scholarly journals Selection of a Bifidobacterium animalis subsp.lactisStrain with a Decreased Ability To Produce Acetic Acid

2012 ◽  
Vol 78 (9) ◽  
pp. 3338-3342 ◽  
Author(s):  
Abelardo Margolles ◽  
Borja Sánchez
Keyword(s):  
Acetic Acid ◽  
Specific Activity ◽  
Skim Milk ◽  
Acetate Kinase ◽  
Resting Cells ◽  
Wild Type ◽  
Kinase Gene ◽  
Content Type ◽  
Bifidobacterium Animalis ◽  
De Man

ABSTRACTWe have characterized a new strain,Bifidobacterium animalissubsp.lactisCECT 7953, obtained by random UV mutagenesis, which produces less acetic acid than the wild type (CECT 7954) in three different experimental settings: De Man-Rogosa-Sharpe broth without sodium acetate, resting cells, and skim milk. Genome sequencing revealed a single Phe-Ser substitution in the acetate kinase gene product that seems to be responsible for the strain's reduced acid production. Accordingly, acetate kinase specific activity was lower in the low acetate producer. Strain CECT 7953 produced less acetate, less ethanol, and more yoghourt-related volatile compounds in skim milk than the wild type did. Thus, CECT 7953 shows promising potential for the development of dairy products fermented exclusively by a bifidobacterial strain.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals A Single-Nucleotide Insertion in a Drug Transporter Gene Induces a Thermotolerance Phenotype inGluconobacter frateuriiby Increasing the NADPH/NADP+Ratio via Metabolic Change

2018 ◽  
Vol 84 (10) ◽  
Author(s):  
Nami Matsumoto ◽  
Hiromi Hattori ◽  
Minenosuke Matsutani ◽  
Chihiro Matayoshi ◽  
Hirohide Toyama ◽  
...  
Keyword(s):  
Acetic Acid ◽  
High Temperatures ◽  
Type Strain ◽  
Wild Type Strain ◽  
Metabolic Change ◽  
Drug Transporter ◽  
Transporter Gene ◽  
Wild Type ◽  
Content Type ◽  
In The Wild

ABSTRACTThermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adaptedGluconobacter frateuriistrain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The ΔotsABstrain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The ΔotsABmutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP+ratio was higher in strain Wild-G, and much higher in the ΔotsABstrain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance inG. frateurii.IMPORTANCEThe biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show thatGluconobacterfrateurii, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP+ratios and reductions in ROS levels. We concluded that inG. frateurii, trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP+ratio to enhance thermotolerance.

scholarly journals Molecular Clues To Understand the Aerotolerance Phenotype of Bifidobacterium animalis subsp. lactis

2011 ◽  
Vol 78 (3) ◽  
pp. 644-650 ◽  
Author(s):  
Lorena Ruiz ◽  
Miguel Gueimonde ◽  
Patricia Ruas-Madiedo ◽  
Angela Ribbera ◽  
Clara G. de los Reyes-Gavilán ◽  
...  
Keyword(s):  
Nadh Oxidase ◽  
Specific Activity ◽  
Abiotic Factors ◽  
Molecular Response ◽  
Aerobic Conditions ◽  
Content Type ◽  
Bifidobacterium Animalis ◽  
Expression Of Genes ◽  
Coproporphyrinogen Iii Oxidase ◽  
And Storage

ABSTRACTOxygen is one of the abiotic factors negatively affecting the survival ofBifidobacteriumstrains used as probiotics, mainly due to the induction of lethal oxidative damage. Aerobic conditions are present during the process of manufacture and storage of functional foods, and aerotolerance is a desired trait for bifidobacteria intended for use in industry. In the present study, the molecular response ofBifidobacterium animalissubsp.lactisIPLA4549 to aerobic conditions is presented. Molecular targets affected by oxygen were studied using two-dimensional electrophoresis (2DE) and quantitative reverse transcriptase (qRT) PCR. Globally, oxygen stress induced a shift in the glycolytic pathway toward the production of acetic acid with a concomitant increase in ATP formation. Several changes in the expression of genes coding for enzymes involved in redox reactions were detected, although the redox ratio remained unaltered. Interestingly, cells grown under aerobic conditions were characterized by higher activity of coproporphyrinogen III oxidase, which can directly detoxify molecular oxygen, and by higher NADH oxidase specific activity, which can oxidize NADH using hydrogen peroxide. In turn, this is in agreement with the glycolytic shift toward acetate production, in that more NADH molecules may be available due to the lower level of lactic acid formation. These findings further our ability to elucidate the mechanisms by whichB. animaliscopes with an oxygen-containing atmosphere.

