Biosynthesis of fragrance 2-phenylethanol from sugars by Pseudomonas putida.

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 (< 3 ppm). To understand the limiting factors, we tested the addition of phenylalanine and intermediates from the Ehrlich and shikimate pathways. The results identified intracellular L-phenylalanine as a key limiting factor for 2-PE production. To overcame this limitation, a chorismate mutase/prephenate dehydratase variant-insentive to feedback inhibition by aromatic amino acids-was introduced in the producing strains. This led to increased L-phenylalanine production and subsequently produced more 2-PE (100 ppm). Random mutagenesis of the strains also produced strains with higher L-phenylalanine titers and increased 2-PE production (up to 120 ppm). The improvements resulted from preventing dead-end product accumulation from shikimate and limiting the catabolism of potential pathway intermediates in the Ehrlich pathway. The study explored agricultural waste substrates, such as corn stover, sugarcane straw and corn-syrup as potential C sources. The best results were obtained using 2G substrates at 3% (between 82 and 100 ppm 2-PE), with glucose being the preferred sugar for 2-PE production among the monomeric sugars in these substrates. CONCLUSIONS: The findings of this study offer strategies to enhance phenylalanine production, a key substrate for the synthesis of aromatic compounds. The ability of P. putida DOT-T1E to thrive with various C-sources and its tolerance to substrates, products, and potential toxicants in industrial wastes, are highlighted. The study identified and overcome possible bottlenecks for 2-PE production. Ultimately, the strains have potential to become efficient microbial platforms for synthesizing 2-PE from agro-industrial waste materials.

Characterization of an extremophile bacterial acid phosphatase derived from metagenomics analysis.

Acid phosphatases are enzymes that play a crucial role in the hydrolysis of various organophosphorous molecules. A putative acid phosphatase called FS6 was identified using genetic profiles and sequences from different environments. FS6 showed high sequence similarity to type C acid phosphatases and retained more than 30% of consensus residues in its protein sequence. A histidine-tagged recombinant FS6 produced in Escherichia coli exhibited extremophile properties, functioning effectively in a broad pH range between 3.5 and 8.5. The enzyme demonstrated optimal activity at temperatures between 25 and 50°C, with a melting temperature of 51.6°C. Kinetic parameters were determined using various substrates, and the reaction catalysed by FS6 with physiological substrates was at least 100-fold more efficient than with p-nitrophenyl phosphate. Furthermore, FS6 was found to be a decamer in solution, unlike the dimeric forms of crystallized proteins in its family.

Top-Down Genomic Surveillance Approach To Investigate the Genomic Epidemiology and Antibiotic Resistance Patterns of Enterococcus faecium Detected in Cancer Patients in Arkansas.

Control of hospital-associated Enterococcus faecium infection is a strenuous task due to the difficulty of identifying transmission routes and the persistence of this nosocomial pathogen despite the implementation of infection control measures that have been successful with other important nosocomial pathogens. This study provides a comprehensive analysis of over 100 E. faecium isolates collected from 66 cancer patients at the University of Arkansas for Medical Sciences (UAMS) between June 2018 and May 2019. In the top-down approach used in this study, we employed, in addition to the 106 E. faecium UAMS isolates, a filtered set of 2,167 E. faecium strains from the GenBank database to assess the current population structure of E. faecium species and, consequently, to identify the lineages associated with our clinical isolates. We then evaluated the antibiotic resistance and virulence profiles of hospital-associated strains from the species pool, focusing on antibiotics of last resort, to establish an updated classification of high-risk and multidrug-resistant nosocomial clones. Further investigation of the clinical isolates collected from UAMS patients using whole-genome sequencing analytical methodologies (core genome multilocus sequence typing [cgMLST], core single nucleotide polymorphism [coreSNP] analysis, and phylogenomics), with the addition of patient epidemiological data, revealed a polyclonal outbreak of three sequence types occurring simultaneously in different patient wards. The integration of genomic and epidemiological data collected from the patients increased our understanding of the relationships and transmission dynamics of the E. faecium isolates. Our study provides new insights into genomic surveillance of E. faecium to assist in monitoring and further limiting the spread of multidrug-resistant E. faecium. IMPORTANCE Enterococcus faecium is a member of the gastrointestinal microbiota. Although its virulence is low in healthy, immunocompetent individuals, E. faecium has become the third leading cause of health care-associated infections in the United States. This study provides a comprehensive analysis of over 100 E. faecium isolates collected from cancer patients at the University of Arkansas for Medical Sciences (UAMS). We employed a top-down analytical approach (from population genomics to molecular biology) to classify our clinical isolates into their genetic lineages and thoroughly evaluate their antibiotic resistance and virulence profiles. The addition of patient epidemiological data to the whole-genome sequencing analytical methodologies performed in the study allowed us to increase our understanding of the relationships and transmission dynamics of the E. faecium isolates. This study provides new insights into genomic surveillance of E. faecium to help monitor and further limit the spread of multidrug-resistant E. faecium.

