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Current Microbiology Apr 2024This manuscript reports the whole genome sequence of a conditionally pathogenic rhizobacterial strain, Pseudomonas putida AKMP7, which has been previously reported by us...
This manuscript reports the whole genome sequence of a conditionally pathogenic rhizobacterial strain, Pseudomonas putida AKMP7, which has been previously reported by us to be beneficial to Arabidopsis thaliana under well-watered conditions and pathogenic to the plant under water stress. As part of a study to understand this unique behavior, the whole genome sequence of this strain was analyzed. Based on the results, it was identified that the total length of the AKMP7 genome is 5,764,016 base pairs, and the total GC content of the genome is 62.93% (typical of P. putida). Using RAST annotation pipeline, it was identified that the genome has 5605 coding sequences, 80 repeat regions, 71 tRNA genes, and 22 rRNA genes. A total of 4487 functional proteins and 1118 hypothetical proteins were identified. Phylogenetic analysis has classified it as P. putida species, with a P value of 0.03. In order to identify close relatives of this strain, comparative genomics was performed with 30 other P. putida strains, taken from publicly available genome databases, using Average Nucleotide Identity (ANI) analysis. Whole genome comparison with these strains reveals that AKMP7 possesses Type-IV Secretion System (T4SS) with conjugative transfer functionality. Interestingly, the T4SS feature is absent in all the beneficial/harmless strains of P. putida that we analyzed. All the plant pathogenic bacteria that were analyzed had the T4SS feature in their genome, indicating its role in pathogenesis. This study aims to address important gaps in understanding the molecular mechanisms involved in the conditional/opportunistic pathogenesis of plant-associated, beneficial soil bacteria, using genomics approaches.
Topics: Pseudomonas putida; Genome, Bacterial; Phylogeny; Base Composition; Arabidopsis; Bacterial Proteins; Plant Diseases; Whole Genome Sequencing; Sequence Analysis, DNA
PubMed: 38658394
DOI: 10.1007/s00284-024-03677-x -
Methods in Enzymology 2024Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a...
Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to produce fluorometabolites (such as fluorinated amino acids, key building blocks for synthesis) are relatively scarce. This chapter discusses the use of L-threonine aldolase enzymes (LTAs), a class of enzymes that catalyze reversible aldol addition to the α-carbon of glycine. The C-C bond formation ability of LTAs, together with their known substrate promiscuity, make them ideal for in vitro F biocatalysis. Here, we describe protocols to harness the activity of the low-specificity LTAs isolated from Escherichia coli and Pseudomonas putida on 2-fluoroacetaldehyde to efficiently synthesize 4-fluoro-L-threonine in vitro. This chapter also provides a comprehensive account of experimental protocols to implement these activities in vivo. These methods are illustrative and can be adapted to produce other fluorometabolites of interest.
Topics: Substrate Specificity; Escherichia coli; Pseudomonas putida; Halogenation; Biocatalysis; Amino Acids; Glycine Hydroxymethyltransferase; Threonine; Fluorine; Aldehydes
PubMed: 38658080
DOI: 10.1016/bs.mie.2024.02.016 -
Molecules (Basel, Switzerland) Mar 2024Alongside fermentable sugars, weak acids, and furan derivatives, lignocellulosic hydrolysates contain non-negligible amounts of lignin-derived aromatic compounds. The...
Alongside fermentable sugars, weak acids, and furan derivatives, lignocellulosic hydrolysates contain non-negligible amounts of lignin-derived aromatic compounds. The biological funnel of lignin offers a new strategy for the "natural" production of protocatechuic acid (PCA). Herein, KT2440 was engineered to produce PCA from lignin-derived monomers in hydrolysates by knocking out protocatechuate 3,4-dioxygenase and overexpressing vanillate--demethylase endogenously, while acetic acid was used for cell growth. The sugar catabolism was further blocked to prevent the loss of fermentable sugar. Using the engineered strain, a total of 253.88 mg/L of PCA was obtained with a yield of 70.85% from corncob hydrolysate 1. The highest titer of 433.72 mg/L of PCA was achieved using corncob hydrolysate 2 without any additional nutrients. This study highlights the potential ability of engineered strains to address the challenges of PCA production from lignocellulosic hydrolysate, providing novel insights into the utilization of hydrolysates.
