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1.
J Agric Food Chem ; 69(46): 13895-13903, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34757739

RESUMO

Bio-based propionate is widely welcome in the food additive industry. The current anaerobic process by Propionibacteria endures low titers and a long fermentation time. In this study, a new route for propionate production from l-threonine was designed. 2-Ketobutyrate, deaminated from l-threonine, is cleaved into propionaldehyde and CO2 and then be oxidized into propionic acid, which is neutralized by ammonia released from the first deamination step. This CoA-independent pathway with only CO2 as a byproduct boosts propionate production from l-threonine with high productivity and purity. The key enzyme for 2-ketobutyrate decarboxylation was selected, and its expression was optimized. The engineered Pseudomonas putida strain, harboring 2-ketoisovalerate decarboxylase from Lactococcus lactis could produce 580 mM (43 g/L) pure propionic acid from 600 mM l-threonine in 24 h in the batch biotransformation process. Furthermore, a high titer of 62 g/L propionic acid with a productivity of 1.07 g/L/h and a molar yield of >0.98 was achieved in the fed-batch pattern. Finally, an efficient sequential fermentation-biotransformation process was demonstrated to produce propionate directly from the fermentation broth containing l-threonine, which further reduces the costs since no l-threonine purification step is required.


Assuntos
Propionatos , Pseudomonas putida , Biotransformação , Fermentação , Pseudomonas putida/metabolismo , Treonina/metabolismo
2.
Metab Eng ; 67: 373-386, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34343699

RESUMO

Pseudomonas putida is evolutionarily endowed with features relevant for bioproduction, especially under harsh operating conditions. The rich metabolic versatility of this species, however, comes at the price of limited formation of acetyl-coenzyme A (CoA) from sugar substrates. Since acetyl-CoA is a key metabolic precursor for a number of added-value products, in this work we deployed an in silico-guided rewiring program of central carbon metabolism for upgrading P. putida as a host for acetyl-CoA-dependent bioproduction. An updated kinetic model, integrating fluxomics and metabolomics datasets in addition to manually-curated information of enzyme mechanisms, identified targets that would lead to increased acetyl-CoA levels. Based on these predictions, a set of plasmids based on clustered regularly interspaced short palindromic repeats (CRISPR) and dead CRISPR-associated protein 9 (dCas9) was constructed to silence genes by CRISPR interference (CRISPRi). Dynamic reduction of gene expression of two key targets (gltA, encoding citrate synthase, and the essential accA gene, encoding subunit A of the acetyl-CoA carboxylase complex) mediated an 8-fold increase in the acetyl-CoA content of rewired P. putida. Poly(3-hydroxybutyrate) (PHB) was adopted as a proxy of acetyl-CoA availability, and two synthetic pathways were engineered for biopolymer accumulation. By including cell morphology as an extra target for the CRISPRi approach, fully rewired P. putida strains programmed for PHB accumulation had a 5-fold increase in PHB titers in bioreactor cultures using glucose. Thus, the strategy described herein allowed for rationally redirecting metabolic fluxes in P. putida from central metabolism towards product biosynthesis-especially relevant when deletion of essential pathways is not an option.


Assuntos
Pseudomonas putida , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Citrato (si)-Sintase/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica , Plasmídeos , Pseudomonas putida/genética , Pseudomonas putida/metabolismo
3.
Sci Rep ; 11(1): 16445, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385565

RESUMO

Predation contributes to the structure and diversity of microbial communities. Predatory myxobacteria are ubiquitous to a variety of microbial habitats and capably consume a broad diversity of microbial prey. Predator-prey experiments utilizing myxobacteria have provided details into predatory mechanisms and features that facilitate consumption of prey. However, prey resistance to myxobacterial predation remains underexplored, and prey resistances have been observed exclusively from predator-prey experiments that included the model myxobacterium Myxococcus xanthus. Utilizing a predator-prey pairing that instead included the myxobacterium, Cystobacter ferrugineus, with Pseudomonas putida as prey, we observed surviving phenotypes capable of eluding predation. Comparative transcriptomics between P. putida unexposed to C. ferrugineus and the survivor phenotype suggested that increased expression of efflux pumps, genes associated with mucoid conversion, and various membrane features contribute to predator avoidance. Unique features observed from the survivor phenotype when compared to the parent P. putida include small colony variation, efflux-mediated antibiotic resistance, phenazine-1-carboxylic acid production, and increased mucoid conversion. These results demonstrate the utility of myxobacterial predator-prey models and provide insight into prey resistances in response to predatory stress that might contribute to the phenotypic diversity and structure of bacterial communities.


