemical families. We present the development of a microbial bioreporter for the detection of cocaine, the nervous system stimulant that is the second-most widely used illegal drug in the US. Escherichia coli was transformed with a plasmid containing a bacterial luxCDABEG bioluminescence gene cassette, activated by a cocaine-responsive signaling cascade. The engineered bioreporter is demonstrated to be a sensitive and specific first-generation detection system for cocaine, with detection thresholds of 17 ± 8 μg/L and 130 ± 50 μg/L in a buffer solution and in urine, respectively. Further improvement of the sensor's performance was achieved by altering the nucleotide sequence of the PBen gene promoter, the construct's sensing element, using accelerated site-directed evolution. The applicability of ready-to-use paper strips with immobilized bioreporter cells was demonstrated for cocaine detection in aqueous solutions. [ABSTRACT FROM AUTHOR]

cis,cis-Muconic acid (MA) is a bio-based chemical that can be converted to direct replacement chemicals or performance-advantaged bioproducts. We recently engineered the bacterium Pseudomonas putida KT2440 for the co-utilization of glucose and xylose to produce MA. This study evaluates the effect of additional genetic modifications, media composition, and bioprocess strategy on MA titer, productivity, and yield in bioreactor cultivations. We achieve a MA titer of 47.2 g L−1, a productivity of 0.49 g L−1 h−1, and a yield of 0.50 C-mol C-mol−1 from glucose and xylose supplemented with 5% (v/v) corn steep liquor with a P. putida strain harboring the deletion of gacS. Additionally, we demonstrate efficient MA production from corn stover-derived sugars and scalability to 150 L bioreactors. Techno-economic analysis and life cycle assessment predict that adipic acid, derived from catalytic hydrogenation of MA, can achieve a selling price as low as $2.60 per kg, approaching cost parity and reducing greenhouse gas emissions by up to 80% relative to fossil carbon-based adipic acid. [ABSTRACT FROM AUTHOR]

Drought is the most important abiotic stress that restricts the genetically predetermined yield potential of the crops. In the present study, four tomato varieties: Kashi Vishesh, Kashi Aman, Kashi Abhiman, and Kashi Amrit, were used to study the effect of PGPMs (plant growth-promoting microorganisms). PGPM strains, Bacillus megaterium BHUPSB14, Pseudomonas fluorescens BHUPSB06, Pseudomonas aeruginosa BHUPSB01, Pseudomonas putida BHUPSB0, Paenibacillus polymixa BHUPSB17, and Trichoderma horzianum, were used as the consortium. The control group was irrigated up to 80% of field capacity, while 7-, 14-, and 21-day water-deficit-exposed (DWD) plants' pot soil moisture was maintained to 40, 25, and 15% of the field capacity, both with and without the PGPM inoculation condition. The physiological parameters, such as electrolyte leakage, relative water content, photosynthetic efficiency, and chlorophyll color index, were significantly improved by PGPM application under progressive drought stress, compared to the control. PGPM application enhanced the proline accumulation and reduced the formation of hydrogen peroxide and lipid peroxidation under drought stress. The plant growth attributes were significantly increased by PGPM application. The Kashi Amrit variety showed the highest fruit yield among the four varieties under all the treatments. The PGPM consortium application also improved the soil physico-biological properties and nutrient availability in the soil. The PGPM consortium used in this study can potentially mitigate drought stress on tomato in drought-prone regions and act as a biofertilizer. The present study will open a new avenue of drought stress management in tomato. [ABSTRACT FROM AUTHOR]

