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Infection, Genetics and Evolution :... Dec 2021The Pseudomonas putida group (P. putida G) is composed of at least 21 species associated with a wide range of environments, including the clinical setting. Here, we...
The Pseudomonas putida group (P. putida G) is composed of at least 21 species associated with a wide range of environments, including the clinical setting. Here, we characterized 13 carbapenem-resistant P. putida G clinical isolates bearing class 1 integrons/transposons (class 1 In/Tn) carrying bla metallo-β-lactamase gene cassettes obtained from hospitals of Argentina. Multilocus sequencing (MLSA) and phylogenetic analyses based on 16S rDNA, gyrB and rpoD sequences distinguished 7 species among them. bla was found in three different cassette arrays: In41 (bla-aacA4), In899 (only bla), and In528 (dfrB1-aacA4-bla). In41 and In899 were associated with complete tniABQC transposition modules and IRi/IRt boundaries characteristic of the Tn5053/Tn402 transposons, which were designated Tn6335 and Tn6336, respectively. The class 1 In/Tn element carrying In528, however, exhibited a defective tni module bearing only the tniC (transposase) gene, associated with a complete IS6100 bounded with two oppositely-oriented IRt end regions. In some P. putida G isolates including P. asiatica, P. juntendi, P. putida G/II, and P. putida G/V, Tn6335/Tn6336 were carried by pLD209-type conjugative plasmids capable of self-mobilization to P. aeruginosa or Escherichia coli. In other isolates of P. asiatica, P. putida G/II, and P. monteiliieilii, however, these bla-containing class 1 In/Tn elements were found inserted into the res regions preceding the tnpR (resolvase) gene of particular Tn21 subgroup members of Tn3 transposons. The overall results reinforce the notion of P. putida G members as bla reservoirs, and shed light on the mechanisms of dissemination of carbapenem resistance genes to other pathogenic bacteria in the clinical setting.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Carbapenems; DNA Transposable Elements; Drug Resistance, Bacterial; Integrons; Pseudomonas putida; beta-Lactamases
PubMed: 34748986
DOI: 10.1016/j.meegid.2021.105131 -
ACS Synthetic Biology Nov 2022KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. However, tools for studying,...
KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. However, tools for studying, engineering, and modulating protein complexes and biosynthetic enzymes in this organism are largely underdeveloped. Genetic code expansion for the incorporation of unnatural amino acids (unAAs) into proteins can advance such efforts and, furthermore, enable additional controls of biological processes of the strain. In this work, we established the orthogonality of two widely used archaeal tRNA synthetase and tRNA pairs in KT2440. Following the optimization of decoding systems, four unAAs were incorporated into proteins in response to a UAG stop codon at 34.6-78% efficiency. In addition, we demonstrated the utility of genetic code expansion through the incorporation of a photocross-linking amino acid, -benzoyl-l-phenylalanine (pBpa), into glutathione -transferase (GstA) and a chemosensory response regulator (CheY) for protein-protein interaction studies in KT2440. This work reported the successful genetic code expansion in KT2440 for the first time. Given the diverse structure and functions of unAAs that have been added to protein syntheses using the archaeal systems, our research lays down a solid foundation for future work to study and enhance the biological functions of KT2440.
Topics: Pseudomonas putida; Genetic Code; Amino Acyl-tRNA Synthetases; RNA, Transfer; Amino Acids
PubMed: 36287825
DOI: 10.1021/acssynbio.2c00325 -
Microbes and Environments 2023Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they...
Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they potentially differ from the genetically defined "true Pseudomonas putida" because many were classified as P. putida based on their phenotypic and metabolic characteristics. A phylogenetic ana-lysis based on the concatenated sequences of the 16S rRNA and rpoD genes revealed that 46 strains of P. putida deposited in Japanese culture collections were classified into nine operational taxonomic units (OTUs) and eleven singletons. The OTU7 strain produces N-acylhomoserine lactone as a quorum-sensing signal. One of the OTU7 strains, JCM 20066, exhibited a ppuI-rsaL-ppuR quorum-sensing system that controls biofilm formation and motility. The P. putida type strain JCM 13063 and six other strains were classified as OTU4. Classification based on the calculation of whole-genome similarity revealed that three OTU4 strains, JCM 20005, 21368, and 13061, were regarded as the same species as JCM 13063 and defined as true P. putida. When orthologous genes in the whole-genome sequences of true P. putida strains were screened, PP4_28660 from P. putida NBRC 14164 (=JCM 13063) was present in all true P. putida genome sequences. The internal region of PP4_28660 was successfully amplified from all true P. putida strains using the specific primers designed in this study.
Topics: Bacterial Typing Techniques; DNA, Bacterial; Fatty Acids; Genomics; Phylogeny; Pseudomonas putida; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 37286511
DOI: 10.1264/jsme2.ME23019 -
Scientific Reports Sep 2021Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter...
Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/P was also efficiently induced by LA. LvaR/P and XutR/P systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/P showed weak GFP expression. These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.
Topics: Flow Cytometry; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Reporter; Genetic Engineering; Genetic Vectors; Glucose; Lactic Acid; Promoter Regions, Genetic; Pseudomonas putida; Recombinant Proteins
PubMed: 34508142
DOI: 10.1038/s41598-021-97550-7 -
Biotechnology and Bioengineering Dec 2021The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be...
The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen. Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrial-scale bioprocesses.
Topics: Biomass; Bioreactors; Carbon; Glycolipids; Metabolic Engineering; Oxygen; Pseudomonas putida
PubMed: 34506651
DOI: 10.1002/bit.27938 -
Journal of Oleo Science Apr 2021A total of 100 environmental samples were investigated for their ability to degrade 1 g/L surfactin as a substrate. Among them, two enrichment cultures, which exhibited...
A total of 100 environmental samples were investigated for their ability to degrade 1 g/L surfactin as a substrate. Among them, two enrichment cultures, which exhibited microbial growth as well as surfactin degradation, were selected and further investigated. After several successive cultivations, nanopore sequencing of full-length 16S rRNA genes with MinION was used to analyze the bacterial species in the enrichment cultures. Variovorax spp., Caulobacter spp., Sphingopyxis spp., and Pseudomonas spp. were found to be dominant in these surfactin-degrading mixed cultures. Finally, one strain of Pseudomonas putida was isolated as a surfactin-degrading bacterium. This strain degraded 1 g/L surfactin below a detectable level within 14 days, and C surfactin was degraded faster than C surfactin.
Topics: Biodegradation, Environmental; Caulobacter; Comamonadaceae; Lipopeptides; Peptides, Cyclic; Pseudomonas putida; Sphingomonadaceae; Surface-Active Agents
PubMed: 33692244
DOI: 10.5650/jos.ess20331 -
Bioresource Technology Mar 2023Preventing catastrophic climate events warrants prompt action to delay global warming, which threatens health and food security. In this context, waste management using... (Review)
Review
Preventing catastrophic climate events warrants prompt action to delay global warming, which threatens health and food security. In this context, waste management using engineered microbes has emerged as a long-term eco-friendly solution for addressing the global climate crisis and transitioning to clean energy. Notably, Pseudomonas putida can valorize industry-derived synthetic wastes including plastics, oils, food, and agricultural waste into products of interest, and it has been extensively explored for establishing a fully circular bioeconomy through the conversion of waste into bio-based products, including platform chemicals (e.g., cis,cis-muconic and adipic acid) and biopolymers (e.g., medium-chain length polyhydroxyalkanoate). However, the efficiency of waste pretreatment technologies, capability of microbial cell factories, and practicability of synthetic biology tools remain low, posing a challenge to the industrial application of P. putida. The present review discusses the state-of-the-art, challenges, and future prospects for divergent biosynthesis of versatile products from waste-derived feedstocks using P. putida.
