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Infectious Diseases and Therapy Aug 2022Pseudomonas putida group are described as low-incidence opportunistic pathogens, but also as a significant reservoir of antimicrobial resistance (AMR) genes, including...
INTRODUCTION
Pseudomonas putida group are described as low-incidence opportunistic pathogens, but also as a significant reservoir of antimicrobial resistance (AMR) genes, including those of metallo-β-lactamases (MBLs). Our objective was the molecular and genomic characterization of MBL-producing P. putida (MPPP) group isolates from Poland, focusing on population structures, successful genotypes and MBL-encoding integrons.
METHODS
During a country-wide MBL surveillance in Pseudomonas spp., 59 non-duplicate MPPP isolates were collected from 36 hospitals in 23 towns from 2003 to 2016. All of the isolates were subjected to whole-genome sequencing (WGS), followed by species identification, multi-locus sequence typing (MLST), single-nucleotide polymorphism (SNP)-based phylogenetic/clonality analysis, resistome determination, and susceptibility testing.
RESULTS
The study collection comprised 12 species, of which P. alloputida (n = 19), P. monteilii (n = 15), and P. asiatica (n = 11) prevailed, while the others were P. kurunegalensis, P. putida, P. soli, P. mosselii, P. juntendi, and four potentially new species. MLST classified the isolates into 23 sequence types (STs) of which 21 were new, with three main clones, namely P. alloputida ST69, P.monteilii ST95 and P. asiatica ST15. The isolates produced VIM-like MBLs only, largely VIM-2 (n = 40), encoded by 24 different class 1 integrons (ten new), a number of which occurred also in P. aeruginosa and/or Enterobacterales in Poland. The plasmid pool was dominated by IncP-9, IncP-2, and pMOS94-like types. Multiple isolates were extensively drug-resistant.
CONCLUSIONS
This study, being one of the most comprehensive analyses of MPPP so far, has shown high diversity of the isolates in general, with three apparently international lineages, each internally diversified by MBL-encoding structures.
PubMed: 35689153
DOI: 10.1007/s40121-022-00659-z -
Heliyon Mar 2023This research was designed to analyze the interactive effects of and salicylic acid on the growth of canola in stress and non-stress conditions. Salicylic acid is a...
This research was designed to analyze the interactive effects of and salicylic acid on the growth of canola in stress and non-stress conditions. Salicylic acid is a phenolic derivative, that has a direct involvement in various plant stages like growth, and inflorescence. While is a drought-tolerant strain having plant growth-promoting characteristics like phosphate solubilization, indole acetic acid, and catalase production. Combined application of and salicylic acid has the ability to develop stress tolerance in plants and also improve growth of plants. They have significant (p < 0.05) effects on germination and morphological, physiological, and biochemical parameters. The plants that received the co-application of and salicylic acid gave more significant results than their alone application. They showed enhanced germination percentage, germination index, promptness index and, seedling vigor index by 19%, 18%, 34% and, 27%, respectively. There was a substantial increase of 25%, 27%, and 39% in shoot length, root length, and leaf area, respectively. The synergistic effect of both treatments has caused a 14% and 12% increase in the Canola plants' relative water content and membrane stability index respectively. A substantial increase of 18% in proline content was observed by the inoculation of whereas proline content was increased by 28% by the exogenous application of salicylic acid. The content of flavonoids (39%) and phenol (40%) was significantly increased by the co-application. The increase in superoxide dismutase (46%), ascorbate peroxidase (43%), and glutathione (19%) were also significant. The present research demonstrated that the combined application of and salicylic acid induces drought tolerance in canola and significantly improves its growth.
PubMed: 36950648
DOI: 10.1016/j.heliyon.2023.e14193 -
Applied and Environmental Microbiology Oct 2023have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon...
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 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 (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 expression via CRISPRi led to faster growth and glucose and -coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+-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 strains, were lower under strong CCR (glucose+-coumarate) condition compared to when repression was absent (-coumarate or glucose only).IMPORTANCEA newly isolated strain, 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 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 gene and small RNAs in carbon catabolite repression.
