scholarly journals Screening of Thermotolerant Gluconobacter Strains for Production of 5-Keto-d-Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing d-Gluconate Dehydrogenase

2009 ◽  
Vol 75 (13) ◽  
pp. 4240-4247 ◽  
Author(s):  
Ittipon Saichana ◽  
Duangtip Moonmangmee ◽  
Osao Adachi ◽  
Kazunobu Matsushita ◽  
Hirohide Toyama
Keyword(s):  
Gluconic Acid ◽  
Flavin Adenine Dinucleotide ◽  
Gluconobacter Oxydans ◽  
Gene Cassette ◽  
Kanamycin Resistance ◽  
Major Product ◽  
Inverse Pcr ◽  
Glycerol Dehydrogenase ◽  
Mutant Strains

ABSTRACT We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-d-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl2 in vitro when cells were grown at 37°C. Addition of 5 mM CaCl2 to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production.

scholarly journals Regulation of Carbon and Electron Flow inClostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures

2001 ◽  
Vol 183 (5) ◽  
pp. 1748-1754 ◽  
Author(s):  
Sylvie Saint-Amans ◽  
Laurence Girbal ◽  
Jose Andrade ◽  
Kerstin Ahrens ◽  
Philippe Soucaille
Keyword(s):  
Electron Flow ◽  
Sharp Decrease ◽  
Major Product ◽  
Glycerol Dehydrogenase ◽  
Hydrogenase Activity ◽  
Specific Rate ◽  
Oxygen Sensitive ◽  
High Level

ABSTRACT The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146–6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H+ ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.

scholarly journals Evidence for the Involvement of the Glc7-Reg1 Phosphatase and the Snf1-Snf4 Kinase in the Regulation of INO1 Transcription in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 73-87 ◽  
Author(s):  
Margaret K Shirra ◽  
Karen M Arndt
Keyword(s):  
Rna Polymerase Ii ◽  
Glucose Repression ◽  
Snf1 Kinase ◽  
Glucose Levels ◽  
Recessive Mutations ◽  
Mutant Strains ◽  
Rna Polymerase Ii Transcription ◽  
Two Hybrid

AbstractBinding of the TATA-binding protein (TBP) to the promoter is a pivotal step in RNA polymerase II transcription. To identify factors that regulate TBP, we selected for suppressors of a TBP mutant that exhibits promoter-specific defects in activated transcription in vivo and severely reduced affinity for TATA boxes in vitro. Dominant mutations in SNF4 and recessive mutations in REG1, OPI1, and RTF2 were isolated that specifically suppress the inositol auxotrophy of the TBP mutant strains. OPI1 encodes a repressor of INO1 transcription. REG1 and SNF4 encode regulators of the Glc7 phosphatase and Snf1 kinase, respectively, and have well-studied roles in glucose repression. In two-hybrid assays, one SNF4 mutation enhances the interaction between Snf4 and Snf1. Suppression of the TBP mutant by our reg1 and SNF4 mutations appears unrelated to glucose repression, since these mutations do not alleviate repression of SUC2, and glucose levels have little effect on INO1 transcription. Moreover, mutations in TUP1, SSN6, and GLC7, but not HXK2 and MIG1, can cause suppression. Our data suggest that association of TBP with the TATA box may be regulated, directly or indirectly, by a substrate of Snf1. Analysis of INO1 transcription in various mutant strains suggests that this substrate is distinct from Opi1.

scholarly journals The Role of Prophage for Genome Diversification within a Clonal Lineage of Lactobacillus johnsonii: Characterization of the Defective Prophage LJ771

2008 ◽  
Vol 190 (17) ◽  
pp. 5806-5813 ◽  
Author(s):  
Emmanuel Denou ◽  
Raymond David Pridmore ◽  
Marco Ventura ◽  
Anne-Cécile Pittet ◽  
Marie-Camille Zwahlen ◽  
...  
Keyword(s):  
Methionine Sulfoxide ◽  
Gene Cassette ◽  
Methionine Sulfoxide Reductase ◽  
Toxin Gene ◽  
Clonal Lineage ◽  
Lactobacillus Johnsonii ◽  
Reductase Gene ◽  
Phage Dna

ABSTRACT Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE (“antitoxin”)/pemK (“toxin”) gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5′ end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage.