As the habitats of bacteria, soil pore network and surface properties control the distribution, adhesion, and motility of bacteria in soils. These physical processes in turn influence bacterial accesses to nutrients and bacterial interactions. Our understanding on the pore- and surface-mediated bacterial interactions is currently limited. In this research, we evaluated the effects of soil pore confinement and surface adhesion on conjugation-based bacterial interactions. The interaction was measured by plasmid transfer between donor and recipient cells within the population of soil bacterium Pseudomonas putida. We found that the presence of porous sand media led to a net increase in conjugation frequency compared to sand-free liquid control. The increase is attributed to the facilitated effect of pore confinement on the collision of bacteria within pores. In contrast, bacterial adhesion to sand surfaces under elevated ionic strength conditions decreased the conjugation frequency as a result of mobility reduction on the surface. Such collision and adhesion mechanisms jointly drive the conjugation as a function of pore and surface properties of porous media. These results provide valuable insights into the roles of soil pores and surfaces in regulating horizontal gene transfer, an essential cell-to-cell interaction sustaining key processes of soil ecology and health. [ABSTRACT FROM AUTHOR]

Sustainable agriculture is reliable, acceptable and trustworthy way to maintain a healthy soil and thus to produce good quality and quantity of food. To achieve this task there is often use of biofertilizers which contain nitrogen, phosphorous and potassium as major nutrients. Phosphate is one of the major nutrients required by plants but not easily available to them because of its insoluble nature. A cost-effective way of availing P to plants is by use of Phosphate Solubilizing Bacteria (PSB). Among different types of Plant growth-promoting rhizobacteria (PGPR), Phosphate solubilizing bacteria constitute an important class. A cost-effective way of availing P to plants is by use of Phosphate Solubilizing Bacteria. These are known for plant growth promotion by enhancing P and N uptake. The PSB are nowadays used as biofertilizers to restore the soil health. There is a wide range of PSB suited for diverse agro-ecologies. Phosphate solubilizing bacteria make it soluble thus play important role in sustainable agriculture. Our data focus on in vitro study to isolate the phosphate solubilizing bacteria from different soils. Four isolates were recovered from different districts of Vidarbha region on Pikovskaya (PVK) agar. Prominent halo zone of clearance was developed around the colonies of four different isolates, indicating phosphate solubilization activity. Identification was done by gDNA and 16S Amplicon QC data. The results indicated that S1, S2, S3 and S4 were related to Bacillus vallismortis, Pseudomonas putida, Bacillus subtilis and Bacillus megaterium, respectively. [ABSTRACT FROM AUTHOR]

This study investigated the germination response to temperature of seeds of nine Arabidopsis thaliana ecotypes. They are characterized by a similar temperature dependency of seed germination, and 10 °C and 29 °C were found to be suboptimal low and high temperatures for all nine ecotypes, even though they originated from regions with diverse climates. We tested the potential of four PGPR strains from the genera Pseudomonas and Bacillus to stimulate seed germination in the two ecotypes under these suboptimal conditions. Biopriming of seeds with only the biofilm-forming strain Pseudomonas putida KT2440 significantly increased the germination of Cape Verde Islands (Cvi-0) seeds at 10 °C. However, biopriming did not significantly improve the germination of seeds of the widely utilized ecotype Columbia 0 (Col-0) at any of the two tested temperatures. To functionally investigate the role of KT2440's biofilm formation in the stimulation of seed germination, we used mutants with compromised biofilm-forming abilities. These bacterial mutants had a reduced ability to stimulate the germination of Cvi-0 seeds compared to wild-type KT2440, highlighting the importance of biofilm formation in promoting germination. These findings highlight the potential of PGPR-based biopriming for enhancing seed germination at low temperatures. [ABSTRACT FROM AUTHOR]

