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Microbial Biotechnology Jan 2020Over the past few decades, considerable interest has been shown in developing nano- and microcarriers with biocompatible and biodegradable materials for medical and...
Over the past few decades, considerable interest has been shown in developing nano- and microcarriers with biocompatible and biodegradable materials for medical and biotechnological applications. Microencapsulation is a technology capable of enhancing the survival rate of bacteria, providing stability in harsh environments. In the present paper, we developed a technology to encapsulate microorganisms within polyhydroxyalkanoate (PHA)-based microcapsules (MPs), employing a modified double emulsion solvent evaporation technique, with Pseudomonas putida KT2440 as a biotechnological model strain. The resulting MPs display a spherical morphology and an average particle size of 10 μm. The stability of the MPs was monitored under different conditions of storage and stress. The MPs remained stable for at least 24 days stored at 4°C in a water suspension. They exhibited greater tolerance to stress conditions; encapsulated cells remained viable for 2 h in alkaline solution and after 24 h of H O exposure at 10 and 20 mM. Results suggested the potential of MPs as a microcontainer of bacterial cells, even for biotechnological applications requiring high alkaline conditions and oxidative stress. We validated the potential applicability of the PHA-based microencapsulation method in other microorganisms by encapsulating the predatory bacterium Bdellovibrio bacteriovorus.
Topics: Polyhydroxyalkanoates; Pseudomonas putida
PubMed: 31714682
DOI: 10.1111/1751-7915.13492 -
Environmental Microbiology May 2021Interkingdom communication is of particular relevance in polymicrobial biofilms. In this work, the ability of the fungus Ophiostoma piceae to form biofilms individually...
Interkingdom communication is of particular relevance in polymicrobial biofilms. In this work, the ability of the fungus Ophiostoma piceae to form biofilms individually and in consortium with the bacterium Pseudomonas putida, as well as the effect of fungal and bacterial signal molecules on the architecture of the biofilms was evaluated. Pseudomonas putida KT2440 is able to form biofilms through the secretion of exopolysaccharides and two large extracellular adhesion proteins, LapA and LapF. It has two intercellular signalling systems, one mediated by dodecanoic acid and an orphan LuxR receptor that could participate in the response to AHL-type quorum sensing molecules (QSMs). Furthermore, the dimorphic fungus O. piceae uses farnesol as QSM to control its yeast to hyphae morphological transition. Results show for the first time the ability of this fungus to form biofilms alone and in mixed cultures with the bacterium. Biofilms were induced by bacterial and fungal QSMs. The essential role of LapA-LapF proteins in the architecture of biofilms was corroborated, LapA was induced by farnesol and dodecanol, while LapF by 3-oxo-C6-HSL and 3-oxo-C12-HSL. Our results indicate that fungal signals can induce a transient rise in the levels of the secondary messenger c-di-GMP, which control biofilm formation and architecture.
Topics: Biofilms; Fungi; Ophiostoma; Pseudomonas putida; Quorum Sensing
PubMed: 33615654
DOI: 10.1111/1462-2920.15444 -
Microbial Biotechnology Jan 2016Pseudomonas putida BIRD-1 has the potential to be used for the industrial production of butanol due to its solvent tolerance and ability to metabolize low-cost...
Pseudomonas putida BIRD-1 has the potential to be used for the industrial production of butanol due to its solvent tolerance and ability to metabolize low-cost compounds. However, the strain has two major limitations: it assimilates butanol as sole carbon source and butanol concentrations above 1% (v/v) are toxic. With the aim of facilitating BIRD-1 strain design for industrial use, a genome-wide mini-Tn5 transposon mutant library was screened for clones exhibiting increased butanol sensitivity or deficiency in butanol assimilation. Twenty-one mutants were selected that were affected in one or both of the processes. These mutants exhibited insertions in various genes, including those involved in the TCA cycle, fatty acid metabolism, transcription, cofactor synthesis and membrane integrity. An omics-based analysis revealed key genes involved in the butanol response. Transcriptomic and proteomic studies were carried out to compare short and long-term tolerance and assimilation traits. Pseudomonas putida initiates various butanol assimilation pathways via alcohol and aldehyde dehydrogenases that channel the compound to central metabolism through the glyoxylate shunt pathway. Accordingly, isocitrate lyase - a key enzyme of the pathway - was the most abundant protein when butanol was used as the sole carbon source. Upregulation of two genes encoding proteins PPUBIRD1_2240 and PPUBIRD1_2241 (acyl-CoA dehydrogenase and acyl-CoA synthetase respectively) linked butanol assimilation with acyl-CoA metabolism. Butanol tolerance was found to be primarily linked to classic solvent defense mechanisms, such as efflux pumps, membrane modifications and control of redox state. Our results also highlight the intensive energy requirements for butanol production and tolerance; thus, enhancing TCA cycle operation may represent a promising strategy for enhanced butanol production.
Topics: Acetate-CoA Ligase; Acyl-CoA Dehydrogenases; Bacterial Proteins; Butanols; Proteomics; Pseudomonas putida
PubMed: 26986205
DOI: 10.1111/1751-7915.12328 -
Microbial Cell Factories Jun 2022Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET)....