Assuntos
Genes Bacterianos , Genômica , Myxococcales/fisiologia , Comportamento Predatório , Pseudomonas putida/genética , Animais , Meios de Cultura , Farmacorresistência Bacteriana/genética , Oligopeptídeos/biossíntese , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/metabolismo
4.
Nat Commun ; 12(1): 4347, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301933

RESUMO

Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.


Assuntos
Produtos Biológicos/metabolismo , Burkholderiales/genética , Genoma Bacteriano/genética , Família Multigênica/genética , Proteobactérias/genética , Burkholderiaceae/genética , Burkholderiaceae/metabolismo , Burkholderiales/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Engenharia Genética/métodos , Mutação , Policetídeos/metabolismo , Proteobactérias/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo
5.
FEBS Lett ; 595(16): 2113-2126, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34245008

RESUMO

Histidine is an important carbon and nitrogen source of γ-proteobacteria and can affect bacteria-host interactions. The mechanisms of histidine uptake are only partly understood. Here, we analyze functional properties of the putative histidine transporter HutT of the soil bacterium Pseudomonas putida. The hutT gene is part of the histidine utilization operon, and the gene product belongs to the amino acid-polyamine-organocation (APC) family of secondary transporters. Deletion of hutT severely impairs growth of P. putida on histidine, suggesting that the encoded transporter is the major histidine uptake system of P. putida. Transport experiments with cells and purified and reconstituted protein indicate that HutT functions as a high-affinity histidine : proton symporter with high specificity for the amino acid. Substitution analyses identified amino acids crucial for HutT function.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Pseudomonas putida/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Pseudomonas putida/genética
6.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205200

RESUMO

Excessive use of nitrogen fertilizer in intensively managed agriculture has resulted in abundant accumulation of nitrate in soil, which limits agriculture sustainability. How to reduce nitrate content is the key to alleviate secondary soil salinization. However, the microorganisms used in soil remediation cause some problems such as weak efficiency and short survival time. In this study, seaweed polysaccharides were used as stimulant to promote the rapid growth and safer nitrate removal of denitrifying bacteria. Firstly, the growth rate and NO3--N removal capacity of three kinds of denitrifying bacteria, Bacillus subtilis (BS), Pseudomonas stutzeri (PS) and Pseudomonas putida (PP), were compared. The results showed that Bacillus subtilis (BS) had a faster growth rate and stronger nitrate removal ability. We then studied the effects of Enteromorpha linza polysaccharides (EP), carrageenan (CA), and sodium alginate (AL) on growth and denitrification performance of Bacillus subtilis (BS). The results showed that seaweed polysaccharides obviously promoted the growth of Bacillus subtilis (BS), and accelerated the reduction of NO3--N. More importantly, the increased NH4+-N content could avoid excessive loss of nitrogen, and less NO2--N accumulation could avoid toxic effects on plants. This new strategy of using denitrifying bacteria for safely remediating secondary soil salinization has a great significance.


Assuntos
Bactérias/crescimento & desenvolvimento , Nitratos/metabolismo , Polissacarídeos/farmacologia , Alga Marinha/química , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Bactérias/metabolismo , Biodegradação Ambiental , Desnitrificação , Pseudomonas putida/crescimento & desenvolvimento , Pseudomonas putida/metabolismo , Pseudomonas stutzeri/crescimento & desenvolvimento , Pseudomonas stutzeri/metabolismo , Solo/química , Microbiologia do Solo
7.
Nat Commun ; 12(1): 4554, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315891