Synthesis and turnover of proteins and mRNA in germinating wheat embryos

1982 ◽  
Vol 60 (3) ◽  
pp. 389-397 ◽  
Author(s):  
Zbyszko F. Grzelczak ◽  
Mark H. Sattolo ◽  
Linda K. Hanley-Bowdoin ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Growth Phase ◽  
Time Course ◽  
Phase 1 ◽  
Major Product ◽  
Lag Phase ◽  
Wheat Embryo ◽  
Phase Development ◽  
Wheat Embryos

The most prominent methionine-labeled protein made when cell-free systems are programmed with bulk mRNA from dry wheat embryos has been identified with what may be the most abundant protein in dry wheat embryos. The protein has been brought to purity and has a distinctive amino acid composition, Gly and Glx accounting for almost 40% of the total amino acids. Designated E because of its conspicuous association with early imbibition of dry wheat embryos, the protein and its mRNA are abundant during the "early" phase (0–1 h) of postimbibition development, and easily detected during "lag" phase (1–5 h), but they are almost totally degraded soon after entry into the "growth" phase of development, by about 10 h postimbibition.The most prominent methionine-labeled protein peculiar to the cell-free translational capacity of bulk mRNA from "growth" phase embryos is not detected as a product of in vivo synthesis. Its electrophoretic properties and its time course of emergence, after 5 h postimbibition development, suggest that this major product of cell-free synthesis may be an in vitro counterpart to a prominent methionine-labeled protein made only in vivo, by "growth" phase embryos. Designated G because of its conspicuous association with "growth" phase development, the cell-free product does not comigrate with any prominent dye-stained band in electrophoretic distributions of wheat proteins. The suspected cellular counterpart to G, also, does not comigrate with a prominent dye-stained wheat protein during electrophoresis, and although found in particulate as well as soluble fractions of wheat embryo homogenates it is not concentrated in either nuclei or mitochondria, as isolated.

Enhanced fitness of a Helicobacter pylori babA mutant in a murine model

2021 ◽  
Author(s):  
M. Lorena Harvey ◽  
Aung Soe Lin ◽  
Lili Sun ◽  
Tatsuki Koyama ◽  
Jennifer H. B. Shuman ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Outer Membrane Proteins ◽  
Population Diversity ◽  
Fitness Advantage ◽  
Neutral Locus ◽  
Mutant Strains ◽  
Bacterial Fitness ◽  
H Pylori

Helicobacter pylori genomes encode >60 predicted outer membrane proteins (OMPs). Several OMPs in the Hop family act as adhesins, but the functions of most Hop proteins are unknown. To identify hop mutant strains that exhibit altered fitness in vivo compared to fitness in vitro , we used a genetic barcoding method that allowed us to track changes in the proportional abundance of H. pylori strains within a mixed population. We generated a library of hop mutant strains, each containing a unique nucleotide barcode, as well as a library of control strains, each containing a nucleotide barcode in an intergenic region predicted to be a neutral locus unrelated to bacterial fitness. We orogastrically inoculated each of the libraries into mice and analyzed compositional changes in the populations over time in vivo compared to changes detected in the populations during library passage in vitro . The control library proliferated as a relatively stable community in vitro, but there was a reduction in the population diversity of this library in vivo and marked variation in the dominant strains recovered from individual animals, consistent with the existence of a non-selective bottleneck in vivo . We did not identify any OMP mutants exhibiting fitness defects exclusively in vivo without corresponding fitness defects in vitro . Conversely, a babA mutant exhibited a strong fitness advantage in vivo but not in vitro . These findings, when taken together with results of other studies, suggest that production of BabA may have differential effects on H. pylori fitness depending on the environmental conditions.

In vitro characterization of Salmonella typhi mutant strains for live oral vaccines

Vaccine ◽  
1990 ◽  
Vol 8 (3) ◽  
pp. 263-268 ◽  
Author(s):  
Evgenia M. Dragunsky ◽  
Evelyn Rivera ◽  
H. Donald Hochstein ◽  
Inessa S. Levenbook
Keyword(s):  
Salmonella Typhi ◽  
Oral Vaccines ◽  
In Vitro Characterization ◽  
Mutant Strains

d-Gluconic acid–based methotrexate prodrug–loaded mixed micelles composed of MDR reversing copolymer: in vitro and in vivo results