Scientific novelty beyond the experiment.

Practical experiments drive important scientific discoveries in biology, but theory-based research studies also contribute novel-sometimes paradigm-changing-findings. Here, we appraise the roles of theory-based approaches focusing on the experiment-dominated wet-biology research areas of microbial growth and survival, cell physiology, host-pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome-sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most-direct and most-scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non-mainstream; and the importance of theory-based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more-recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID-19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology-related papers and-although we ourselves are involved in laboratory experiments and practical fieldwork-also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment.

Herbicolin A production and its modulation by quorum sensing in a Pantoea agglomerans rhizobacterium bioactive against a broad spectrum of plant-pathogenic fungi.

Global population growth makes it necessary to increase agricultural production yields. However, climate change impacts and diseases caused by plant pathogens are challenging modern agriculture. Therefore, it is necessary to look for alternatives to the excessive use of chemical fertilizers and pesticides. The plant microbiota plays an essential role in plant nutrition and health, and offers enormous potential to meet future challenges of agriculture. In this context, here we characterized the antifungal properties of the rhizosphere bacterium Pantoea agglomerans 9Rz4, which is active against a broad spectrum of plant pathogenic fungi. Chemical analyses revealed that strain 9Rz4 produces the antifungal herbicolin A and its biosynthetic gene cluster was identified and characterized. We found that the only acyl-homoserine lactone-based quorum sensing system of 9Rz4 modulates herbicolin A gene cluster expression. No role of plasmid carriage in the production of herbicolin A was observed. Plant assays revealed that herbicolin A biosynthesis does not affect the root colonization ability of P. agglomerans 9Rz4. Current legislative restrictions are aimed at reducing the use of chemical pesticides in agriculture, and the results derived from this study may lay the foundations for the development of novel biopesticides from rhizosphere microorganisms.

Virulence Induction in Pseudomonas aeruginosa under Inorganic Phosphate Limitation: a Proteomics Perspective.

Inorganic phosphate (Pi) is a central nutrient and signal molecule for bacteria. Pi limitation was shown to increase the virulence of several phylogenetically diverse pathogenic bacteria with different lifestyles. Hypophosphatemia enhances the risk of death in patients due to general bacteremia and was observed after surgical injury in humans. Phosphate therapy, or the reduction of bacterial virulence by the administration of Pi or phosphate-containing compounds, is a promising anti-infective therapy approach that will not cause cytotoxicity or the emergence of antibiotic-resistant strains. The proof of concept of phosphate therapy has been obtained using primarily Pseudomonas aeruginosa (PA). However, a detailed understanding of Pi-induced changes at protein levels is missing. Using pyocyanin production as proxy, we show that the Pi-mediated induction of virulence is a highly cooperative process that occurs between 0.2 to 0.6 mM Pi. We present a proteomics study of PA grown in minimal medium supplemented with either 0.2 mM or 1 mM Pi and rich medium. About half of the predicted PA proteins could be quantified. Among the 1,471 dysregulated proteins comparing growth in 0.2 mM to 1 mM Pi, 1,100 were depleted under Pi-deficient conditions. Most of these proteins are involved in general and energy metabolism, different biosynthetic and catabolic routes, or transport. Pi depletion caused accumulation of proteins that belong to all major families of virulence factors, including pyocyanin synthesis, secretion systems, quorum sensing, chemosensory signaling, and the secretion of proteases, phospholipases, and phosphatases, which correlated with an increase in exoenzyme production and antibacterial activity. IMPORTANCE Antibiotics are our main weapons to fight pathogenic bacteria, but the increase in antibiotic-resistant strains and their consequences represents a major global health challenge, revealing the necessity to develop alternative antimicrobial strategies that do not involve the bacterial killing or growth inhibition. P. aeruginosa has been placed second on the global priority list to guide research on the development of new antibiotics. One of the most promising alternative strategies is the phosphate therapy for which the proof of concept has been obtained for P. aeruginosa. This article reports the detailed changes at the protein levels comparing P. aeruginosa grown under Pi-abundant and Pi-depleted conditions. These data describe in detail the molecular mechanisms underlying phosphate therapy. Apart from Pi, several other phosphate-containing compounds have been used for phosphate therapy and this study will serve as a reference for comparative studies aimed at evaluating the effect of alternative compounds.