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are semi-crystalline elastomers with a low melting point and high elongation at break, allowing for a wide range of applications in domestic, agricultural, industrial, and mainly medical fields. Utilizing low-cost cellulose hydrolyzed sugar as a carbon source and metabolic engineering to enhance synthesis in Pseudomonas putida is a promising strategy for commercializing mcl-PHAs, but little has been attempted to improve the utilization of glucose for synthesizing mcl-PHAs. In this study, a multi-pathway modification was performed to improve the utilization of substrate glucose and the synthesis capacity of PHAs. To enhance glucose metabolism to flow to acetyl-CoA, which is an important precursor of mcl-PHA, multiple genes in glucose metabolism were inactive (branch pathway and negative regulatory) and overexpressed (positive regulatory) in this study. The two genes, gcd (encoding glucose dehydrogenase) and gltA (encoding citrate synthase), involved in glucose peripheral pathways and TCA cycles were separately and jointly knocked out in Pseudomonas putida QSRZ6 (ΔphaZΔhsdR), and the mcl-PHA synthesis was improved in the mutants; particularly, the mcl-PHA titer of QSRZ603 (ΔgcdΔgltA) was increased by 33.7%. Based on the glucose branch pathway truncation, mcl-PHA synthesis was further improved with hexR-inactivation (encoding a negative regulator in glucose metabolism). Compared with QSRZ603 and QSRZ6, the mcl-PHA titer of QSRZ607 (ΔgcdΔgltAΔhexR) was increased by 62.8% and 117.5%, respectively. The mutant QSRZ609 was constructed by replacing the endogenous promoter of gltB encoding a transcriptional activator of the two-component regulatory system GltR/GltS with the ribosome subunit promoter P33. The final mcl-PHA content and titers of QSRZ609 reached 57.3 wt% and 2.5 g/L, an increase of and 20.9% and 27.3% over that of the parent strain QSRZ605 and an increase of 110.4% and 159.9% higher as compared to QSRZ6, respectively. The fermentation was optimized with a feeding medium in shaker flacks; then, the mcl-PHA contents and titer of QSRZ609 were 59.1 wt% and 6.8 g/L, respectively. The results suggest that the regulation from glucose to acetyl-CoA by polygenic modification is an effective strategy for enhancing mcl-PHA synthesis, and the mutants obtained in this study can be used as chassis to further increase mcl-PHA production.

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.

Abstract Salinity stress negatively affects the growth and yield of crops worldwide. Onion (Allium cepa L.) is moderately sensitive to salinity. Beneficial microorganisms can potentially confer salinity tolerance. This study investigated the effects of endomycorrhizal fungi (M), Pseudomonas putida (Ps) and their combination (MPs) on onion growth under control (0 ppm), moderate (2000 ppm) and high (4000 ppm) NaCl salinity levels. A pot experiment was conducted with sandy loam soil and onion cultivar Giza 20. Results showed that salinity reduced growth attributes, leaf pigments, biomass and bulb yield while increasing oxidative stress markers. However, individual or combined inoculations significantly increased plant height, bulb diameter and biomass production compared to uninoculated plants under saline conditions. MPs treatment provided the highest stimulation, followed by Pseudomonas and mycorrhizae alone. Overall, dual microbial inoculation showed synergistic interaction, conferring maximum benefits for onion growth, bulbing through integrated physiological and biochemical processes under salinity. Bulb yield showed 3.5, 36 and 83% increase over control at 0, 2000 and 4000 ppm salinity, respectively. In conclusion, combined application of mycorrhizal-Pseudomonas inoculations (MPs) effectively mitigate salinity stress. This approach serves as a promising biotechnology for ensuring sustainable onion productivity under saline conditions.

Strawberry cultivation holds significant economic and social promise within Peruvian fruit production. However, conventional management practices have led to the excessive use of agrochemicals in this crop. This study proposes an organic approach to strawberry production, integrating less environmentally harmful technologies. The aim was to assess microbial inoculation by using Trichoderma sp. and Pseudomonas putida and the application of organic amendments on strawberry seedlings of the commercial cultivar “San Andreas”. A field experiment was established with evaluations in the vegetative and productive stages. Results indicate that the co-inoculation of Trichoderma sp. and Pseudomonas putida increased leaf area by 7%, and enhanced the aerial part’s fresh and dry biomass by 13% and 28%, respectively, compared to treatment without microbial inoculation. Concurrently, compost application increased the leaf number and aerial dry biomass by 22% and 19% at the end of the vegetative stage, respectively, compared to treatment without organic amendment. In addition, it reduced the days for flowering, maintaining the fruit’s physicochemical attributes. Regarding yield, the amendments application significantly enhanced fruit weight per plant by 40%, especially when applied together with Trichoderma sp., and co-inoculation increased the number of fruits per meter square by 22%. These findings highlight the potential of technologies such as microbial inoculation and organic amendments to enhance strawberry yields and to gradually reduce the use of synthetic fertilizers.