BACKGROUND
Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.
RESULTS
P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.
CONCLUSION
Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.
Topics: Biocatalysis; Hydrolases; Plastics; Polyethylene Terephthalates; Pseudomonas putida
PubMed: 35717313
DOI: 10.1186/s12934-022-01849-7 -
Microbial Biotechnology Sep 2021Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in...
Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two-step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan-genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6-phosphogluconate and subsequently metabolizes it through the Entner-Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked-out to avoid the production of the dead-end product xylonate. We generated a set of DOT-T1E-derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT-T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes.
Topics: Amino Acids, Aromatic; Glucose; Lignin; Pseudomonas putida; Xylose
PubMed: 34403199
DOI: 10.1111/1751-7915.13844 -
FEMS Microbiology Letters Jan 2023Amino acids are crucial in nitrogen cycling and to shape the metabolism of microorganisms. Among them, arginine is a versatile molecule able to sustain nitrogen, carbon,...
Amino acids are crucial in nitrogen cycling and to shape the metabolism of microorganisms. Among them, arginine is a versatile molecule able to sustain nitrogen, carbon, and even ATP supply and to regulate multicellular behaviors such as biofilm formation. Arginine modulates the intracellular levels of 3'-5'cyclic diguanylic acid (c-di-GMP), a second messenger that controls biofilm formation, maintenance and dispersion. In Pseudomonas putida, KT2440, a versatile microorganism with wide biotechnological applications, modulation of c-di-GMP levels by arginine requires the transcriptional regulator ArgR, but the connections between arginine metabolism and c-di-GMP are not fully characterized. It has been recently demonstrated that arginine can be perceived by the opportunistic human pathogen Pseudomonas aeruginosa through the transducer RmcA protein (Redox regulator of c-di-GMP), which can directly decrease c-di-GMP levels and possibly affect biofilm architecture. A RmcA homolog is present in P. putida, but its function and involvement in arginine perceiving or biofilm life cycle had not been studied. Here, we present a preliminary characterization of the RmcA-dependent response to arginine in P. putida in modulating biofilm formation, c-di-GMP levels, and energy metabolism. This work contributes to further understanding the molecular mechanisms linking biofilm homeostasis and environmental adaptation.
Topics: Humans; Bacterial Proteins; Pseudomonas putida; Phosphoric Diester Hydrolases; Cyclic GMP; Biofilms; Arginine; Pseudomonas aeruginosa; Gene Expression Regulation, Bacterial
PubMed: 37550221
DOI: 10.1093/femsle/fnad077 -
PloS One 2015By shedding light on variation in time as well as in space, long-term biogeographic studies can help us define organisms' distribution patterns and understand their... (Clinical Trial)
Clinical Trial
By shedding light on variation in time as well as in space, long-term biogeographic studies can help us define organisms' distribution patterns and understand their underlying drivers. Here we examine distributions of Pseudomonas in and around 15 human homes, focusing on the P. putida and P. fluorescens species groups. We describe recovery from 10,941 samples collected during up to 8 visits per home, occurring on average 2.6 times per year. We collected a mean of 141 samples per visit, from sites in most rooms of the house, from the surrounding yards, and from human and pet occupants. We recovered Pseudomonas in 9.7% of samples, with the majority of isolates being from the P. putida and P. fluorescens species groups (approximately 62% and 23% of Pseudomonas samples recovered respectively). Although representatives of both groups were recovered from every season, every house, and every type of environment sampled, recovery was highly variable across houses and samplings. Whereas recovery of P. putida group was higher in summer and fall than in winter and spring, P. fluorescens group isolates were most often recovered in spring. P. putida group recovery from soils was substantially higher than its recovery from all other environment types, while higher P. fluorescens group recovery from soils than from other sites was much less pronounced. Both species groups were recovered from skin and upper respiratory tract samples from healthy humans and pets, although this occurred infrequently. This study indicates that even species that are able to survive under a broad range of conditions can be rare and variable in their distributions in space and in time. For such groups, determining patterns and causes of stochastic and seasonal variability may be more important for understanding the processes driving their biogeography than the identity of the types of environments in which they can be found.
Topics: Adult; Animals; Base Sequence; Female; Housing; Humans; Kentucky; Male; Molecular Sequence Data; Pets; Pseudomonas fluorescens; Pseudomonas putida; Respiratory System; Seasons; Skin; Soil Microbiology
PubMed: 26023929
DOI: 10.1371/journal.pone.0127704 -
International Journal of Molecular... Aug 2023A novel group of conjugative plasmids of is characterized. The prototype plasmid pPPUT-Tik1-1 (153,663 bp), isolated from a permafrost strain of Tik1, carries a...