Pseudomonas aeruginosa Pangenome: Core and Accessory Genes of a Highly Resourceful Opportunistic Pathogen.

In this chapter, we leverage a novel approach to assess the seamless population structure of Pseudomonas aeruginosa, using the full repertoire of genomes sequenced to date (GenBank, April 6, 2020). In order to assess the set of core functions that represents the species as well as the differences in these core functions among the phylogroups observed in the population structure analysis, we performed pangenome analyses at the species level and at the phylogroup level. The existence of the phylogroups described in the population structure analyses was supported by their different profiles of antibiotic-resistant determinants. Finally, we utilized a presence/absence matrix of protein families from the entire species to evaluate if P. aeruginosa phylogroups can be differentiated according to their accessory genomic content. Our analysis shows that the core genome of P. aeruginosa is approximately 62% of the average gene content for the species, and it is highly enriched with pathways related to the metabolism of carbohydrates and amino acids as well as cellular processes and cell maintenance. The analysis of the accessory genome of P. aeruginosa performed in this chapter confirmed not only the existence of the three phylogroups previously described in the population structure analysis, but also of 29 genetic substructures (subgroups) within the main phylogroups. Our work illustrates the utility of populations genomics pipelines to better understand highly complex bacterial species such as P. aeruginosa.

Insertion sequences and other mobile elements associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteraemia.

Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in the dissemination and modification of the antibiotic-resistant phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over 5 days from an inpatient with prolonged bacteraemia. Isolates from both species harboured similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events and IS-mediated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the ISs in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype-phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium, offering a new paradigm for the study of daptomycin resistance and novel insights into its dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in the rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.

Complete Genome Sequence of a Non-Carbapenemase-Producing Carbapenem-Resistant Providencia rettgeri Strain Isolated from a Clinical Urine Sample in Arkansas.

Here, we report the complete genome sequence of Providencia rettgeri isolate PROV_UAMS_01, which was recovered in 2021 from a urine sample from a hospitalized patient in Arkansas, USA. The genome sequence of P. rettgeri isolate PROV_UAMS_01 comprises a single chromosomal replicon with a G+C content of 40.51% and a total of 3,887 genes.

Providing octane degradation capability to Pseudomonas putida KT2440 through the horizontal acquisition of oct genes located on an integrative and conjugative element.

The extensive use of petrochemicals has produced serious environmental pollution problems; fortunately, bioremediation is considered an efficient way to fight against pollution. In line with Synthetic Biology is that robust microbial chassis with an expanded ability to remove environmental pollutants are desirable. Pseudomonas putida KT2440 is a robust lab microbe that has preserved the ability to survive in the environment and is the natural host for the self-transmissible TOL plasmid, which allows metabolism of toluene and xylenes to central metabolism. We show that the P. putida KT2440 (pWW0) acquired the ability to use octane as the sole C-source after acquisition of an almost 62-kb ICE from a microbial community that harbours an incomplete set of octane metabolism genes. The ICE bears genes for an alkane monooxygenase, a PQQ-dependent alcohol dehydrogenase and aldehyde dehydrogenase but lacks the electron donor enzymes required for the monooxygenase to operate. Host rubredoxin and rubredoxin reductase allow metabolism of octane to octanol. Proteomic assays and mutants unable to grow on octane or octanoic acid revealed that metabolism of octane is mediated by redundant host and ICE enzymes. Octane is oxidized to octanol, octanal and octanoic acid, the latter is subsequently acylated and oxidized to yield acetyl-CoA that is assimilated via the glyoxylate shunt; in fact, a knockout mutant in the aceA gene, encoding isocitrate lyase was unable to grow on octane or octanoic acid.