Topics: Sugars; Catabolite Repression; Xylose; Pseudomonas putida; Glucose; Hexoses; Pentoses; Carbon
PubMed: 37724856
DOI: 10.1128/aem.00852-23 -
Applied Microbiology and Biotechnology Mar 2023Pseudomonas putida KT2440 is a well-known model organism for the medium-chain-length (mcl) polyhydroxyalkanoate (PHA) accumulation. (R)-Specific enoyl-coenzyme A...
Pseudomonas putida KT2440 is a well-known model organism for the medium-chain-length (mcl) polyhydroxyalkanoate (PHA) accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the β-oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC, are annotated in P. putida KT2440. To investigate the relationship of fatty acids-PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in wild type (WT) and mutants under different growth conditions were analysed. PhaJ4 was the main monomer supplier for PHA synthesis with FA as sole carbon and energy source, with preference towards C8 and C10 substrate, whereas PhaJ1 showed preference for the C6 substrate. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7% of the cell dry weight (CDW). The deletion of (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG), which connects de novo FA and PHA synthesis pathways, while causing a further 1.8-fold decrease in PHA content, did not abolish PHA accumulation. Further proteome analysis revealed quinoprotein alcohol dehydrogenases PedE and PedH as potential monomer suppliers, but when these were deleted, the PHA level remained at 2.2-14.8% CDW depending on the fatty acid used and whether nitrogen limitation was applied. Therefore, it is likely that some other non-specific dehydrogenases supply monomers for PHA synthesis, demonstrating the redundancy of PHA metabolism. KEY POINTS: • β-oxidation intermediates are converted to PHA monomers by hydratases PhaJ1, PhaJ4 and MaoC in Pseudomonas putida KT2440. • When these are deleted, the PHA level decreases, but it is not abolished. • PHA non-specific enzyme(s) also contributes to PHA metabolism in KT2440.
Topics: Pseudomonas putida; Polyhydroxyalkanoates; Oxidation-Reduction; Fatty Acids; Oxidoreductases
PubMed: 36763117
DOI: 10.1007/s00253-023-12413-7 -
Microbial Biotechnology Jan 2021Rhamnolipids are biosurfactants with a wide range of industrial applications that entered into the market a decade ago. They are naturally produced by Pseudomonas... (Review)
Review
Rhamnolipids are biosurfactants with a wide range of industrial applications that entered into the market a decade ago. They are naturally produced by Pseudomonas aeruginosa and some Burkholderia species. Occasionally, some strains of different bacterial species, like Pseudomonas chlororaphis NRRL B-30761, which have acquired RL-producing ability by horizontal gene transfer, have been described. P. aeruginosa, the ubiquitous opportunistic pathogenic bacterium, is the best rhamnolipids producer, but Pseudomonas putida has been used as heterologous host for the production of this biosurfactant with relatively good yields. The molecular genetics of rhamnolipids production by P. aeruginosa has been widely studied not only due to the interest in developing overproducing strains, but because it is coordinately regulated with the expression of different virulence-related traits by the quorum-sensing response. Here, we highlight how the research of the molecular mechanisms involved in rhamnolipid production have impacted the development of strains that are suitable for industrial production of this biosurfactant, as well as some perspectives to improve these industrial useful strains.
Topics: Glycolipids; Molecular Biology; Pseudomonas; Pseudomonas aeruginosa; Pseudomonas putida; Surface-Active Agents
PubMed: 33151628
DOI: 10.1111/1751-7915.13700 -
ELife Oct 2019Plant leaves constitute a huge microbial habitat of global importance. How microorganisms survive the dry daytime on leaves and avoid desiccation is not well understood....
Plant leaves constitute a huge microbial habitat of global importance. How microorganisms survive the dry daytime on leaves and avoid desiccation is not well understood. There is evidence that microscopic surface wetness in the form of thin films and micrometer-sized droplets, invisible to the naked eye, persists on leaves during daytime due to deliquescence - the absorption of water until dissolution - of hygroscopic aerosols. Here, we study how such microscopic wetness affects cell survival. We show that, on surfaces drying under moderate humidity, stable microdroplets form around bacterial aggregates due to capillary pinning and deliquescence. Notably, droplet-size increases with aggregate-size, and cell survival is higher the larger the droplet. This phenomenon was observed for 13 bacterial species, two of which - and - were studied in depth. Microdroplet formation around aggregates is likely key to bacterial survival in a variety of unsaturated microbial habitats, including leaf surfaces.