RESUMO

The planktonic synthesis of reduced organophosphorus molecules, such as alkylphosphonates and aminophosphonates, represents one half of a vast global oceanic phosphorus redox cycle. Whilst alkylphosphonates tend to accumulate in recalcitrant dissolved organic matter, aminophosphonates do not. Here, we identify three bacterial 2-aminoethylphosphonate (2AEP) transporters, named AepXVW, AepP and AepSTU, whose synthesis is independent of phosphate concentrations (phosphate-insensitive). AepXVW is found in diverse marine heterotrophs and is ubiquitously distributed in mesopelagic and epipelagic waters. Unlike the archetypal phosphonate binding protein, PhnD, AepX has high affinity and high specificity for 2AEP (Stappia stellulata AepX Kd 23 ± 4 nM; methylphosphonate Kd 3.4 ± 0.3 mM). In the global ocean, aepX is heavily transcribed (~100-fold>phnD) independently of phosphate and nitrogen concentrations. Collectively, our data identifies a mechanism responsible for a major oxidation process in the marine phosphorus redox cycle and suggests 2AEP may be an important source of regenerated phosphate and ammonium, which are required for oceanic primary production.


Assuntos
Ácido Aminoetilfosfônico/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Minerais/metabolismo , Fósforo/metabolismo , Rhodobacteraceae/metabolismo , Água do Mar/microbiologia , Proteínas de Bactérias/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Cinética , Oceanos e Mares , Oxirredução , Filogenia , Proteômica , Pseudomonas putida/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rhodobacteraceae/genética
8.
Nat Commun ; 12(1): 3912, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162838

RESUMO

Biological lignin valorization has emerged as a major solution for sustainable and cost-effective biorefineries. However, current biorefineries yield lignin with inadequate fractionation for bioconversion, yet substantial changes of these biorefinery designs to focus on lignin could jeopardize carbohydrate efficiency and increase capital costs. We resolve the dilemma by designing 'plug-in processes of lignin' with the integration of leading pretreatment technologies. Substantial improvement of lignin bioconversion and synergistic enhancement of carbohydrate processing are achieved by solubilizing lignin via lowering molecular weight and increasing hydrophilic groups, addressing the dilemma of lignin- or carbohydrate-first scenarios. The plug-in processes of lignin could enable minimum polyhydroxyalkanoate selling price at as low as $6.18/kg. The results highlight the potential to achieve commercial production of polyhydroxyalkanoates as a co-product of cellulosic ethanol. Here, we show that the plug-in processes of lignin could transform biorefinery design toward sustainability by promoting carbon efficiency and optimizing the total capital cost.


Assuntos
Carbono/metabolismo , Lignina/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Bioengenharia/economia , Bioengenharia/métodos , Carboidratos/química , Hidrólise , Microbiologia Industrial/economia , Microbiologia Industrial/métodos , Pseudomonas putida/genética , Pseudomonas putida/metabolismo
9.
Ecotoxicol Environ Saf ; 221: 112431, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34146980

RESUMO

Detailed characterization of new Pseudomonas strains that degrade toxic pollutants is required and utterly necessary before their potential use in environmental microbiology and biotechnology applications. Therefore, phenol degradation by Pseudomonas putida KB3 under suboptimal temperatures, pH, and salinity was examined in this study. Parallelly, adaptive mechanisms of bacteria to stressful growth conditions concerning changes in cell membrane properties during phenol exposure as well as the expression level of genes encoding catechol 2,3-dioxygenase (xylE) and cyclopropane fatty acid synthase (cfaB) were determined. It was found that high salinity and the low temperature had the most significant effect on the growth of bacteria and the rate of phenol utilization. Degradation of phenol (300 mg L-1) proceeded 12-fold and seven-fold longer at 10 °C and 5% NaCl compared to the optimal conditions. The ability of bacteria to degrade phenol was coupled with a relatively high activity of catechol 2,3-dioxygenase. The only factor that inhibited enzyme activity by approximately 80% compared to the control sample was salinity. Fatty acid methyl ester (FAMEs) profiling, membrane permeability measurements, and hydrophobicity tests indicated severe alterations in bacteria membrane properties during phenol degradation in suboptimal growth conditions. The highest values of pH, salinity, and temperature led to a decrease in membrane permeability. FAME analysis showed fatty acid saturation indices and cyclopropane fatty acid participation at high temperature and salinity. Genetic data showed that suboptimal growth conditions primarily resulted in down-regulation of xylE and cfaB gene expression.