scholarly journals Metabolic Pathway Involved in 6-Chloro-2-Benzoxazolinone Degradation by Pigmentiphaga sp. Strain DL-8 and Identification of the Novel Metal-Dependent Hydrolase CbaA

2016 ◽  
Vol 82 (14) ◽  
pp. 4169-4179 ◽  
Author(s):  
Weiliang Dong ◽  
Fei Wang ◽  
Fei Huang ◽  
Yicheng Wang ◽  
Jie Zhou ◽  
...  
Keyword(s):  
Metabolic Pathway ◽  
Specific Activity ◽  
Degradation Mechanism ◽  
Draft Genome ◽  
Proton Donor ◽  
Binding Motif ◽  
Resting Cells ◽  
Content Type ◽  
Peptide Mass ◽  
Donor And Acceptor

ABSTRACT6-Chloro-2-benzoxazolinone (CDHB) is a precursor of herbicide, insecticide, and fungicide synthesis and has a broad spectrum of biological activity.Pigmentiphagasp. strain DL-8 can transform CDHB into 2-amino-5-chlorophenol (2A5CP), which it then utilizes as a carbon source for growth. The CDHB hydrolase (CbaA) was purified from strain DL-8, which can also hydrolyze 2-benzoxazolinone (BOA), 5-chloro-2-BOA, and benzamide. The specific activity of purified CbaA was 5,900 U · mg protein−1for CDHB, withKmandkcatvalues of 0.29 mM and 8,500 s−1, respectively. The optimal pH for purified CbaA was 9.0, the highest activity was observed at 55°C, and the inactive metal-free enzyme could be reactivated by Mg2+, Ni2+, Ca2+, or Zn2+. Based on the results obtained for the CbaA peptide mass fingerprinting and draft genome sequence of strain DL-8,cbaA(encoding 339 amino acids) was cloned and expressed inEscherichia coliBL21(DE3). CbaA shared 18 to 21% identity with some metal-dependent hydrolases of the PF01499 family and contained the signature metal-binding motif Q127XXXQ131XD133XXXH137. The conserved amino acid residues His288 and Glu301 served as the proton donor and acceptor.E. coliBL21(DE3-pET-cbaA) resting cells could transform 0.2 mM CDHB into 2A5CP. The mutant strain DL-8ΔcbaAlost the ability to degrade CDHB but retained the ability to degrade 2A5CP, consistent with strain DL-8. These results indicated thatcbaAwas the key gene responsible for CDHB degradation by strain DL-8.IMPORTANCE2-Benzoxazolinone (BOA) derivatives are widely used as synthetic intermediates and are also an important group of allelochemicals acting in response to tissue damage or pathogen attack in gramineous plants. However, the degradation mechanism of BOA derivatives by microorganisms is not clear. In the present study, we reported the identification of CbaA and metabolic pathway responsible for the degradation of CDHB inPigmentiphagasp. DL-8. This will provide microorganism and gene resources for the bioremediation of the environmental pollution caused by BOA derivatives.

scholarly journals Mutations Suppressing the Loss of DegQ Function in Bacillus subtilis (natto) Poly-γ-Glutamate Synthesis

2011 ◽  
Vol 77 (23) ◽  
pp. 8249-8258 ◽  
Author(s):  
Thi-Huyen Do ◽  
Yuki Suzuki ◽  
Naoki Abe ◽  
Jun Kaneko ◽  
Yoshifumi Itoh ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Wild Type ◽  
Two Component System ◽  
Kinase Gene ◽  
Content Type ◽  
Mutant Proteins ◽  
Suppressor Mutations ◽  
Type Mutant ◽  
Glutamate Synthesis

ABSTRACTThedegQgene ofBacillus subtilis(natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out thedegQgene inBacillus subtilis(natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found indegS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His6mutant proteins were expressed inEscherichia colicells and subjected to anin vitrophosphorylation assay. Compared with the wild type, mutant DegS-His6proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSUin vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.

