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Journal of Bacteriology Sep 2019TonB-ExbB-ExbD-like energy transduction systems are widespread among Gram-negative bacteria. While most species have only one copy of -e genes, the species possess more...
TonB-ExbB-ExbD-like energy transduction systems are widespread among Gram-negative bacteria. While most species have only one copy of -e genes, the species possess more TonB-ExbBD homologues. One of them, the TonB3-PocA-PocB complex, was recently shown to be required for polar localization of FlhF and, thus, the flagella in Here, we show that the orthologous TonB-PocA-PocB complex is important for polar localization of FlhF and flagella in as well. Additionally, the system is necessary for maintaining membrane integrity, as the inactivation of the TonB-PocAB complex results in increased membrane permeability, lowered stress tolerance, and conditional cell lysis. Interestingly, the functionality of TonB-PocAB complex is more important for stationary than for exponentially growing bacteria. The whole-cell proteome analysis provided a likely explanation for this growth phase dependence, as extensive reprogramming was disclosed in an exponentially growing deletion strain, while only a few proteomic changes, mostly downregulation of outer membrane proteins, were determined in the stationary-phase strain. We propose that this response in exponential phase, involving, , activation of AlgU and ColR regulons, can compensate for TonB-PocAB's deficiency, while stationary-phase cells are unable to alleviate the lack of TonB-PocAB. Our results suggest that mislocalization of flagella does not cause the membrane integrity problems; rather, the impaired membrane intactness of the TonB-PocAB-deficient strain could be the reason for the random placement of flagella. The ubiquitous species are well adapted to survive in a wide variety of environments. Their success relies on their versatile metabolic, signaling, and transport ability but also on their high intrinsic tolerance to various stress factors. This is why the study of the stress-surviving mechanisms of species is of utmost importance. The stress tolerance of is mainly achieved through the high barrier property of their membranes. Here, we present evidence that the TonB-ExbBD-like TonB-PocAB system is involved in maintaining the membrane homeostasis of , and its deficiency leads to lowered stress tolerance and conditional cell lysis.
Topics: Bacterial Proteins; Cell Membrane; Conserved Sequence; DNA, Bacterial; Down-Regulation; Flagella; Gene Expression Regulation, Bacterial; Membrane Proteins; Proteomics; Pseudomonas putida
PubMed: 31182498
DOI: 10.1128/JB.00303-19 -
Systematic and Applied Microbiology Jul 2019Bacteria of the Pseudomonas putida group are studied for a large panel of properties ranging from plant growth promotion and bioremediation to pathogenicity. To date,...
Genomic, phylogenetic and catabolic re-assessment of the Pseudomonas putida clade supports the delineation of Pseudomonas alloputida sp. nov., Pseudomonas inefficax sp. nov., Pseudomonas persica sp. nov., and Pseudomonas shirazica sp. nov.
Bacteria of the Pseudomonas putida group are studied for a large panel of properties ranging from plant growth promotion and bioremediation to pathogenicity. To date, most of the classification of individual pseudomonads from this group relies on 16S RNA gene analysis, which is insufficient for accurate taxonomic characterization within bacterial species complexes of the Pseudomonas putida group. Here, a collection of 20 of these bacteria, isolated from various soils, was assessed via multi-locus sequence analysis of rpoD, gyrB and rrs genes. The 20 strains clustered in 7 different clades of the P. putida group. One strain per cluster was sequenced and results were compared to complete genome sequences of type strains of the P. putida group. Phylogenetic analyses, average nucleotide identity data and digital DNA hybridizations, combined to phenotypic characteristics, resulted in the proposition and description of four new species i.e. Pseudomonas alloputida Kh7 (= LMG 29756 = CFBP 8484 ) sp. nov., Pseudomonas inefficax JV551A3 (= DSM108619 = CFBP 8493 ) sp. nov., Pseudomonas persica RUB6 (= LMG 29757 = CFBP 8486 ) sp. nov. and Pseudomonas shirazica VM14 (= LMG 29953 = CFBP 8487 ) sp. nov.