Assuntos
Adaptação Fisiológica/genética , Fenol/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Biodegradação Ambiental , Catecol 2,3-Dioxigenase/genética , Membrana Celular/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Metiltransferases/genética , Fenol/toxicidade , Pseudomonas putida/efeitos dos fármacos , Salinidade , Temperatura
10.
Sci Rep ; 11(1): 11991, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099824

RESUMO

L-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-ß-L-rhamnose (dTDP-L-Rha). dTDP-L-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-L-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-L-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-D-glucose to dTDP-4-keto-6-deoxy-L-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-L-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.


Assuntos
Carboidratos Epimerases/metabolismo , Desoxiglucose/análogos & derivados , Escherichia coli/enzimologia , Pseudomonas putida/metabolismo , Carboidratos Epimerases/genética , Catálise , Bases de Dados Factuais , Desoxiglucose/metabolismo , Desoxirribonucleotídeos/metabolismo , Biblioteca Gênica , Açúcares de Nucleosídeo Difosfato/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Nucleotídeos de Timina/metabolismo
11.
J Biol Chem ; 297(2): 100920, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34181948

RESUMO

The Pseudomonas putida F1 genome contains five genes annotated as encoding 3-ketoacyl-acyl carrier protein (ACP) synthases. Four are annotated as encoding FabF (3-ketoacyl-ACP synthase II) proteins, and the fifth is annotated as encoding a FabB (3-ketoacyl-ACP synthase I) protein. Expression of one of the FabF proteins, FabF2, is cryptic in the native host and becomes physiologically important only when the repressor controlling fabF2 transcription is inactivated. When derepressed, FabF2 can functionally replace FabB, and when expressed from a foreign promoter, had weak FabF activity. Complementation of Escherichia coli fabB and fabF mutant strains with high expression showed that P. putida fabF1 restored E. coli fabF function, whereas fabB restored E. coli fabB function and fabF2 restored the functions of both E. coli fabF and fabB. The P. putida ΔfabF1 deletion strain was almost entirely defective in synthesis of cis-vaccenic acid, whereas the ΔfabB strain is an unsaturated fatty acid (UFA) auxotroph that accumulated high levels of spontaneous suppressors in the absence of UFA supplementation. This was due to increased expression of fabF2 that bypasses loss of fabB because of the inactivation of the regulator, Pput_2425, encoded in the same operon as fabF2. Spontaneous suppressor accumulation was decreased by high levels of UFA supplementation, whereas competition by the P. putida ß-oxidation pathway gave increased accumulation. The ΔfabB ΔfabF2 strain is a stable UFA auxotroph indicating that suppressor accumulation requires FabF2 function. However, at low concentrations of UFA supplementation, the ΔfabF2 ΔPput_2425 double-mutant strain still accumulated suppressors at low UFA concentrations.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Ácidos Graxos Insaturados/biossíntese , Pseudomonas putida/metabolismo , Teste de Complementação Genética
12.
Nat Commun ; 12(1): 2261, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859194

RESUMO

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.


Assuntos
Lignina/metabolismo , Engenharia Metabólica/métodos , Pseudomonas putida/metabolismo , Succinatos/metabolismo , Álcalis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Basidiomycota/enzimologia , Basidiomycota/genética , Técnicas Biossensoriais , Burkholderia/enzimologia , Burkholderia/genética , Carbono/metabolismo , Ciclo do Ácido Cítrico/genética , Ácidos Cumáricos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Lignina/química , Estudo de Prova de Conceito , Pseudomonas putida/genética
13.
J Oleo Sci ; 70(4): 581-587, 2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33692244

RESUMO

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 MinIONTM 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 C13 surfactin was degraded faster than C15 surfactin.


Assuntos
Biodegradação Ambiental , Lipopeptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Pseudomonas putida/metabolismo , Tensoativos/metabolismo , Caulobacter/metabolismo , Comamonadaceae/metabolismo , Lipopeptídeos/química , Peptídeos Cíclicos/química , Pseudomonas putida/isolamento & purificação , Sphingomonadaceae/metabolismo , Tensoativos/química
14.
Metab Eng ; 64: 167-179, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33549838

RESUMO

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.