Biosensors are used to detect and quantify chemicals produced in industrial microbiology with high specificity, sensitivity, and portability. Most biosensors, however, are limited by the need for transcription factors engineered to recognize specific molecules. In this study, we overcome the limitations typically associated with traditional biosensors by engineering Pseudomonas putida for whole-cell sensing of a variety of chemicals. Our approach integrates fluorescent reporters with synthetic auxotrophies within central metabolism that can be complemented by target analytes in growth-coupled setups. This platform enables the detection of a wide array of structurally diverse chemicals under various conditions, including co-cultures of producer cell factories and sensor strains. We also demonstrate the applicability of this versatile biosensor platform for monitoring complex biochemical processes, including plastic degradation by either purified hydrolytic enzymes or engineered bacteria. This microbial system provides a rapid, sensitive, and readily adaptable tool for monitoring cell factory performance and for environmental analyzes. Biosensors are powerful tools for quantification of a wide range of molecules but require extensive engineering for each analyte. Here, the authors engineered a robust environmental bacterium for sensing a diverse set of chemicals, such as lactate and PET degradation products, via growth-coupling [ABSTRACT FROM AUTHOR]

There is high market demand for (−)‐menthol because of its unique aroma and cooling properties. The first bottleneck encountered in the biosynthesis of (−)‐menthol in Escherichia coli is the low hydroxylation efficiency of (−)‐limonene‐3‐hydroxylase. Few known enzymes can hydroxylate the third carbon atom of (−)‐limonene. In this work, initially, we searched for new enzymes and screened a library of cytochrome P450BM3 variants aiming to improve catalysis of this reaction, but did not identify any variants with improved activity or regioselectivity. Then, through protein engineering of a cytochrome P450 monooxygenase variant from Pseudomonas putida (PpcamY96F/V247L), we obtained variant PpcamF87W/Y96F/L244I/V247L/V396A with regioselectivity of 93% (starting from 70%) toward (−)‐limonene without losing activity. Because of the improvement in regioselectivity, its turnover number was 1.5 times that of the starting enzyme. We analyzed the reasons for the improvement through molecular dynamics simulations. The new variant also exhibited 95% regioselectivity toward (+)‐limonene. This enzyme variant can be applied in biosynthesis of (−)‐menthol. [ABSTRACT FROM AUTHOR]

The present study aimed to characterize the biosurfactants synthesized by lactic acid bacteria (LAB) obtained from fermented foods, optimize the conditions for increasing the yield of biosurfactants and explore their antimicrobial and antibiofilm potential. Out of the 26 LAB isolates, isolate BS2 showed the highest biosurfactant production as indicated in the oil displacement test, drop collapse and emulsification activity. BS2 was identified as Lactiplantibacillus plantarum 1625 using 16S-rRNA gene sequencing and phylogenetic analysis. The biosurfactant produced by BS2 was identified as an anionic glycol-lipo-proteins by employing Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The biosurfactants produced by L. plantarum 1625 demonstrated strong antibacterial and antibiofilm characteristics against pathogenic strains such as Staphylococcus aureus MTCC 1049, Escherichia coli MTCC 1587, and Pseudomonas putida MTCC 1655. The minimal inhibition concentration value of antibacterial activity was found to be 0.1 mg/mL with the inhibition percentage ranging from 90 to 95%. Further, the effect of temperature, pH, and substrate composition on biosurfactant production was also studied to enhance it production using the Box-Behnken Design approach of Response surface methodology (RSM). Application of biosurfactant led to a considerable decrease in biofilm-forming harmful bacteria, as proven by scanning electron microscopy analysis. The results highlight the potential uses of biosurfactants in distinct industries, and biotechnological contexts, especially in the creation of new antimicrobial and antibiofilm agents. [ABSTRACT FROM AUTHOR]

