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PloS One 2022Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid...
Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid handler systems are increasingly used to overcome these issues for many of the sample preparation steps. Here, we detail a step-by-step protocol to prepare samples for bottom-up proteomic analysis for Gram-negative bacterial and fungal cells. The full modular protocol consists of three optimized protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of protein from 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 Pseudomonas putida and Rhodosporidium toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with protein relative quantity variance (CV%) below 15%.
Topics: Automation; Chromatography, High Pressure Liquid; Mass Spectrometry; Proteome; Proteomics; Pseudomonas putida; Reproducibility of Results; Rhodotorula; Specimen Handling
PubMed: 35213656
DOI: 10.1371/journal.pone.0264467 -
Journal of Microbiology and... Feb 2021Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The...
Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The presence of multiple isozyme-encoding genes is a widespread phenomenon that seems to give the carrying strains a selective advantage in the natural environment over those with only a single copy. In the naphthalene-degrading strain ND6, catechol can be converted into intermediates of the tricarboxylic acid cycle via either the - or -cleavage pathways. In this study, we demonstrated that the catechol ortho-cleavage pathway genes ( and ) on the chromosome play an important role. The and operons are co-transcribed, whereas and are under independent transcriptional regulation. We examined the binding of regulatory proteins to promoters. In the presence of -muconate, a well-studied inducer of the cat gene cluster, CatR and CatR occupy an additional downstream site, designated as the activation binding site. Notably, CatR binds to both the and promoters with high affinity, while CatR binds weakly. This is likely caused by a T to G mutation in the G/T-N-A motif. Specifically, we found that CatR and CatR regulate and in a cooperative manner, which provides new insights into naphthalene degradation.
Topics: Bacterial Proteins; Catechols; Gene Expression Regulation, Bacterial; Multigene Family; Operon; Promoter Regions, Genetic; Pseudomonas putida
PubMed: 33323670
DOI: 10.4014/jmb.2009.09026 -
International Journal of Molecular... May 2022The biofilm of is complexly regulated by several intercellular and extracellular factors. The cell surface adhesin LapA of this bacterium is a central factor for the...
The biofilm of is complexly regulated by several intercellular and extracellular factors. The cell surface adhesin LapA of this bacterium is a central factor for the biofilm and, consequently, the regulation of expression, for example, by Fis. It has been recently shown that peptides in growth media enhance the formation of biofilm, but not as a source of carbon and nitrogen. Moreover, the peptide-dependent biofilm appeared especially clearly in the -overexpression strain, which also has increased LapA. Therefore, we investigate here whether there is a relationship between LapA and peptide-dependent biofilm. The strains with inducible expression and LapA without the vWFa domain, which is described as a domain similar to von Willebrand factor domain A, were constructed. Thereafter, the biofilm of these strains was assessed in growth media containing extracellular peptides in the shape of tryptone and without it. We show that the vWFa domain in LapA is necessary for biofilm enhancement by the extracellular peptides in the growth medium. The importance of vWFa in LapA was particularly evident for the -overexpression strain F15. The absence of the vWFa domain diminished the positive effect of Fis on the F15 biofilm.
Topics: Bacterial Proteins; Biofilms; Gene Expression Regulation, Bacterial; Peptides; Pseudomonas putida
PubMed: 35682576
DOI: 10.3390/ijms23115898 -
The Plant Pathology Journal Feb 2023Previously, Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 from a sequential screening procedure were proven to effectively control Phytophthora blight...
Previously, Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 from a sequential screening procedure were proven to effectively control Phytophthora blight caused by Phytophthora capsici. In this study, we further investigated the anti-oomycete activities of these strains against mycelial growth, zoospore germination, and germ tube elongation of P. capsici. We also investigated root colonization ability of the bacterial strains in square dishes, including cell motility (swimming and swarming motilities) and biofilm formation. Both strains significantly inhibited mycelial growth in liquid and solid V8 juice media and M9 minimal media, zoospore germination, and germ tube elongation compared with Bacillus vallismortis EXTN-1 (positive biocontrol strain), Sphingomonas aquatilis KU408 (negative biocontrol strain), and MgSO4 solution (untreated control). In diluted (nutrient-deficient) V8 juice broth, the tested strain populations were maintained at >108 cells/ml, simultaneously providing mycelial inhibitory activity. Additionally, these strains colonized pepper roots at a 106 cells/ml concentration for 7 days. The root colonization of the strains was supported by strong swimming and swarming activities, biofilm formation, and chemotactic activity towards exudate components (amino acids, organic acids, and sugars) of pepper roots. Collectively, these results suggest that strains YJR13 and YJR92 can effectively suppress Phytophthora blight of pepper through direct anti-oomycete activities against mycelial growth, zoospore germination and germ tube elongation. Bacterial colonization of pepper roots may be mediated by cell motility and biofilm formation together with chemotaxis to root exudates.
PubMed: 36760054
DOI: 10.5423/PPJ.OA.01.2023.0001 -
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 -
Microorganisms Dec 2022Competition for nutrients and niches (CNN) is known to be one of the mechanisms for biocontrol mostly exhibited by strains. Phenotypic and full genome analysis revealed...
Competition for nutrients and niches (CNN) is known to be one of the mechanisms for biocontrol mostly exhibited by strains. Phenotypic and full genome analysis revealed PCL1760 controlling tomato foot and root rot (TFRR) solely through CNN mechanism. Although the availability of nutrients and motility are the known conditions for CNN, persistence of bacteria through dormancy by ribosomal hibernation is a key phenomenon to evade both biotic and abiotic stress. To confirm this hypothesis, S gene knockout mutant of PCL1760 (SB9) was first obtained through genetic constructions and compared with the wild type PCL1760. Primarily, relative expression of S in PCL1760 was conducted on tomato seedlings which showed a higher expression at the apical part (1.02 ± 0.18) of the plant roots than the basal (0.41 ± 0.13). The growth curve and persistence in ceftriaxone after the induction of starvation with rifampicin were performed on both strains. Colonization on the tomato root by CFU and qPCR, including biocontrol ability against was also tested. The growth dynamics of both PCL1760 and SB9 in basal and rich medium statistically did not differ ( ≤ 0.05). There was a significant difference observed in persistence showing PCL1760 to be more persistent than its mutant SB9, while SB9 (pJeM2:S) was 221.07 folds more than PCL1760. In colonization and biocontrol ability tests, PCL1760 was dominant over SB9 colonizing and controlling TFRR (in total, 3.044 × 10 to 6.95 × 10 fg/µL and 55.28% to 30.24%, respectively). The deletion of the S gene in PCL1760 reduced the persistence and effectiveness of the strain, suggesting persistence as one important characteristic of the CNN.
PubMed: 36677311
DOI: 10.3390/microorganisms11010019 -
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