Assuntos
Pseudomonas putida , Simportadores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lignina/metabolismo , Prótons , Pseudomonas putida/genética , Pseudomonas putida/metabolismo
15.
FEBS J ; 288(16): 4955-4972, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33621443

RESUMO

The primary photochemistry is similar among the flavin-bound sensory domains of light-oxygen-voltage (LOV) photoreceptors, where upon blue-light illumination a covalent adduct is formed on the microseconds time scale between the flavin chromophore and a strictly conserved cysteine residue. In contrast, the adduct-state decay kinetics vary from seconds to days or longer. The molecular basis for this variation among structurally conserved LOV domains is not fully understood. Here, we selected PpSB2-LOV, a fast-cycling (τrec 3.5 min, 20 °C) short LOV protein from Pseudomonas putida that shares 67% sequence identity with a slow-cycling (τrec 2467 min, 20 °C) homologous protein PpSB1-LOV. Based on the crystal structure of the PpSB2-LOV in the dark state reported here, we used a comparative approach, in which we combined structure and sequence information with molecular dynamic (MD) simulations to address the mechanistic basis for the vastly different adduct-state lifetimes in the two homologous proteins. MD simulations pointed toward dynamically distinct structural region, which were subsequently targeted by site-directed mutagenesis of PpSB2-LOV, where we introduced single- and multisite substitutions exchanging them with the corresponding residues from PpSB1-LOV. Collectively, the data presented identify key amino acids on the Aß-Bß, Eα-Fα loops, and the Fα helix, such as E27 and I66, that play a decisive role in determining the adduct lifetime. Our results additionally suggest a correlation between the solvent accessibility of the chromophore pocket and adduct-state lifetime. The presented results add to our understanding of LOV signaling and will have important implications in tuning the signaling behavior (on/off kinetics) of LOV-based optogenetic tools.


Assuntos
Proteínas de Bactérias/química , Oxigênio/química , Pseudomonas putida/metabolismo , Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Oxigênio/metabolismo , Processos Fotoquímicos , Conformação Proteica
16.
Phys Chem Chem Phys ; 23(2): 1566-1576, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33404558

RESUMO

The bacterial cell envelope, in particular the cell wall, is considered the main controlling factor in the biosorption of aqueous uranium(vi) by microorganisms. However, the specific roles of the cell wall, associated biomolecules, and other components of the cell envelope are not well defined. Here we report findings on the biosorption of uranium by isolated cell envelope components and associated biomolecules, with P. putida 33015 and B. subtilis 168 investigated as representative strains for the differences in Gram-negative and Gram-positive cell envelope architecture, respectively. The cell wall and cell surface membrane were isolated from intact cells and characterised by X-ray Photoelectron Spectroscopy (XPS) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FT-IR) spectroscopy; revealing variations in the abundance of functional moieties and biomolecules associated with components of the cell envelope. Uranium biosorption was investigated as a function of cell envelope component and pH, comparing with intact cells. The isolated cell wall from both strains exhibited the greatest uranium biosorption capacity. Deprotonation of favourable functional groups on the biomass as the pH increased from 3 to 5.5 increased their uranium biosorption capacity by approximately 3 fold. The results from ATR-FT-IR indicated that uranium(vi) biosorption was mediated by phosphate and carboxyl groups associated with proteins and phosphorylated biopolymers of the cell envelope. This includes outer membrane phospholipids and LPS of Gram-negative bacteria and teichoic acids, surface proteins and peptidoglycan from Gram-positive bacteria. As a result, the biosorption process of uranium(vi) to microorganisms is controlled by surface interactions, resulting in higher accumulation of uranium in the cell envelope. This demonstrates the importance of bacterial cell wall as the key mediator of uranium biosorption with microorganisms.