Food contamination has emerged as a significant global health concern, posing substantial challenges to the food industry. Bacteria are the primary cause of foodborne diseases. Consequently, it is crucial to develop accurate and efficient sensing platforms to detect foodborne bacteria in food products. Among various detection methods, biosensors have emerged as a promising solution due to their portability, affordability, simplicity, selectivity, sensitivity, and rapidity. Electrospun nanofibers have gained increasing popularity in enhancing biosensor performance. These nanofibers possess a distinctive three-dimensional structure, providing a large surface area and ease of preparation. This review provides an overview of the electrospinning technique, nanofibers and nanofiber-based biosensors. It also explores their mechanisms and applications in the detection of foodborne bacteria such as Salmonella, Listeria monocytogenes (L. monocytogenes), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas putida (P. putida). [ABSTRACT FROM AUTHOR]

Salinity stress negatively affects the growth and yield of crops worldwide. Onion (Allium cepa L.) is moderately sensitive to salinity. Beneficial microorganisms can potentially confer salinity tolerance. This study investigated the effects of endomycorrhizal fungi (M), Pseudomonas putida (Ps) and their combination (MPs) on onion growth under control (0 ppm), moderate (2000 ppm) and high (4000 ppm) NaCl salinity levels. A pot experiment was conducted with sandy loam soil and onion cultivar Giza 20. Results showed that salinity reduced growth attributes, leaf pigments, biomass and bulb yield while increasing oxidative stress markers. However, individual or combined inoculations significantly increased plant height, bulb diameter and biomass production compared to uninoculated plants under saline conditions. MPs treatment provided the highest stimulation, followed by Pseudomonas and mycorrhizae alone. Overall, dual microbial inoculation showed synergistic interaction, conferring maximum benefits for onion growth, bulbing through integrated physiological and biochemical processes under salinity. Bulb yield showed 3.5, 36 and 83% increase over control at 0, 2000 and 4000 ppm salinity, respectively. In conclusion, combined application of mycorrhizal-Pseudomonas inoculations (MPs) effectively mitigate salinity stress. This approach serves as a promising biotechnology for ensuring sustainable onion productivity under saline conditions. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production. Results: When incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol). Conclusions: By integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida. [ABSTRACT FROM AUTHOR]

The study was conducted at the Mycotoxin Laboratory, Department of Plant Protection / College of Agriculture / University of Basrah for the period 2023-2024. A set of experiments were performed to evaluate the effectiveness of Beltanol (BT), Carbendazim (MBC) and Thiophanate- methyl (TM) fungicides in controlling Rhizoctonia solani, which causes eggplant root rot. The genetic matching using BLAST revealed that the bacterial isolates that isolated from non-infected eggplant soils were Aeromonas caviae, Enterobacter cloacae and Pseudomonas putida. The toxicity results of fungicides on R. solani also showed the efficiency of the BT in inhibiting the pathogenic fungus by 100% for the tested concentrations of 2, 4, 8, 10, 15, 20, 100, 250, 300, 350, 400 and 500 mg L-1. Both the MBC and TM fungicides demonstrated variation in their efficiency in controlling the pathogen. The toxicity lines confirmed the values of the median effective concentrations (EC50), as they reached 100 and 489.7 mg L-1 for both MBC and TM, respectively. The 50% inhibitory concentration (I50) of BT was estimated at 7.94 mg L-1 for A. caviae, E. cloacae and P. putida respectively. While it was 12.58 mg L-1 for MBC in A. caviae, E. cloacae and P. putida respectively. While it was 31.62 mg L-1 for TM in the case of A. caviae, E. cloacae and P. putida respectively. [ABSTRACT FROM AUTHOR]