Topics: Lignin; Pseudomonas putida; Acetic Acid; Sugars; Hydroxybenzoates
PubMed: 38611834
DOI: 10.3390/molecules29071555 -
Communications Biology Apr 2024In their natural habitats, microbes rarely exist in isolation; instead, they thrive in consortia, where various interactions occur. In this study, a defined synthetic...
In their natural habitats, microbes rarely exist in isolation; instead, they thrive in consortia, where various interactions occur. In this study, a defined synthetic co-culture of the cyanobacterium S. elongatus cscB, which supplies sucrose to the heterotrophic P. putida cscRABY, is investigated to identify potential interactions. Initial experiments reveal a remarkable growth-promoting effect of the heterotrophic partner on the cyanobacterium, resulting in an up to 80% increase in the growth rate and enhanced photosynthetic capacity. Vice versa, the presence of the cyanobacterium has a neutral effect on P. putida cscRABY, highlighting the resilience of pseudomonads against stress and their potential as co-culture partners. Next, a suitable reference process reinforcing the growth-promoting effect is established in a parallel photobioreactor system, which sets the basis for the analysis of the co-culture at the transcriptome, proteome, and metabolome levels. In addition to several moderate changes, including alterations in the metabolism and stress response in both microbes, this comprehensive multi-OMICs approach strongly hints towards the exchange of further molecules beyond the unidirectional feeding with sucrose. Taken together, these findings provide valuable insights into the complex dynamics between both co-culture partners, indicating multi-level interactions, which can be employed for further streamlining of the co-cultivation system.
Topics: Pseudomonas putida; Coculture Techniques; Multiomics; Sucrose; Synechococcus
PubMed: 38609451
DOI: 10.1038/s42003-024-06098-5 -
Scientific Reports Apr 2024The recent expansion of multidrug-resistant (MDR) pathogens poses significant challenges in treating healthcare-associated infections. Although antibacterial resistance...
The recent expansion of multidrug-resistant (MDR) pathogens poses significant challenges in treating healthcare-associated infections. Although antibacterial resistance occurs by numerous mechanisms, active efflux of the drugs is a critical concern. A single species of efflux pump can produce a simultaneous resistance to several drugs. One of the best-studied efflux pumps is the TtgABC: a tripartite resistance-nodulation-division (RND) efflux pump implicated in the intrinsic antibiotic resistance in Pseudomonas putida DOT-T1E. The expression of the TtgABC gene is down-regulated by the HTH-type transcriptional repressor TtgR. In this context, by employing quantum chemistry methods based on the Density Functional Theory (DFT) within the Molecular Fragmentation with Conjugate Caps (MFCC) approach, we investigate the coupling profiles of the transcriptional regulator TtgR in complex with quercetin (QUE), a natural polyphenolic flavonoid, tetracycline (TAC), and chloramphenicol (CLM), two broad-spectrum antimicrobial agents. Our quantum biochemical computational results show the: [i] convergence radius, [ii] total binding energy, [iii] relevance (energetically) of the ligands regions, and [iv] most relevant amino acids residues of the TtgR-QUE/TAC/CLM complexes, pointing out distinctions and similarities among them. These findings improve the understanding of the binding mechanism of effectors and facilitate the development of new chemicals targeting TtgR, helping in the battle against the rise of resistance to antimicrobial drugs. These advances are crucial in the ongoing fight against rising antimicrobial drug resistance, providing hope for a future where healthcare-associated infections can be more beneficially treated.
Topics: Humans; Anti-Bacterial Agents; Chloramphenicol; Amino Acids; Antifibrinolytic Agents; Biological Transport; Cross Infection
PubMed: 38609407
DOI: 10.1038/s41598-024-58441-9 -
Molecular & Cellular Proteomics : MCP May 2024Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material,...
Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.
