scholarly journals Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

2014 ◽  
Vol 80 (20) ◽  
pp. 6539-6548 ◽  
Author(s):  
Jan Volmer ◽  
Christoph Neumann ◽  
Bruno Bühler ◽  
Andreas Schmid
Keyword(s):  
Organic Solvents ◽  
Efflux Pump ◽  
Phenotypic Variability ◽  
Metabolic Energy ◽  
Solvent Tolerance ◽  
Second Phase ◽  
Styrene Epoxidation ◽  
Tolerance Mechanisms ◽  
Content Type ◽  
Solvent Tolerant

ABSTRACTThe application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase forin situproduct removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especiallyPseudomonasstrains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified inPseudomonastaiwanensisVLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion ofttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants ofP. taiwanensisVLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of theP. taiwanensisVLB120ΔCparent strain, the maximum specific epoxidation activity ofP. taiwanensisVLB120ΔCΔttgVdoubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.

scholarly journals Novel Toxin-Antitoxin Module SlvT-SlvA Regulates Megaplasmid Stability and Incites Solvent Tolerance in Pseudomonas putida S12

2020 ◽  
Vol 86 (13) ◽  
Author(s):  
Hadiastri Kusumawardhani ◽  
David van Dijk ◽  
Rohola Hosseini ◽  
Johannes H. de Winde
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Efflux Pump ◽  
Gene Clusters ◽  
Host Cells ◽  
Solvent Tolerance ◽  
Content Type ◽  
Solvent Tolerant

ABSTRACT Pseudomonas putida S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies revealed that P. putida S12 contains the single-copy 583-kbp megaplasmid pTTS12. pTTS12 carries several important operons and gene clusters facilitating P. putida S12 survival and growth in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major determinant of solvent tolerance on the megaplasmid pTTS12. In addition, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA, respectively) encoded on pTTS12 which contributes to the solvent tolerance phenotype and is important for conferring stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with the slvAT gene pair created a solvent tolerance phenotype in non-solvent-tolerant strains, such as P. putida KT2440, Escherichia coli TG1, and E. coli BL21(DE3). IMPORTANCE Sustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during the bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates, or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant Pseudomonas putida S12 for molecular adaptation to an organic solvent. In addition to the solvent extrusion pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to short-term tolerance in moderate solvent concentrations, as well as to the stability of pTTS12. These two gene clusters were successfully expressed in non-solvent-tolerant strains of P. putida and Escherichia coli strains to confer and enhance solvent tolerance.

scholarly journals Intensity and Mechanisms of Fluoroquinolone Resistance within theH30 andH30Rx Subclones of Escherichia coli Sequence Type 131 Compared with Other Fluoroquinolone-Resistant E. coli

2015 ◽  
Vol 59 (8) ◽  
pp. 4471-4480 ◽  
Author(s):  
James R. Johnson ◽  
Brian Johnston ◽  
Michael A. Kuskowski ◽  
Evgeni V. Sokurenko ◽  
Veronika Tchesnokova
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Sequence Type ◽  
Fluoroquinolone Resistance ◽  
Solvent Tolerance ◽  
Organic Solvent Tolerance ◽  
Content Type ◽  
E Coli ◽  
Pump Activity

ABSTRACTThe recent expansion of theH30 subclone ofEscherichia colisequence type 131 (ST131) and its CTX-M-15-associatedH30Rx subset remains unexplained. Although ST131H30 typically exhibits fluoroquinolone resistance, so do multiple otherE. colilineages that have not expanded similarly. To determine whetherH30 isolates have more intense fluoroquinolone resistance than other fluoroquinolone-resistantE. coliisolates and to identify possible mechanisms, we determined the MICs for four fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin, and norfloxacin) among 89 well-characterized, genetically diverse fluoroquinolone-resistantE. coliisolates (48 non-H30 and 41H30 [23H30Rx and 18H30 non-Rx]). We compared the MICs with theH30 andH30Rx status, the presence/number of nonsynonymous mutations ingyrA,parC, andparE, the presence ofaac(6′)-1b-cr(an aminoglycoside/fluoroquinolone agent-modifying enzyme), and the efflux pump activity (measured as organic solvent tolerance [OST]). Among 1,518 recentE. coliclinical isolates, ST131H30 predominated clonally, both overall and among the fluoroquinolone-resistant isolates. Among the 89 study isolates, compared with non-H30 isolates,H30 isolates exhibited categorically higher MICs for all four fluoroquinolone agents, higher absolute ciprofloxacin and norfloxacin MICs, more nonsynonymous mutations ingyrA,parC, andparE(specificallygyrAD87N,parCE84V, andparEI529L), and a numerically higher prevalence of (H30Rx-associated)aac(6′)-1b-crbut lower OST scores. All putative resistance mechanisms were significantly associated with the MICs [foraac(6′)-1b-cr: ciprofloxacin and norfloxacin only].parCD87N corresponded with ST131H30 andparEI529L with ST131 generally. Thus, more intense fluoroquinolone resistance may provide ST131H30, especiallyH30Rx [ifaac(6′)-1b-crpositive], with subtle fitness advantages over other fluoroquinolone-resistantE. colistrains. This urges both parsimonious fluoroquinolone use and a search for other fitness-enhancing traits within ST131H30.