Topics: Microbial Viability; Plant Leaves; Water Microbiology
PubMed: 31610846
DOI: 10.7554/eLife.48508 -
AMB Express May 2022Psychrotrophic Pseudomonas is one of the significant microbes that lead to putrefaction in chilled meat. One of the biggest problems in the detection of Pseudomonas is...
Psychrotrophic Pseudomonas is one of the significant microbes that lead to putrefaction in chilled meat. One of the biggest problems in the detection of Pseudomonas is that several species are seemingly identical. Currently, antibiotic resistance is one of the most significant challenges facing the world's health and food security. Therefore, this study was designed to apply an accurate technique for eliminating the identification discrepancy of Pseudomonas species and to study their resistance against various antimicrobials. A total of 320 chicken meat specimens were cultivated, and the isolated bacteria' were phenotypically recognized. Protein analysis was carried out for cultured isolates via Microflex LT. The resistance of Pseudomonas isolates was recorded through Vitek® 2 AST-GN83 cards. Overall, 69 samples were identified as Pseudomonas spp. and included 18 Pseudomonas lundensis (P. lundensis), 16 Pseudomonas fragi (P. fragi), 13 Pseudomonas oryzihabitans (P. oryzihabitans), 10 Pseudomonas stutzeri (P. stutzeri), 5 Pseudomonas fluorescens (P. fluorescens), 4 Pseudomonas putida (P. putida), and 3 Pseudomonas aeruginosa (P. aeruginosa) isolates. Microflex LT identified all Pseudomonas isolates (100%) correctly with a score value ≥ 2.00. PCA positively discriminated the identified isolates into various groups. The antimicrobial resistance levels against Pseudomonas isolates were 81.16% for nitrofurantoin, 71% for ampicillin and ampicillin/sulbactam, 65.22% for cefuroxime and ceftriaxone, 55% for aztreonam, and 49.28% for ciprofloxacin. The susceptibilities were 100% for cefotaxime, 98.55% for ceftazidime, 94.20% for each piperacillin/tazobactam and cefepime, 91.3% for cefazolin. In conclusion, chicken meat was found to be contaminated with different Pseudomonas spp., with high incidence rates of P. lundensis. Microflex LT is a potent tool for distinguishing Pseudomonads at the species level.
PubMed: 35532863
DOI: 10.1186/s13568-022-01390-1 -
Metabolic Engineering Sep 2023Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield...
Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. The periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P. putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we adopted a dynamic C- and H-metabolic flux analysis strategy, termed D-fluxomics. D-fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P. putida, characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate-unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P. putida towards efficient, sugar-based bioprocesses.
Topics: Pseudomonas putida; Sugars; Deuterium; Gluconates; Glucose
PubMed: 37454792
DOI: 10.1016/j.ymben.2023.07.004 -
Metabolic Engineering Nov 2022Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO-but formatotrophy has been engineered in only a few...
Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO-but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.
Topics: Pseudomonas putida; Metabolic Engineering; Formates; Methylobacterium extorquens; Glycine
PubMed: 36328297
DOI: 10.1016/j.ymben.2022.10.008 -
Current Opinion in Biotechnology Feb 2024The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental... (Review)
Review
The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental interest. The foundations of such performance include its robust redox metabolism, ability to tolerate a wide range of physicochemical stresses, rapid growth, versatile metabolism, nonpathogenic nature, and the availability of molecular tools for advanced genetic programming. These attributes have been leveraged for hosting engineered pathways for production of valuable chemicals or degradation/valorization of environmental pollutants. This has in turn pushed the boundaries of conventional enzymology toward previously unexplored reactions in nature. Furthermore, modifications to the physical properties of the cells have been made to enhance their catalytic performance. These advancements establish P. putida as bona fide chassis for synthetic biology, on par with more traditional metabolic engineering platforms.
Topics: Metabolic Engineering; Pseudomonas putida; Synthetic Biology; Biotransformation; Oxidation-Reduction
PubMed: 38061264
DOI: 10.1016/j.copbio.2023.103025