scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in Lactococcus lactis MG1363

2003 ◽  
Vol 185 (5) ◽  
pp. 1564-1571 ◽  
Author(s):  
Christian Solem ◽  
Brian J. Koebmann ◽  
Peter R. Jensen
Keyword(s):  
Lactococcus Lactis ◽  
Reference Strain ◽  
Enzyme Level ◽  
Activity Level ◽  
Glycolytic Flux ◽  
Resting Cells ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gapdh Activity ◽  
Absolute Control

ABSTRACT Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in Lactococcus lactis (B. Poolman, B. Bosman, J. Kiers, and W. N. Konings, J. Bacteriol. 169:5887-5890, 1987). Those studies were based on inhibitor titrations with iodoacetate, which specifically inhibits GAPDH, and the data suggested that it should be possible to increase the glycolytic flux by overproducing GAPDH activity. To test this hypothesis, we constructed a series of mutants with GAPDH activities from 14 to 210% of that of the reference strain MG1363. We found that the glycolytic flux was unchanged in the mutants overproducing GAPDH. Also, a decrease in the GAPDH activity had very little effect on the growth rate and the glycolytic flux until 25% activity was reached. Below this activity level, the glycolytic flux decreased proportionally with decreasing GAPDH activity. These data show that GAPDH activity has no control over the glycolytic flux (flux control coefficient = 0.0) at the wild-type enzyme level and that the enzyme is present in excess capacity by a factor of 3 to 4. The early experiments by Poolman and coworkers were performed with cells resuspended in buffer, i.e., nongrowing cells, and we therefore analyzed the control by GAPDH under similar conditions. We found that the glycolytic flux in resting cells was even more insensitive to changes in the GAPDH activity; in this case GAPDH was also present in a large excess and had no control over the glycolytic flux.

scholarly journals Functional Analysis of the Lactococcus lactis galU and galE Genes and Their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis

2001 ◽  
Vol 67 (7) ◽  
pp. 3033-3040 ◽  
Author(s):  
Ingeborg C. Boels ◽  
Ana Ramos ◽  
Michiel Kleerebezem ◽  
Willem M. de Vos
Keyword(s):  
Lactococcus Lactis ◽  
Sole Carbon Source ◽  
Northern Blot Analysis ◽  
Fold Increase ◽  
Activity Level ◽  
Wild Type ◽  
Degenerate Primers ◽  
Poor Growth ◽  
Wild Type Level ◽  
Exopolysaccharide Biosynthesis

ABSTRACT We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.

scholarly journals CHOLINE ACETYLTRANSFERASE-DEFICIENT MUTANTS OF THE NEMATODE CAENORHABDITIS ELEGANS

Genetics ◽  
1984 ◽  
Vol 106 (2) ◽  
pp. 227-248
Author(s):  
James B Rand ◽  
Richard L Russell
Keyword(s):  
Caenorhabditis Elegans ◽  
Choline Acetyltransferase ◽  
Cholinesterase Inhibitors ◽  
Gene Dosage ◽  
Enzyme Level ◽  
Group Iv ◽  
Wild Type ◽  
Nematode Caenorhabditis Elegans ◽  
Wild Type Enzyme ◽  
Single Complex

ABSTRACT We have identified five independent allelic mutations, defining the gene cha-1, that result in decreased choline acetyltransferase (ChAT) activity in Caenorhabditis elegans. Four of the mutant alleles, when homozygous, lead to ChAT reductions of>98%, as well as recessive phenotypes of uncoordinated behavior, small size, slow growth and resistance to cholinesterase inhibitors. Animals homozygous for the fifth allele retain approximately 10% of the wild-type enzyme level; purified enzyme from this mutant has altered K  m values for both choline and acetyl-CoA and is more thermolabile than the wild-type enzyme. These qualitative alterations, together with gene dosage data, argue that cha-1 is the structural gene for ChAT. cha-1 has been mapped to the left arm of linkage group IV and is within 0.02 map unit of the gene unc-17, mutant alleles of which lead to all of the phenotypes of cha-1 mutants except for the ChAT deficiency. Extensive complementation studies of cha-1 and unc-17 alleles reveal a complex complementation pattern, suggesting that both loci may be part of a single complex gene.

scholarly journals Twofold Reduction of Phosphofructokinase Activity in Lactococcus lactis Results in Strong Decreases in Growth Rate and in Glycolytic Flux

2001 ◽  
Vol 183 (11) ◽  
pp. 3458-3467 ◽  
Author(s):  
Heidi W. Andersen ◽  
Christian Solem ◽  
Karin Hammer ◽  
Peter R. Jensen
Keyword(s):  
Growth Rate ◽  
Lactococcus Lactis ◽  
Defined Medium ◽  
Glycolytic Flux ◽  
Wild Type ◽  
Synthetic Promoters ◽  
Phosphofructokinase Activity ◽  
Sugar Phosphates ◽  
Strong Control ◽  
Reduced Activity

ABSTRACT Two mutant strains of Lactococcus lactis in which the promoter of the las operon, harboring pfk, pyk, and ldh, were replaced by synthetic promoters were constructed. These las mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions. One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two- to fourfold in the las mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.

scholarly journals The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

2004 ◽  
Vol 70 (9) ◽  
pp. 5477-5484 ◽  
Author(s):  
Johan Palmfeldt ◽  
Marco Paese ◽  
Bärbel Hahn-Hägerdal ◽  
Ed W. J. van Niel
Keyword(s):  
Growth Rate ◽  
Alcohol Dehydrogenase ◽  
Lactococcus Lactis ◽  
Product Formation ◽  
Glycolytic Flux ◽  
Resting Cells ◽  
Acid Product ◽  
Mixed Acid ◽  
Intracellular Concentrations ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of Pi was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.

scholarly journals The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Yuhao Dong ◽  
Qing Li ◽  
Jinzhu Geng ◽  
Qing Cao ◽  
Dan Zhao ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Efflux Pump ◽  
Fold Increase ◽  
Activity Level ◽  
Macrolide Resistance ◽  
Wild Type ◽  
Iron Deprivation ◽  
Pump Activity ◽  
Tonb System ◽  
Increased Sensitivity

AbstractThe TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

2021 ◽  
Vol 11 (12) ◽  
pp. 5521
Author(s):  
Li Liu ◽  
Shuhuai Yu ◽  
Wei Zhao
Keyword(s):  
Ph Value ◽  
Wild Type ◽  
Michaelis Constant ◽  
Wild Type Enzyme ◽  
Maximum Reaction Rate ◽  
E Coli ◽  
Maximum Reaction ◽  
Conversion Rates ◽  
Raoultella Terrigena ◽  
Sucrose Isomerase

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

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