Topics: DNA, Bacterial; Genes, Bacterial; Genes, Essential; Genome, Bacterial; Nucleic Acid Hybridization; Phenotype; Phylogeny; Pseudomonas putida; Sequence Analysis, DNA; Soil Microbiology; Species Specificity
PubMed: 31122691
DOI: 10.1016/j.syapm.2019.04.004 -
Brazilian Journal of Microbiology :... Oct 2019Degradation or the removal of aflatoxin B from agriculture commodities is very important because of its acute toxicity and economic loss due to rejection of about 25%...
Degradation or the removal of aflatoxin B from agriculture commodities is very important because of its acute toxicity and economic loss due to rejection of about 25% contaminated agri produce. The present study aimed at using Pseudomonas putida for the aflatoxin B (AFB) degradation and to understand the mechanism involved. AFB degradation was studied with P. putida culture, culture supernatant, cell lysate, cell lysate in the presence of protease inhibitor, and heat-inactivated cell lysate. The remaining AFB was qualitatively and quantitatively measured by thin-layer chromatography and HPLC with a UV detector. P. putida culture and culture supernatant showed 80% reduction in AFB within 24 h of incubation. Cell lysate and the lysate in the presence of protease inhibitor showed the same reduction in 6 and 4 h respectively. The protease-inhibited lysate showed greater thermostability, broad pH range, and tolerance to some of the solvents and detergents in terms of aflatoxin B degrading activity. The heat-inactivated lysate showed only 20% reduction in 24 h of incubation indicating loss of activity on heating. As cell-free supernatant and cell lysate are capable of reducing AFB effectively, actively growing cells are not necessary for degradation. The active principle for degradation might be proteinaceous; therefore, heat-inactivated lysate is ineffective for reducing the AFB. These results showed that degradation of aflatoxin B by P. putida might be an enzymatic process and could be used in a broad range of conditions.
Topics: Aflatoxin B1; Bacterial Proteins; Biodegradation, Environmental; Hot Temperature; Hydrogen-Ion Concentration; Peptide Hydrolases; Pseudomonas putida
PubMed: 31401783
DOI: 10.1007/s42770-019-00134-x -
Journal of Bacteriology Mar 2009Pseudomonas putida harbors two ferredoxin-NADP(+) reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally...
Pseudomonas putida harbors two ferredoxin-NADP(+) reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k(cat)/K(m) value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.
Topics: Culture Media; FMN Reductase; Ferredoxin-NADP Reductase; Gene Expression Regulation, Bacterial; Heat-Shock Response; Iron; Kinetics; Models, Molecular; Mutation; Pseudomonas putida
PubMed: 19114475
DOI: 10.1128/JB.01473-08 -
Microbial Biotechnology Nov 2016Currently, chlorpyrifos (CP) and carbofuran are often applied together to control major agricultural pests in many developing countries, in most cases, they are...
Currently, chlorpyrifos (CP) and carbofuran are often applied together to control major agricultural pests in many developing countries, in most cases, they are simultaneously detected in agricultural soils. Some cost-effective techniques are required for the remediation of combined pollution caused by multiple pesticides. In this work, we aim at constructing a detectable recombinant microorganism with the capacity to simultaneously degrade CP and carbofuran. To achieve this purpose, CP/carbofuran hydrolase genes and gfp were integrated into the chromosome of a biosafety strain Pseudomonas putida KT2440 using a chromosomal scarless modification strategy with upp as a counter-selectable marker. The toxicity of the hydrolysis products was significantly lower compared with the parent compounds. The recombinant strain could utilize CP or carbofuran as the sole source of carbon for growth. The inoculation of the recombinant strain to soils treated with carbofuran and CP resulted in a higher degradation rate than in noninoculated soils. Introduced green fluorescent protein can be employed as a biomarker to track the recombinant strain during bioremediation. Therefore, the recombinant strain has potential to be applied for in situ bioremediation of soil co-contaminated with carbofuran and CP.