Isolation of AmpC- and extended spectrum ß-lactamase-producing Enterobacterales from fresh vegetables in the United States.

Vegetables may serve as a reservoir for antibiotic resistant bacteria and resistance genes. AmpC ß-lactamases and extended spectrum beta-lactamases (ESBL) inactivate commonly used ß-lactam antibiotics, including penicillins and cephalosporins. In this study, we determined the prevalence of AmpC and ESBL-producing Enterobacterales in retail vegetables in the United States. A total of 88 vegetable samples were collected for the screening of AmpC and ESBL-producing Enterobacterales using CHROMagar ESBL agar. These vegetables included washed ready-to-eat salad (23), microgreens/sprouts (13), lettuce (11), herbs (11), spinach (5), mushrooms (5), brussels sprouts (4), kale (3), and other vegetable samples (13). AmpC and ESBL activity in these isolates were determined using double disk combination tests. Two vegetable samples (2.27%), organic basil and brussels sprouts, were positive for AmpC-producing Enterobacterales and eight samples (9.09%), including bean sprouts, organic parsley, organic baby spinach, and several mixed salads, were positive for ESBL-producing Enterobacterales. Whole genome sequencing was used to identify the bacterial species and resistance genes in these isolates. Genes encoding AmpC ß-lactamases were found in Enterobacter hormaechei strains S43-1 and 74-2, which were consistent with AmpC production phenotypes. Multidrug-resistant E. hormaechei strains S11-1, S17-1, and S45-4 possess an ESBL gene, blaSHV66 , whereas five Serratia fonticola isolates contain genes encoding a minor ESBL, FONA-5. In addition, we used shotgun metagenomic sequencing approach to examine the microbiome and resistome profiles of three spinach samples. We found that Pseudomonas was the most prevalent bacteria genus in the spinach samples. Within the Enterobacteriaceae family, Enterobacter was the most abundant genus in the spinach samples. Moreover, antibiotic resistance genes encoding 12 major classes of antibiotics, including ß-lactam antibiotics, aminoglycoside, macrolide, fluoroquinolone, and others, were found in these spinach samples. Therefore, vegetables can serve as an important vehicle for transmitting antibiotic resistance. The study highlights the need for antibiotic resistance surveillance in vegetable products.

Plasma Metabolomics in a Nonhuman Primate Model of Abdominal Radiation Exposure.

The acute radiation syndrome is defined in large part by radiation injury in the hematopoietic and gastrointestinal (GI) systems. To identify new pathways involved in radiation-induced GI injury, this study assessed dose- and time-dependent changes in plasma metabolites in a nonhuman primate model of whole abdominal irradiation. Male and female adult Rhesus monkeys were exposed to 6 MV photons to the abdomen at doses ranging between 8 and 14 Gy. At time points from 1 to 60 days after irradiation, plasma samples were collected and subjected to untargeted metabolomics. With the limited sample size of females, different discovery times after irradiation between males and females were observed in metabolomics pattern. Detailed analyses are restricted to only males for the discovery power. Radiation caused an increase in fatty acid oxidation and circulating levels of corticosteroids which may be an indication of physiological stress, and amino acids, indicative of a cellular repair response. The largest changes were observed at days 9 and 10 post-irradiation, with most returning to baseline at day 30. In addition, dysregulated metabolites involved in amino acid pathways, which might indicate changes in the microbiome, were detected. In conclusion, abdominal irradiation in a nonhuman primate model caused a plasma metabolome profile indicative of GI injury. These results point to pathways that may be targeted for intervention or used as early indicators of GI radiation injury. Moreover, our results suggest that effects are sex-specific and that interventions may need to be tailored accordingly.

Comparative Analysis of PacBio and Oxford Nanopore Sequencing Technologies for Transcriptomic Landscape Identification of Penaeus monodon.

With the advantages that long-read sequencing platforms such as Pacific Biosciences (Menlo Park, CA, USA) (PacBio) and Oxford Nanopore Technologies (Oxford, UK) (ONT) can offer, various research fields such as genomics and transcriptomics can exploit their benefits. Selecting an appropriate sequencing platform is undoubtedly crucial for the success of the research outcome, thus there is a need to compare these long-read sequencing platforms and evaluate them for specific research questions. This study aims to compare the performance of PacBio and ONT platforms for transcriptomic analysis by utilizing transcriptome data from three different tissues (hepatopancreas, intestine, and gonads) of the juvenile black tiger shrimp, Penaeus monodon. We compared three important features: (i) main characteristics of the sequencing libraries and their alignment with the reference genome, (ii) transcript assembly features and isoform identification, and (iii) correlation of the quantification of gene expression levels for both platforms. Our analyses suggest that read-length bias and differences in sequencing throughput are highly influential factors when using long reads in transcriptome studies. These comparisons can provide a guideline when designing a transcriptome study utilizing these two long-read sequencing technologies.