scholarly journals Role of the Multidrug Efflux Systems ofPseudomonas aeruginosa in Organic Solvent Tolerance

1998 ◽  
Vol 180 (11) ◽  
pp. 2987-2991 ◽  
Author(s):  
Xian-Zhi Li ◽  
Li Zhang ◽  
Keith Poole
Keyword(s):  
Organic Solvent ◽  
Organic Solvents ◽  
Efflux Pump ◽  
Solvent Tolerance ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Organic Solvent Tolerance ◽  
Multidrug Efflux Pumps ◽  
Energy Dependent

ABSTRACT Multidrug efflux pumps with a broad substrate specificity make a major contribution to intrinsic and acquired multiple antibiotic resistance in Pseudomonas aeruginosa. Using genetically defined efflux pump mutants, we investigated the involvement of the three known efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, in organic solvent tolerance in this organism. Our results showed that all three systems are capable of providing some level of tolerance to organic solvents such as n-hexane andp-xylene. Expression of MexAB-OprM was correlated with the highest levels of tolerance, and indeed, this efflux system was a major contributor to the intrinsic solvent tolerance of P. aeruginosa. Intrinsic organic solvent tolerance was compromised by a protonophore, indicating that it is substantially energy dependent. These data suggest that the efflux of organic solvents is a factor in the tolerance of P. aeruginosa to these compounds and that the multidrug efflux systems of this organism can accommodate organic solvents, as well as antibiotics.

scholarly journals A novel toxin-antitoxin module SlvT–SlvA governs megaplasmid stability and incites solvent tolerance in Pseudomonas putida S12

2020 ◽  
Author(s):  
Hadiastri Kusumawardhani ◽  
David van Dijk ◽  
Rohola Hosseini ◽  
Johannes H. de Winde
Keyword(s):  
Pseudomonas Putida ◽  
Genetic Stability ◽  
Aromatic Compounds ◽  
Efflux Pump ◽  
Gene Clusters ◽  
Host Cells ◽  
Solvent Tolerance ◽  
E Coli ◽  
Solvent Tolerant

AbstractPseudomonas putida S12 is highly tolerant towards organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies reveal that P. putida S12 contains a single-copy 583 kbp megaplasmid pTTS12. This pTTS12 encodes several important operons and gene clusters facilitating P. putida S12 to survive and grow in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major contributor of solvent tolerance on the megaplasmid pTTS12. Importantly, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA respectively) encoded on pTTS12 which contributes to the solvent tolerant phenotype and is essential in conferring genetic stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with slvAT gene pair created a solvent tolerance phenotype in non-solvent tolerant strains such as P. putida KT2440, E. coli TG1, and E. coli BL21(DE3).ImportanceSustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent-tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant P. putida S12 for molecular adaptation to organic solvent. In addition to the RND efflux pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to tolerance in low solvent concentration as well as to genetic stability of pTTS12. These two gene clusters were successfully transferred to non-solvent tolerant strains of P. putida and to E. coli strains to confer and enhance solvent tolerance.

Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance

1999 ◽  
Vol 45 (1) ◽  
pp. 18-22 ◽  
Author(s):  
Xian-Zhi Li ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Organic Solvent ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Environmental Pollutants ◽  
Multidrug Resistant ◽  
Solvent Tolerance ◽  
Organic Solvent Tolerance ◽  
Organic Solvent Tolerant ◽  
Solvent Tolerant

Organic solvent-tolerant mutants of Pseudomonas aeruginosa selected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical of nalB multidrug-resistant mutants. Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems in P. aeruginosa demonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system. Sequence analysis of mexR, the repressor gene of mexAB-oprM efflux operon, identified a nonsense mutation and a point mutation in the mexR genes of two solvent-tolerant mutants. These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.Key words: Pseudomonas aeruginosa, organic solvent tolerance, multidrug resistance, MexAB-OprM efflux pump, mexR gene.