Topics: Biotransformation; Carbofuran; Carbon; Chlorpyrifos; Green Fluorescent Proteins; Insecticides; Metabolic Engineering; Pseudomonas putida; Sequence Analysis, DNA; Soil; Soil Microbiology; Staining and Labeling
PubMed: 27418102
DOI: 10.1111/1751-7915.12381 -
Applied and Environmental Microbiology Apr 2021S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of S12...
S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of S12 carries several gene clusters involved in solvent tolerance, and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent tolerance of this strain. Whole-genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% [vol/vol]). We identified mutations in an RND efflux pump regulator, , that resulted in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β', a global two-component regulatory system (GacA/GacS), and a putative AraC family transcriptional regulator, Afr. Transcriptomic analysis further revealed a constitutive downregulation of energy-consuming activities in ALE-derived strains, such as flagellar assembly, FF ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of the solvent tolerance trait in S12 lacking its megaplasmid. Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of ; however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured S12 strain to regain its solvent tolerance. Through these adapted strains, we began to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in This work sheds light on the possible genetic engineering targets to enhance solvent tolerance in as well as other bacteria.
Topics: Laboratories; Mutation; Plasmids; Polymorphism, Single Nucleotide; Pseudomonas putida; Solvents; Toluene
PubMed: 33674430
DOI: 10.1128/AEM.00041-21 -
Microbial Cell Factories May 2016Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas...
BACKGROUND
Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas putida KT2440 can use glycerol to synthesize medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA), a class of biopolymers of industrial interest. Here, glycerol metabolism in P. putida KT2440 was studied on the level of gene expression (transcriptome) and metabolic fluxes (fluxome), using precisely adjusted chemostat cultures, growth kinetics and stoichiometry, to gain a systematic understanding of the underlying metabolic and regulatory network.
RESULTS
Glycerol-grown P. putida KT2440 has a maintenance energy requirement [0.039 (mmolglycerol (gCDW h)(-1))] that is about sixteen times lower than that of other bacteria, such as Escherichia coli, which provides a great advantage to use this substrate commercially. The shift from carbon (glycerol) to nitrogen (ammonium) limitation drives the modulation of specific genes involved in glycerol metabolism, transport electron chain, sensors to assess the energy level of the cell, and PHA synthesis, as well as changes in flux distribution to increase the precursor availability for PHA synthesis (Entner-Doudoroff pathway and pyruvate metabolism) and to reduce respiration (glyoxylate shunt). Under PHA-producing conditions (N-limitation), a higher PHA yield was achieved at low dilution rate (29.7 wt% of CDW) as compared to a high rate (12.8 wt% of CDW). By-product formation (succinate, malate) was specifically modulated under these regimes. On top of experimental data, elementary flux mode analysis revealed the metabolic potential of P. putida KT2440 to synthesize PHA and identified metabolic engineering targets towards improved production performance on glycerol.
CONCLUSION
This study revealed the complex interplay of gene expression levels and metabolic fluxes under PHA- and non-PHA producing conditions using the attractive raw material glycerol as carbon substrate. This knowledge will form the basis for the development of future metabolically engineered hyper-PHA-producing strains derived from the versatile bacterium P. putida KT2440.
Topics: Biomass; Carbon; Gene Expression Regulation, Bacterial; Glycerol; Isocitrate Dehydrogenase; Metabolic Flux Analysis; Metabolic Networks and Pathways; Nitrogen; Polyhydroxyalkanoates; Pseudomonas putida; Transcriptome
PubMed: 27142075
DOI: 10.1186/s12934-016-0470-2 -
Metabolic Engineering Jul 2022Polyethylene terephthalate (PET), the most common synthetic polyester today, is largely produced from fossil resources, contributing to global warming. Consequently,...