Clover Root Exudates Favor Novosphingobium sp. HR1a Establishment in the Rhizosphere and Promote Phenanthrene Rhizoremediation.

Rhizoremediation is based on the ability of microorganisms to metabolize nutrients from plant root exudates and, thereby, to cometabolize or even mineralize toxic environmental contaminants. Novosphingobium sp. HR1a is a bacterial strain able to degrade a wide variety of polycyclic aromatic hydrocarbons (PAHs). Here, we have demonstrated that the number of CFU in microcosms vegetated with clover was almost 2 orders of magnitude higher than that in nonvegetated microcosms or microcosms vegetated with rye-grass or grass. Strain HR1a was able to eliminate 92% of the phenanthrene in the microcosms with clover after 9 days. We have studied the molecular basis of the interaction between strain HR1a and clover by phenomic, metabolomic, and transcriptomic analyses. By measuring the relative concentrations of several metabolites exudated by clover both in the presence and in the absence of the bacteria, we identified some compounds that were probably consumed in the rhizosphere; the transcriptomic analyses confirmed the expression of genes involved in the catabolism of these compounds. By using a transcriptional fusion of the green fluorescent protein (GFP) to the promoter of the gene encoding the dioxygenase involved in the degradation of PAHs, we have demonstrated that this gene is induced at higher levels in clover microcosms than in nonvegetated microcosms. Therefore, the positive interaction between clover and Novosphingobium sp. HR1a during rhizoremediation is a result of the bacterial utilization of different carbon and nitrogen sources released during seedling development and the capacity of clover exudates to induce the PAH degradation pathway. IMPORTANCE The success of an eco-friendly and cost-effective strategy for soil decontamination is conditioned by the understanding of the ecology of plant-microorganism interactions. Although many studies have been published about the bacterial metabolic capacities in the rhizosphere and about rhizoremediation of contaminants, there are fewer studies dealing with the integration of bacterial metabolic capacities in the rhizosphere during PAH bioremediation, and some aspects still remain controversial. Some authors have postulated that the presence of easily metabolizable carbon sources in root exudates might repress the expression of genes required for contaminant degradation, while others found that specific rhizosphere compounds can induce such genes. Novosphingobium sp. HR1a, which is our model organism, has two characteristics desirable in bacteria for use in remediation: its ubiquity and the capacity to degrade a wide variety of contaminants. We have demonstrated that this bacterium consumes several rhizospheric compounds without repression of the genes required for the mineralization of PAHs. In fact, some compounds even induced their expression.

PahT regulates carbon fluxes in Novosphingobium sp. HR1a and influences its survival in soil and rhizospheres.

Novosphingobium sp. HR1a is a good biodegrader of PAHs and aromatic compounds, and also a good colonizer of rhizospheric environments. It was previously demonstrated that this microbe is able to co-metabolize nutrients existing in root exudates together with the PAHs. We have revealed here that PahT, a regulator of the IclR-family, regulates the central carbon fluxes favouring the degradation of PAHs and mono-aromatic compounds, the ethanol and acetate metabolism and the uptake, phosphorylation and further degradation of mono- and oligo-saccharides through a phosphoenolpyruvate transferase system (PTS). As final products of these fluxes, pyruvate and acetyl-CoA are obtained. The pahT gene is located within a genomic region containing two putative transposons that carry all the genes for PAH catabolism; PahT also regulates these genes. Furthermore, encoded in this genomic region, there are genes that are involved in the recycling of phosphoenolpyruvate, from the obtained pyruvate, which is the motor molecule involved in the saccharide uptake by the PTS system. The co-metabolism of PAHs with different carbon sources, together with the activation of the thiosulfate utilization and an alternative cytochrome oxidase system, also regulated by PahT, represents an advantage for Novosphingobium sp. HR1a to survive in rhizospheric environments.