Assuntos
Parede Celular/metabolismo , Urânio/metabolismo , Bacillus subtilis/metabolismo , Membrana Celular/metabolismo , Espectroscopia Fotoeletrônica , Pseudomonas putida/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
17.
BMC Microbiol ; 21(1): 9, 2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407113

RESUMO

BACKGROUND: Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions but does not lead to growth or survival under anoxic conditions. RESULTS: Here, a data-driven approach was used to develop a rational design for a P. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa. CONCLUSIONS: The results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 49 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Metabólica/métodos , Pseudomonas putida/crescimento & desenvolvimento , Anaerobiose , Simulação por Computador , Bases de Dados Genéticas , Fermentação , Perfilação da Expressão Gênica , Viabilidade Microbiana , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Pirimidinas/metabolismo
18.
Environ Microbiol ; 23(3): 1608-1619, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33393180

RESUMO

Despite its environmental robustness Pseudomonas putida strain KT2440 is very sensitive to DNA damage and displays poor homologous recombination efficiencies. To gain an insight into this deficiency isogenic ∆recA and ∆lexA1 derivatives of prophage-free strain P. putida EM173 were generated and responses of the recA and lexA1 promoters to DNA damage tested with GFP reporter technology. Basal expression of recA and lexA1 of P. putida were high in the absence of DNA damage and only moderately induced by norfloxacin. A similar behaviour was observed when equivalent GFP fusions to the recA and lexA promoters of E. coli were placed in P. putida EM173. In contrast, all SOS promoters were subject to strong repression in E. coli, which was released only when cells were treated with the antibiotic. Replacement of P. putida's native LexA1 and RecA by E. coli homologues did not improve the responsiveness of the indigenous functions to DNA damage. Taken together, it seems that P. putida fails to mount a strong SOS response due to the inefficacy of the crucial RecA-LexA interplay largely tractable to the weakness of the corresponding promoters and the inability of the repressor to shut them down entirely in the absence of DNA damage.


Assuntos
Pseudomonas putida , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Resposta SOS em Genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
19.
Environ Microbiol ; 23(1): 174-189, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33089610

RESUMO

The role of archetypal ribonucleases (RNases) in the physiology and stress endurance of the soil bacterium and metabolic engineering platform Pseudomonas putida KT2440 has been inspected. To this end, variants of this strain lacking each of the most important RNases were constructed. Each mutant lacked either one exoribonuclease (PNPase, RNase R) or one endoribonuclease (RNase E, RNase III, RNase G). The global physiological and metabolic costs of the absence of each of these enzymes were then analysed in terms of growth, motility and morphology. The effects of different oxidative chemicals that mimic the stresses endured by this microorganism in its natural habitats were studied as well. The results highlighted that each ribonuclease is specifically related with different traits of the environmental lifestyle that distinctively characterizes this microorganism. Interestingly, the physiological responses of P. putida to the absence of each enzyme diverged significantly from those known previously in Escherichia coli. This exposed not only species-specific regulatory functions for otherwise known RNase activities but also expanded the panoply of post-transcriptional adaptation devices that P. putida can make use of for facing hostile environments.


Assuntos
Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Pseudomonas putida/metabolismo , Ecossistema , Endorribonucleases/genética , Escherichia coli/metabolismo , Exorribonucleases/genética , Oxirredução , Pseudomonas putida/genética , Microbiologia do Solo
20.
Mol Microbiol ; 115(1): 131-141, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32945019

RESUMO

The coexistence of multiple homologous resistance-nodulation-division (RND) efflux pumps in bacteria is frequently described with overlapping substrate profiles. However, it is unclear how bacteria balance their transcription in response to the changing environment. Here, we characterized a repressor, SrpR, in Pseudomonas putida B6-2 (DSM 28064), whose coding gene is adjacent to srpS that encodes the local repressor of the RND-type efflux pump SrpABC gene cluster. SrpR was demonstrated as a specific repressor of another RND efflux pump gene cluster ttgABC that is locally repressed by TtgR. SrpR was found to be capable of binding to the ttgABC operator with a higher affinity (KD , 138.0 nM) compared to TtgR (KD , 15.4 µM). EMSA and ß-galactosidase assays were performed to survey possible effectors of SrpR with 35 available chemicals being tested. Only 2,3,4-trichlorophenol was identified as an effector of SrpR. A regulation model was then proposed, representing a novel strategy for balancing the efflux systems with partially overlapping substrate profiles. This study highlights sophisticated interactions among the RND efflux pumps in a Pseudomonas strain, which may endow bacteria with certain advantages in a fluctuant environment.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Pseudomonas putida/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana Transportadoras/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Pseudomonas putida/genética , Proteínas Repressoras/genética , Transcrição Genética/genética
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