scholarly journals Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming

2015 ◽  
Vol 81 (17) ◽  
pp. 5714-5723 ◽  
Author(s):  
Cheng Zhou ◽  
Jintong Ye ◽  
Yanfen Xue ◽  
Yanhe Ma
Keyword(s):  
Directed Evolution ◽  
Textile Industry ◽  
Specific Activity ◽  
High Specific Activity ◽  
Pectate Lyase ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Content Type ◽  
Alkaline Pectate Lyase

ABSTRACTThermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase fromBacillussp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca2+. However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in thet50(time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in thet50value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.

scholarly journals Rezafungin In Vitro Activity against Contemporary Nordic Clinical Candida Isolates and Candida auris Determined by the EUCAST Reference Method

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Marie Helleberg ◽  
Karin Meinike Jørgensen ◽  
Rasmus Krøger Hare ◽  
Raluca Datcu ◽  
Anuradha Chowdhary ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Target Genes ◽  
Specific Activity ◽  
Significant Proportion ◽  
Reference Method ◽  
Cross Resistance ◽  
Wild Type ◽  
Candida Auris ◽  
Content Type

ABSTRACT Rezafungin (formerly CD101) is a novel echinocandin in clinical development. EUCAST epidemiological cutoff values (ECOFFs) have not yet been established. We determined the in vitro activity of rezafungin and comparators against 1,293 Nordic yeast isolates and 122 Indian Candida auris isolates and established single-center wild-type upper limits (WT-UL). The isolates (19 Candida spp. and 13 other yeast species) were identified using Chromagar; matrix-assisted laser desorption ionization–time of flight (MALDI-TOF); and, when needed, internal transcribed spacer sequencing. EUCAST E.Def 7.3.1 susceptibility testing included rezafungin, anidulafungin, micafungin, amphotericin B, and fluconazole. WT-UL were established following EUCAST principles for visual and statistical ECOFF setting. fks target genes were sequenced for rezafungin non-wild-type isolates. EUCAST clinical breakpoints for fungi version 9.0 were adopted for susceptibility classification. Rezafungin had species-specific activity similar to that of anidulafungin and micafungin. On a milligram-per-liter basis, rezafungin was overall less active than anidulafungin and micafungin but equally or more active than fluconazole and amphotericin B against the most common Candida species, except C. parapsilosis. We identified 37 (3.1%) rezafungin non-wild-type isolates of C. albicans (1.9%), C. glabrata (3.0%), C. tropicalis (2.7%), C. dubliniensis (2.9%), C. krusei (1.2%), and C. auris (14.8%). Alterations in Fks hot spots were found in 26/26 Nordic and 8/18 non-wild-type C. auris isolates. Rezafungin displayed broad in vitro activity against Candida spp., including C. auris. Adopting WT-UL established here, few Nordic strains, but a significant proportion of C. auris isolates, had elevated MICs with mutations in fks target genes that conferred echinocandin cross-resistance. fks1 mutations raised rezafungin MICs notably less than anidulafungin and micafungin MICs in C. auris.

scholarly journals Enhancement of Acetic Acid Tolerance in Saccharomyces cerevisiae by Overexpression of theHAA1Gene, Encoding a Transcriptional Activator

2012 ◽  
Vol 78 (22) ◽  
pp. 8161-8163 ◽  
Author(s):  
Koichi Tanaka ◽  
Yukari Ishii ◽  
Jun Ogawa ◽  
Jun Shima
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Transcriptional Activator ◽  
Wild Type ◽  
Acetic Acid Tolerance ◽  
Content Type ◽  
In The Wild ◽  
Overexpressing Strain

ABSTRACTHaa1 is a transcriptional activator required forSaccharomyces cerevisiaeadaptation to weak acids. Here we show that the constitutiveHAA1-overexpressing strain acquired a higher level of acetic acid tolerance. Under conditions of acetic acid stress, the intracellular level of acetic acid was significantly lower inHAA1-overexpressing cells than in the wild-type cells.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals The Serratia marcescens Siderophore Serratiochelin Is Necessary for Full Virulence during Bloodstream Infection

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Danelle R. Weakland ◽  
Sara N. Smith ◽  
Bailey Bell ◽  
Ashootosh Tripathi ◽  
Harry L. T. Mobley
Keyword(s):  
Serratia Marcescens ◽  
Bloodstream Infection ◽  
Iron Acquisition ◽  
Gene Clusters ◽  
Clinical Isolates ◽  
Wild Type ◽  
Serratia Plymuthica ◽  
Content Type ◽  
Cas Assay ◽  
Essential Nutrient

ABSTRACT Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica. Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.

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