Topics: Pseudomonas putida; Biopolymers; Polyhydroxyalkanoates; Industrial Waste
PubMed: 36638894
DOI: 10.1016/j.biortech.2023.128607 -
Preparative Biochemistry & Biotechnology 2021Biobased chemicals are gaining popularity and market in attempts to mitigate the deteriorating environmental and sustainability issues. Components of renewable...
Biobased chemicals are gaining popularity and market in attempts to mitigate the deteriorating environmental and sustainability issues. Components of renewable agricultural and forest biomass residues are projected to serve as abundant precursors to synthesis of expanding range of products. Agroindustrial wastes comprises of several phenolic compounds associated with lignin via ether linkages such as ferulic acid, p-coumaric, syringic acid and vanillin. These aromatic chemicals have myriad industrial applications. In this study, p-coumaric acid and ferulic acid were found to be two major components in corn bran derived lignin hydrolysate. Engineered KT2440 was constructed and found to convert p-coumaric acid and vanillic acid to protocatechuic acid in >90% and >50% yields, respectively. Engineering the strain included deletion of the gene encoding protocatechuate 3,4-dioxygenase, and overexpression of vanillate-O-demethylase gene from . ADP1.
Topics: Coumaric Acids; Hydroxybenzoates; Industrial Microbiology; Lignin; Phenols; Pseudomonas putida; Zea mays
PubMed: 33583338
DOI: 10.1080/10826068.2021.1881908 -
Microbiology (Reading, England) Jan 2023The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive...
The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS-GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.
Topics: Type VI Secretion Systems; Bacterial Proteins; Pseudomonas putida; Sigma Factor; Multigene Family; Gene Expression Regulation, Bacterial
PubMed: 36748579
DOI: 10.1099/mic.0.001295 -
Microbial Cell Factories Jan 2023Pseudomonas putida has received increasing interest as a cell factory due to its remarkable features such as fast growth, a versatile and robust metabolism, an extensive...
BACKGROUND
Pseudomonas putida has received increasing interest as a cell factory due to its remarkable features such as fast growth, a versatile and robust metabolism, an extensive genetic toolbox and its high tolerance to oxidative stress and toxic compounds. This interest is driven by the need to improve microbial performance to a level that enables biologically possible processes to become economically feasible, thereby fostering the transition from an oil-based economy to a more sustainable bio-based one. To this end, one of the current strategies is to maximize the product-substrate yield of an aerobic biocatalyst such as P. putida during growth on glycolytic carbon sources, such as glycerol and xylose. We demonstrate that this can be achieved by implementing the phosphoketolase shunt, through which pyruvate decarboxylation is prevented, and thus carbon loss is minimized.
RESULTS
In this study, we introduced the phosphoketolase shunt in the metabolism of P. putida KT2440. To maximize the effect of this pathway, we first tested and selected a phosphoketolase (Xfpk) enzyme with high activity in P. putida. Results of the enzymatic assays revealed that the most efficient Xfpk was the one isolated from Bifidobacterium breve. Using this enzyme, we improved the P. putida growth rate on glycerol and xylose by 44 and 167%, respectively, as well as the biomass yield quantified by OD by 50 and 30%, respectively. Finally, we demonstrated the impact on product formation and achieved a 38.5% increase in mevalonate and a 25.9% increase in flaviolin yield from glycerol. A similar effect was observed on the mevalonate-xylose and flaviolin-xylose yields, which increased by 48.7 and 49.4%, respectively.
CONCLUSIONS
Pseudomonas putida with the implemented Xfpk shunt grew faster, reached a higher final OD and provided better product-substrate yields than the wild type. By reducing the pyruvate decarboxylation flux, we significantly improved the performance of this important workhorse for industrial applications. This work encompasses the first steps towards full implementation of the non-oxidative glycolysis (NOG) or the glycolysis alternative high carbon yield cycle (GATCHYC), in which a substrate is converted into products without CO loss These enhanced properties of P. putida will be crucial for its subsequent use in a range of industrial processes.
Topics: Pseudomonas putida; Xylose; Glycerol; Mevalonic Acid; Pyruvates; Carbon
PubMed: 36658566
DOI: 10.1186/s12934-022-02015-9