A novel group of conjugative plasmids of is characterized. The prototype plasmid pPPUT-Tik1-1 (153,663 bp), isolated from a permafrost strain of Tik1, carries a defective mercury transposon, Tn, and a streptomycin resistance transposon, Tn Ten plasmids and 34 contigs with backbone regions closely related to pPPUT-Tik1-1 have been found in GenBank. Two of these plasmids from clinical strains of and are almost identical to the ancient plasmid. A characteristic feature of this group of plasmids is the presence of two genes encoding the initiators of replication ( and ). None of these genes have high similarity with plasmid replication genes belonging to known incompatibility groups. It has been demonstrated that while pPPUT-Tik1-1-like plasmids have homologous backbone regions, they significantly differ by the molecular structure and the predicted functions of their accessory regions. Some of the pPPUT-Tik1-1-related plasmids carry determinants of antibiotic resistance and/or heavy metal salts. Some plasmids are characterized by the ability to degrade xenobiotics. Plasmids related to pPPUT-Tik1-1 are characterized by a narrow host range and are found in various species of the genus. Interestingly, we also found shorter plasmid variants containing the same replication module, but lacking conjugation genes and containing other structural changes that strongly distinguish them from plasmids related to pPPUT-Tik1-1, indicating that the structure of the replication module cannot be used as the sole criterion for classifying plasmids. Overall, the results suggest that the plasmids of the novel group can be spread using conjugation in environmental and clinical strains of and may play diverse adaptive functions due to the presence of various accessory regions.
Topics: Pseudomonas putida; Permafrost; Pseudomonas; Databases, Nucleic Acid; Host Specificity
PubMed: 37686323
DOI: 10.3390/ijms241713518 -
Nature Microbiology Oct 2022Many bacteria utilize contact-dependent killing machineries to eliminate rivals in their environmental niches. Here we show that the plant root colonizer Pseudomonas...
Many bacteria utilize contact-dependent killing machineries to eliminate rivals in their environmental niches. Here we show that the plant root colonizer Pseudomonas putida strain IsoF is able to kill a wide range of soil and plant-associated Gram-negative bacteria with the aid of a type IVB secretion system (T4BSS) that delivers a toxic effector into bacterial competitors in a contact-dependent manner. This extends the range of targets of T4BSSs-so far thought to transfer effectors only into eukaryotic cells-to prokaryotes. Bioinformatic and genetic analyses showed that this killing machine is entirely encoded by the kib gene cluster located within a rare genomic island, which was recently acquired by horizontal gene transfer. P. putida IsoF utilizes this secretion system not only as a defensive weapon to kill bacterial competitors but also as an offensive weapon to invade existing biofilms, allowing the strain to persist in its natural environment. Furthermore, we show that strain IsoF can protect tomato plants against the phytopathogen Ralstonia solanacearum in a T4BSS-dependent manner, suggesting that IsoF can be exploited for pest control and sustainable agriculture.
Topics: Biofilms; Solanum lycopersicum; Pseudomonas putida; Ralstonia solanacearum; Soil
PubMed: 36123439
DOI: 10.1038/s41564-022-01209-6 -
MBio Jun 2019Nicotine, a toxic and addictive alkaloid from tobacco, is an environmental pollutant in areas near cigarette production facilities. Over the last decade, our group has...
Nicotine, a toxic and addictive alkaloid from tobacco, is an environmental pollutant in areas near cigarette production facilities. Over the last decade, our group has studied, in depth, the pyrrolidine pathway of nicotine degradation in S16. However, little is known regarding whole mechanism(s) regulating transcription of the nicotine degradation pathway gene cluster. In the present study, we comprehensively elucidate an overall view of the NicR2-mediated two-step mechanism regulating 3-succinoyl-pyridine (SP) biotransformation, which involves the association of free NicR2 with two promoters and the dissociation of NicR2 from the NicR2-promoter complex. NicR2 can bind to another promoter, , and regulate expression of the nicotine-degrading genes in the middle of gene cluster, which are not controlled by the previously reported promoter. We identified the function of the inverted repeat bases on the two promoters responsible for NicR2 binding and found out that the -35/-10 motif for RNA polymerase is overlapped by the NicR2 binding site. We clarify the exact role of 6-hydroxy-3-succinoyl-pyridine (HSP), which acts as an antagonist and may prevent binding of free NicR2 to the promoters but cannot release NicR2 from the promoters. Finally, a regulatory model is proposed, which consists of three parts: the interaction between NicR2 and two promoters ( and ), the interaction between NicR2 and two effectors (HSP and SP), and the interaction between NicR2 and RNA polymerase. We report the entire process underlying the NicR2 regulatory mechanism from association between free NicR2 and two promoters to dissociation of the NicR2-promoter complex. NicR2 can bind to another promoter, , which controls expression of nicotine-degrading genes that are not controlled by the promoter. We identified specific nucleotides of the promoter responsible for NicR2 binding. HSP was further demonstrated as an antagonist, which prevents the binding of NicR2 to the and promoters, by locking NicR2 in the derepression conformation. The competition between NicR2 and RNA polymerase is essential to initiate transcription of nicotine-degrading genes. This study extends our understanding of molecular mechanisms in biodegradation of environmental pollutants and toxicants.
Topics: Biodegradation, Environmental; Gene Expression Regulation, Bacterial; Metabolic Networks and Pathways; Multigene Family; Nicotine; Promoter Regions, Genetic; Pseudomonas putida; Pyridines; Succinates
PubMed: 31164460
DOI: 10.1128/mBio.00602-19