Topics: Polyhydroxyalkanoates; Pseudomonas putida; Proteomics; Lipoproteins; Bacterial Outer Membrane Proteins; Bacterial Proteins; Nitrogen; Plant Lectins
PubMed: 38608840
DOI: 10.1016/j.mcpro.2024.100765 -
Cellulitis with Pseudomonas putida bacteremia in a patient with autoimmune hepatitis: A case report.The Journal of Dermatology Apr 2024
PubMed: 38605436
DOI: 10.1111/1346-8138.17225 -
Journal of Microbiology & Biology... Apr 2024Nicotine is a major alkaloid in tobacco plants and an addictive component of tobacco products. Some bacteria grow on tobacco plants and have evolved the ability to...
Nicotine is a major alkaloid in tobacco plants and an addictive component of tobacco products. Some bacteria grow on tobacco plants and have evolved the ability to metabolize nicotine. As part of our microbiology teaching lab, we used minimal media with nicotine as the sole carbon source to isolate nicotine-degrading bacteria from tobacco leaves and commercial tobacco products. Students then identified these bacteria using 16S rRNA sequencing and biochemical assays and assessed their ability to catabolize nicotine using UV spectroscopy. Students were able to isolate and identify 14 distinct genera that can metabolize nicotine. This modification of the commonly used unknown project gave students firsthand experience using selective media, and students got the opportunity to work with largely uncharacterized microbes with a real-world connection to public health, which increased student engagement. Students had the opportunity to think critically about why nicotine-degrading microorganisms associate with tobacco plants, why there are different bacteria that use the same specialized metabolism, and how these organisms are isolated from other bacteria using selective media.
PubMed: 38602406
DOI: 10.1128/jmbe.00152-23 -
Journal of Applied Microbiology Apr 2024This study explores the biocontrol potential of Pseudomonas putida Z13 against Botrytis cinerea in tomato plants, addressing challenges posed by the pathogen's fungicide...
AIMS
This study explores the biocontrol potential of Pseudomonas putida Z13 against Botrytis cinerea in tomato plants, addressing challenges posed by the pathogen's fungicide resistance. The aims of the study were to investigate the in vitro and in silico biocontrol traits of Z13, identify its plant-colonizing efficacy, evaluate the efficacy of different application strategies against B. cinerea in planta, and assess the capacity of Z13 to trigger induced systemic resistance (ISR) in plants.
METHODS AND RESULTS
The in vitro experiments revealed that Z13 inhibits the growth of B. cinerea, produces siderophores, and exhibits swimming and swarming activity. Additionally, the Z13 genome harbors genes that encode compounds triggering ISR, such as pyoverdine and pyrroloquinoline quinone. The in planta experiments demonstrated Z13's efficacy in effectively colonizing the rhizosphere and leaves of tomato plants. Therefore, three application strategies of Z13 were evaluated against B. cinerea: root drenching, foliar spray, and the combination of root drenching and foliar spray. It was demonstrated that the most effective treatment of Z13 against B. cinerea was the combination of root drenching and foliar spray. Transcriptomic analysis showed that Z13 upregulates the expression of the plant defense-related genes PR1 and PIN2 upon B. cinerea inoculation.
CONCLUSION
The results of the study demonstrated that Z13 possesses significant biocontrol traits, such as the production of siderophores, resulting in significant plant protection against B. cinerea when applied as a single treatment to the rhizosphere or in combination with leaf spraying. Additionally, it was shown that Z13 root colonization primes plant defenses against the pathogen.
Topics: Solanum lycopersicum; Botrytis; Pseudomonas putida; Plant Diseases; Siderophores; Plant Roots; Rhizosphere; Biological Control Agents; Plant Leaves; Disease Resistance
PubMed: 38599633
DOI: 10.1093/jambio/lxae094 -
Biotechnology For Biofuels and... Apr 2024Petrochemicals contribute to environmental issues, with concerns ranging from energy consumption and carbon emission to pollution. In contrast, microbial biorefineries...
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.
PubMed: 38566218
DOI: 10.1186/s13068-024-02498-1