Strawberry cultivation holds significant economic and social promise within Peruvian fruit production. However, conventional management practices have led to the excessive use of agrochemicals in this crop. This study proposes an organic approach to strawberry production, integrating less environmentally harmful technologies. The aim was to assess microbial inoculation by using Trichoderma sp. and Pseudomonas putida and the application of organic amendments on strawberry seedlings of the commercial cultivar "San Andreas". A field experiment was established with evaluations in the vegetative and productive stages. Results indicate that the co-inoculation of Trichoderma sp. and Pseudomonas putida increased leaf area by 7%, and enhanced the aerial part's fresh and dry biomass by 13% and 28%, respectively, compared to treatment without microbial inoculation. Concurrently, compost application increased the leaf number and aerial dry biomass by 22% and 19% at the end of the vegetative stage, respectively, compared to treatment without organic amendment. In addition, it reduced the days for flowering, maintaining the fruit's physicochemical attributes. Regarding yield, the amendments application significantly enhanced fruit weight per plant by 40%, especially when applied together with Trichoderma sp., and co-inoculation increased the number of fruits per meter square by 22%. These findings highlight the potential of technologies such as microbial inoculation and organic amendments to enhance strawberry yields and to gradually reduce the use of synthetic fertilizers. [ABSTRACT FROM AUTHOR]

In recent years, the design–build–test–learn (DBTL) cycle has become a key concept in strain engineering. Modern biofoundries enable automated DBTL cycling using robotic devices. However, both highly automated facilities and semi-automated facilities encounter bottlenecks in clone selection and screening. While fully automated biofoundries can take advantage of expensive commercially available colony pickers, semi-automated facilities have to fall back on affordable alternatives. Therefore, our clone selection method is particularly well-suited for academic settings, requiring only the basic infrastructure of a biofoundry. The automated liquid clone selection (ALCS) method represents a straightforward approach for clone selection. Similar to sophisticated colony-picking robots, the ALCS approach aims to achieve high selectivity. Investigating the time analogue of five generations, the model-based set-up reached a selectivity of 98 ± 0.2% for correctly transformed cells. Moreover, the method is robust to variations in cell numbers at the start of ALCS. Beside Escherichia coli, promising chassis organisms, such as Pseudomonas putida and Corynebacterium glutamicum, were successfully applied. In all cases, ALCS enables the immediate use of the selected strains in follow-up applications. In essence, our ALCS approach provides a 'low-tech' method to be implemented in biofoundry settings without requiring additional devices. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis and associated bacteria are important urogenital pathogens. This study aimed to investigate the effect of T. vaginalis and its associated bacteria on semen and sperm parameters related to male fertility. Urine and semen samples were collected from 97 males, whose ages between 18-50 years. Semen and sperm parameters, such as semen volume, sperm count, sperm concentration, sperm motility, leucocytes/pus cells, and erythrocytes, were evaluated according to the WHO instructions. Conventional-PCR and real-time quantitative PCR were used to detect T. vaginalis infection. Amie's-transport, blood-agar, MacConkey-agar, Chocolate-agar, Thayer-Martin-agar, and Mannitol-salt-agar were used to culture bacteria in urine, and the Vitek2 system was used to differentiate bacteria. Samples that showed positive microscopic examination for bacteria but were not culturable were diagnosed using PCR. T. vaginalis and bacterial infection rates were 31.8% and 17.5%, respectively. T. vaginalis, bacteria, and both together caused a decrease in some qualitative and quantitative parameters of semen and sperm such as a decline in sperm total count by 46.5, 63.46, and 65.67% respectively, sperm concentration by 6.27, 15.37, and 44.27% respectively, sperm motility by 67.63, 66.18 and 66.75% respectively, semen volume by 42.2, 36.4 and 63.86% respectively. They caused increases of 100.92, 16.89, and 84.56%, respectively, and sperm death by 36.58, 80.0%, 93%, and 60.77%, respectively. In addition, they caused an increase in WBCs/pus cells and RBCs in the semen and urine. Four bacteria species, Pantoea spp., Sphingomonas paucimobilis, Staphylococcus aureus, Methylobacterium spp. were detected in urine, and 10 species, Enterobacter aerogenes, Serratia marcescens, Pseudomonas putida, Sphingomonas paucimobilis, Aeromonas sobria, Burkholderia cepacia, Staphylococcus aureus, Aerococcus viridans, Pseudomonas androgens and Staphylococcus epidermidis were detected in semen. In addition, PCR identified three unculturable species: one in urine (Holomonas spp.), and two in semen (Lactobacillus jensenii and Holomonas spp.), S. aureus had the highest frequency in urine and semen of (32%). [ABSTRACT FROM AUTHOR]