Polyethylene terephthalate (PET), the most common synthetic polyester today, is largely produced from fossil resources, contributing to global warming. Consequently, sustainable sources must be developed to meet the increasing demand for this useful polymer. Here, we demonstrate a cascaded value chain that provides green PET from lignin, the world's most underutilized renewable, via fermentative production of cis, cis-muconate (MA) from lignin-based aromatics as a central step. Catechol, industrially the most relevant but apparently also a highly toxic lignin-related aromatic, strongly inhibited MA-producing Pseudomonas putida MA-1. Assessed by C metabolic flux analysis, the microbe substantially redirected its carbon core fluxes, resulting in enhanced NADPH supply for stress defense but causing additional ATP costs. The reconstruction of MA production in a genome-reduced P. putida chassis yielded novel producers with superior pathway fluxes and enhanced robustness to catechol and a wide range of other aromatics. Using the advanced producer P. putida MA-10 catechol, MA could be produced in a fed-batch process from catechol (plus glucose as additional growth substrate) up to an attractive titer of 74 g L and a space-time-yield of 1.4 g L h. In terms of co-consumed sugar, the further streamlined strain MA-11 achieved the highest yield of 1.4 mol MA (mol glucose), providing a striking economic advantage. Following fermentative production, bio-based MA was purified and used to chemically synthetize the PET monomer terephthalic acid and the comonomer diethylene glycol terephthalic acid through five steps, which finally enabled the first green PET from lignin.
Topics: Catechols; Glucose; Lignin; Oxidation-Reduction; Polyethylene Terephthalates; Pseudomonas putida
PubMed: 35545205
DOI: 10.1016/j.ymben.2022.05.001 -
Microbial Biotechnology Sep 2019Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties....
Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties. The P. putida strain KT2440 is often described as Generally Regarded as Safe, or GRAS, indicating the strain is safe to use as food additive. This description is incorrect. P. putida KT2440 is classified by the FDA as HV1 certified, indicating it is safe to use in a P1 or ML1 environment.
Topics: Food Microbiology; Food Safety; Industrial Microbiology; Pseudomonas putida; United States; United States Food and Drug Administration
PubMed: 31199068
DOI: 10.1111/1751-7915.13443 -
Journal of Applied Microbiology Jul 2007The main focus of this study was to gain an overall view of Pseudomonas putida 06909 genes involved in the Pseudomonas-Phytophthora interaction as a biological control...
AIMS
The main focus of this study was to gain an overall view of Pseudomonas putida 06909 genes involved in the Pseudomonas-Phytophthora interaction as a biological control mechanism, and to understand the roles of these genes.
METHODS AND RESULTS
Sixteen Ps. putida genes with increased expression on Phytophthora mycelial surfaces were identified using in vivo expression technology (IVET) screening. Sequence analysis of these Phytophthora mycelium-induced (pmi) genes revealed that many of them display similarity to genes known or predicted to be involved in carbohydrate catabolism, energy metabolism, amino acid/nucleotide metabolism, and membrane transport processes. Disruption of three pmi genes encoding succinate semialdehyde dehydrogenase, a dicarboxylic acid transporter, and glyceraldehyde-3-phosphate dehydrogenase showed significant phenotypic differences involved in the colonization processes, including motility, biofilm formation on abiotic surfaces, colony morphology, and competitive colonization of fungal mycelia. All three of these pmi genes were induced by glycogen and other substances, such as organic acids and amino acids utilized by Ps. putida.
CONCLUSIONS
The IVET screening and mutant characterization can be used to identify bacterial genes that are induced on the mycelial surface and provide insight into the possible mechanisms of mycelial colonization by this bacterium.
SIGNIFICANCE AND IMPACT OF THE STUDY
The IVET screening through a bacterial genome library might be a huge task. However, because the genes involved in direct interaction with Phytophthora and in bacterial adaptation can be identified, the IVET system will be a valuable tool in studying biocontrol bacteria at the molecular and ecological levels.
Topics: Bacterial Proteins; DNA, Bacterial; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genomic Library; Mutagenesis; Mutation; Mycelium; Pest Control, Biological; Phenotype; Phytophthora; Promoter Regions, Genetic; Pseudomonas putida
PubMed: 17584458
DOI: 10.1111/j.1365-2672.2006.03232.x