2018 ◽  
Vol 296 (12) ◽  
pp. 1971-1981 ◽  
Author(s):  
Popat S. Kumbhar ◽  
Swapnil Birange ◽  
Mahesh Atavale ◽  
John I. Disouza ◽  
Arehalli S. Manjappa
Keyword(s):  
Gluconic Acid ◽  
Mixed Micelles

scholarly journals Phycobilins as Potent Food Bioactive Broad-Spectrum Inhibitors Against Proteases of SARS-CoV-2 and Other Coronaviruses: A Preliminary Study

2021 ◽  
Vol 12 ◽  
Author(s):  
Brahmaiah Pendyala ◽  
Ankit Patras ◽  
Chandravanu Dash
Keyword(s):  
Binding Affinity ◽  
Broad Spectrum ◽  
In Silico ◽  
Research Area ◽  
Therapeutic Drug ◽  
Inhibitor Activity ◽  
Current Vaccine ◽  
Mutant Strains ◽  
The Impact

In the 21st century, we have witnessed three coronavirus outbreaks: SARS in 2003, MERS in 2012, and the ongoing pandemic coronavirus disease 2019 (COVID-19). The search for efficient vaccines and development and repurposing of therapeutic drugs are the major approaches in the COVID-19 pandemic research area. There are concerns about the evolution of mutant strains (e.g., VUI – 202012/01, a mutant coronavirus in the United Kingdom), which can potentially reduce the impact of the current vaccine and therapeutic drug development trials. One promising approach to counter the mutant strains is the “development of effective broad-spectrum antiviral drugs” against coronaviruses. This study scientifically investigates potent food bioactive broad-spectrum antiviral compounds by targeting main protease (Mpro) and papain-like protease (PLpro) proteases of coronaviruses (CoVs) using in silico and in vitro approaches. The results reveal that phycocyanobilin (PCB) shows potential inhibitor activity against both proteases. PCB had the best binding affinity to Mpro and PLpro with IC50 values of 71 and 62 μm, respectively. Also, in silico studies with Mpro and PLpro enzymes of other human and animal CoVs indicate broad-spectrum inhibitor activity of the PCB. As with PCB, other phycobilins, such as phycourobilin (PUB), phycoerythrobilin (PEB), and phycoviolobilin (PVB) show similar binding affinity to SARS-CoV-2 Mpro and PLpro.

scholarly journals Mouse-Colonizing Helicobacter pylori SS1 Is Unusually Susceptible to Metronidazole Due to Two Complementary Reductase Activities

2000 ◽  
Vol 44 (11) ◽  
pp. 3127-3132 ◽  
Author(s):  
Jin-Yong Jeong ◽  
Douglas E. Berg
Keyword(s):  
Helicobacter Pylori ◽  
Kanamycin Resistance ◽  
Phenotypic Traits ◽  
Insertion Mutant ◽  
Low Concentrations ◽  
Mutant Strains ◽  
Resistance Markers ◽  
Clinically Significant ◽  
H Pylori ◽  
Derivatives Of

ABSTRACT In most strains of Helicobacter pylori, mutational inactivation of the rdxA (HP0954) gene, which encodes a nitroreductase that converts metronidazole (MTZ) from a harmless prodrug to a mutagenic and bacteriocidal product, is sufficient to make this pathogen resistant to clinically significant levels of MTZ. Here we report that SS1, a strain with the special ability to colonize mice, is unusual in being susceptible to very low concentrations of MTZ (0.5 μg/ml) and in being especially difficult to mutate to MTZ resistance (Mtzr). These phenotypic traits were traced to expression in this strain of the normally quiescent H. pylori frxAgene (HP0642, an rdxA paralog) along with rdxA. Transformation tests using rdxA::camand frxA::kan insertion mutant DNAs, with selection solely for the chloramphenicol and kanamycin resistance markers, and sequence analyses of frxA in spontaneous Mtzr derivatives of rdxA null mutant strains each showed that the development of Mtzr in SS1 required inactivation of both rdxA and frxA. Inactivation of either gene alone left SS1 susceptible to MTZ, although it was readily mutable from an MTZ-susceptible to an Mtzrphenotype. Reverse transcriptase PCR tests showed that frxAmRNA was at least 10-fold more abundant in SS1 than in reference strain 26695. It is proposed that these reductases play primarily nutritional roles during bacterial growth.

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