Capillary assemblies and microfluidic devices used for bacterial chemotaxis assays have certain inherent limitations. This opens opportunities for innovation in the area. The present study describes an innovative economical device called chemotaxis plate and also a method to use this device for chemotaxis assay. Two type cultures, Pseudomonas putida MCC 2989 and Bacillus subtilis MCC 2049, chemotactic to L-aspartate, were used to validate the new device and establish the protocol for assay. 100 to 1000 fold higher number of cells were recovered in presence of chemoattractant as compared to control (p < 0.05). This novel assay technique showed 100% sensitivity and 99.21% specificity for chemotaxis assay of Pseudomonas putida MCC 2989 towards 3 mM L-aspartate over 50 min assay time. The device was also used to isolate bacteria chemotactic to caffeine directly from environmental samples. Very high chemotaxis response indices were reported for the first-time using chemotaxis plate. [ABSTRACT FROM AUTHOR]

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]

Microbial consortia for disease suppression involve combining multiple beneficial microorganisms to enhance their effectiveness in plant disease management. In present study, development of microbial consortia for the management of major diseases in coconut and citrus was carried out using bacteria Pseudomonas fluorescens, Pseudomonas putida (striata), Bacillus subtilis and fungi - Trichoderma reesei, T. harzianum, T. asperellum against major pathogens viz. Ganoderma lucidum, Thielaviopsis paradoxa, Phytopthora palmivora, Lasiodiplodia theobromae isolated from the coconut rhizosphere, and Fusarium solani isolated from the citrus rhizosphere. The promising fungal and bacterial antagonists were identified and studied for compatibility. Non-volatile compounds of consortia inhibited the test pathogens with an increase in concentration from 10 % to 75% with fungal consortia and bacterial consortia and also with mixed consortia which is composed of bacterial consortia + fungal consortia. Superior growth suppression was recorded with mixed consortia even at 10% concentration (59.44% to 65.83%) against the test pathogens in the ascending order of L. theobromae (59.44%) T. paradoxa (63.89%), G. lucidum (65.83%), P. palmivora (63.61%) and F. solani (62.78%). A similar trend was observed in 75% concentration where inhibition observed in the order of Thielaviopsis paradoxa (90.28%), G. lucidum (89.44%), F. solani (82.50%), L. theobromae (81.94%) and P. palmivora (81.39%). Volatile effect by bacterial consortia recorded the superior inhibition on test pathogens in the order of Ganoderma (85.28%), F. solani (75.28%), T. paradoxa (71.94%), P. palmivora (71.67%) and L. theobrome (67.50%) compared to the individual bioagents. Similarly, the fungal consortia showed the superior inhibitory effect on test pathogens in the order of G. lucidum (83.25%), P. palmivora (82.50%), L. theobromae (83.06%), F. solani (80.56%) and T. paradoxa (73.61%). Since there was no zone of inhibition between the strains, the interactions between Pseudomonas and Bacillus strains of Trichoderma spp. were compatible with one another. Neem cake recorded superior CFU population from 9.43 X 106 CFU at seven days by T. asperellum. Shelf life study on mixed consortia with bacterial + fungal bioagents in talc formulation indicated that all the bacterial and fungal CFU count recorded in 106 dilution for 90 days. [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]