scholarly journals Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Alexander Bollinger ◽  
Rebecka Molitor ◽  
Stephan Thies ◽  
Rainhard Koch ◽  
Cristina Coscolín ◽  
...  
Keyword(s):  
Organic Solvent ◽  
Organic Solvents ◽  
Residual Activity ◽  
Water Soluble ◽  
Toxic Waste ◽  
Carboxylic Ester ◽  
Content Type ◽  
Organic Solvent Tolerant ◽  
Solvent Tolerant ◽  
Ester Hydrolases

ABSTRACT Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.

scholarly journals Greater Ciprofloxacin Tolerance as a Possible Selectable Phenotype Underlying the Pandemic Spread of theH30 Subclone of Escherichia coli Sequence Type 131

2015 ◽  
Vol 59 (11) ◽  
pp. 7132-7135 ◽  
Author(s):  
James R. Johnson ◽  
Stephen B. Porter ◽  
Paul Thuras ◽  
Timothy J. Johnson ◽  
Lance B. Price ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Organic Solvent ◽  
Efflux Pump ◽  
Sequence Type ◽  
Solvent Tolerance ◽  
Organic Solvent Tolerance ◽  
Content Type ◽  
E Coli ◽  
Pump Activity

ABSTRACTMinimum bactericidal concentrations (MBCs) for ciprofloxacin were significantly higher among 41 members of theH30 subclone withinEscherichia colisequence type 131 than among 48 other fluoroquinolone-resistantE. coliisolates. This MBC difference, which was not explained by ciprofloxacin MICs,gyrA,parC, andparEmutations, the presence ofaac(6′)-Ib-cr, or organic solvent tolerance (a surrogate for efflux pump activity), conceivably could have promoted the pandemic emergence of theH30 sequence type 131 subclone.

scholarly journals Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

2012 ◽  
Vol 56 (8) ◽  
pp. 4450-4458 ◽  
Author(s):  
Mark Veleba ◽  
Paul G. Higgins ◽  
Gerardo Gonzalez ◽  
Harald Seifert ◽  
Thamarai Schneiders
Keyword(s):  
Multidrug Resistance ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Content Type ◽  
Resistance Phenotype ◽  
Serratia Proteamaculans ◽  
Virulence Potential ◽  
Article Type ◽  
Acrab Efflux Pump

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

scholarly journals Transporters and Efflux Pumps Are the Main Mechanisms Involved in Staphylococcus epidermidis Adaptation and Tolerance to Didecyldimethylammonium Chloride

2020 ◽  
Vol 8 (3) ◽  
pp. 344 ◽  
Author(s):  
Urška Ribič ◽  
Jernej Jakše ◽  
Nataša Toplak ◽  
Simon Koren ◽  
Minka Kovač ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Thioredoxin Reductase ◽  
Multidrug Transporter ◽  
Acid Biosynthesis ◽  
Tolerance Mechanisms ◽  
Down Regulation ◽  
Didecyldimethylammonium Chloride ◽  
First Time

Staphylococcus epidermidis cleanroom strains are often exposed to sub-inhibitory concentrations of disinfectants, including didecyldimethylammonium chloride (DDAC). Consequently, they can adapt or even become tolerant to them. RNA-sequencing was used to investigate adaptation and tolerance mechanisms of S. epidermidis cleanroom strains (SE11, SE18), with S. epidermidis SE11Ad adapted and S. epidermidis SE18To tolerant to DDAC. Adaptation to DDAC was identified with up-regulation of genes mainly involved in transport (thioredoxin reductase [pstS], the arsenic efflux pump [gene ID, SE0334], sugar phosphate antiporter [uhpT]), while down-regulation was seen for the Agr system (agrA, arC, agrD, psm, SE1543), for enhanced biofilm formation. Tolerance to DDAC revealed the up-regulation of genes associated with transporters (L-cysteine transport [tcyB]; uracil permease [SE0875]; multidrug transporter [lmrP]; arsenic efflux pump [arsB]); the down-regulation of genes involved in amino-acid biosynthesis (lysine [dapE]; histidine [hisA]; methionine [metC]), and an enzyme involved in peptidoglycan, and therefore cell wall modifications (alanine racemase [SE1079]). We show for the first time the differentially expressed genes in DDAC-adapted and DDAC-tolerant S. epidermidis strains, which highlight the complexity of the responses through the involvement of different mechanisms.

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