Reverse Genetics of Escherichia coliGlycerol Kinase Allosteric Regulation and Glucose Control of Glycerol Utilization In Vivo

ABSTRACT Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of Escherichia coli glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIAGlc (formerly known as IIIglc), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the glpK gene. Genetics methods based on homologous recombination are used to place glpKalleles with known specific mutations into the chromosomal context of the glpK gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. TheE. coli strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different glpRalleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the glpK gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIAGlc inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.

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Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis

Journal of Bacteriology ◽

10.1128/jb.183.11.3391-3398.2001 ◽

2001 ◽

Vol 183 (11) ◽

pp. 3391-3398 ◽

Cited By ~ 43

Author(s):

Vicente Monedero ◽

Oscar P. Kuipers ◽

Emmanuel Jamet ◽

Josef Deutscher

Keyword(s):

Lactococcus Lactis ◽

Inhibitory Effect ◽

Wild Type ◽

Phosphotransferase System ◽

Inducer Exclusion ◽

Gram Positive Bacteria ◽

In Vivo Experiments ◽

Regulatory Functions

ABSTRACT In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methylβ-d-thiogalactoside (TMG) and 2-deoxy-d-glucose (2-DG). In vivo experiments with theptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

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A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo.

Journal of Bacteriology ◽

10.1128/jb.178.10.2846-2852.1996 ◽

1996 ◽

Vol 178 (10) ◽

pp. 2846-2852 ◽

Cited By ~ 22

Author(s):

D W Pettigrew ◽

W Z Liu ◽

C Holmes ◽

N D Meadow ◽

S Roseman

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Glucose Control ◽

Amino Acid Change ◽

Glycerol Kinase ◽

Single Amino Acid ◽

Single Amino Acid Change ◽

Glycerol Utilization

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Control of glycolysis in vertebrate skeletal muscle during exercise

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1996.270.4.r821 ◽

1996 ◽

Vol 270 (4) ◽

pp. R821-R829 ◽

Cited By ~ 4

Author(s):

U. Krause ◽

G. Wegener

Keyword(s):

Gastrocnemius Muscle ◽

Rana Temporaria ◽

High Capacity ◽

Repeated Exercise ◽

Frog Rana Temporaria ◽

Time Courses ◽

Fructose 1,6 Bisphosphate ◽

Assay Conditions

The gastrocnemius muscle of the frog (Rana temporaria) has a high capacity for anaerobic glycolysis from glycogen. Glycolytic metabolites and effectors of phosphofructokinase, particularly the hexose bisphosphates, were followed in muscle during exercise (swimming between 5 s and 5 min), recovery (rest for up to 2 h after 5 min of swimming), and repeated exercise (swimming for up to 60 s after 2 h of recovery). Glycogen phosphorylase and phosphofructokinase were swiftly activated with exercise. The hexose bisphosphates followed markedly different time courses. Fructose 1,6-bisphosphate was transiently increased in both exercise and repeated exercise. This appears to be an effect rather than a cause of phosphofructokinase activation. Glucose 1,6-biphosphate was accumulated only while phosphofructokinase was active and was unchanged at other times. Fructose 2,6-biphosphate showed a 10-fold transient increase on exercise in rested frogs, almost disappeared from the muscle during recovery, and did not change during repeated exercise. Fructose 2,6-biphosphate is a potent activator of phosphofructokinase in vitro under near physiological assay conditions, and it may serve this function also in vivo during exercise. Glucose 1,6-biphosphate could be an activator of phosphofructokinase in repeated exercise when fructose 2,6-biphosphate is not available.

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PTS 50: Past, Present and Future, or Diauxie Revisited

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000369809 ◽

2015 ◽

Vol 25 (2-3) ◽

pp. 79-93 ◽

Cited By ~ 9

Author(s):

Joseph W. Lengeler

Keyword(s):

Catabolite Repression ◽

Biological Significance ◽

Cellular Growth ◽

Gram Positive ◽

Gram Negative ◽

Inducer Exclusion ◽

Gram Positive Bacteria ◽

Cellular Control

Past: The title ‘PTS 50 or The PTS after 50 years' relies on the first description in 1964 of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) by Kundig, Gosh and Roseman [Proc Natl Acad Sci USA 1964;52:1067-1074]. The system comprised proteins named Enzyme I, HPr and Enzymes II, as part of a novel PTS for carbohydrates in Gram-negative and Gram-positive bacteria, whose ‘biological significance remained unclear'. In contrast, studies which would eventually lead to the discovery of the central role of the PTS in bacterial metabolism had been published since before 1942. They are primarily linked to names like Epps and Gale, J. Monod, Cohn and Horibata, and B. Magasanik, and to phenomena like ‘glucose effects', ‘diauxie', ‘catabolite repression' and carbohydrate transport. Present: The pioneering work from Roseman's group initiated a flood of publications. The extraordinary progress from 1964 to this day in the qualitative and in vitro description of the genes and enzymes of the PTS, and of its multiple roles in global cellular control through ‘inducer exclusion', gene induction and ‘catabolite repression', in cellular growth, in cell differentiation and in chemotaxis, as well as the differences of its functions between Gram-positive and Gram-negative bacteria, was one theme of the meeting and will not be treated in detail here. Future: At the 1988 Paris meeting entitled ‘The PTS after 25 years', Saul Roseman predicted that ‘we must describe these interactions [of the PTS components] in a quantitative way [under] in vivo conditions'. I will present some results obtained by our group during recent years on the old phenomenon of diauxie by means of very fast and quantitative tests, measured in vivo, and obtained from cultures of isogenic mutant strains growing under chemostat conditions. The results begin to hint at the problems relating to future PTS research, but also to the ‘true science' of Roseman.

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Mammalian deubiquitinating enzyme inhibitors display in vitro and in vivo activity against malaria parasites and potentiate artemisinin action

10.1101/2020.08.13.249425 ◽

2020 ◽

Author(s):

Nelson V. Simwela ◽

Katie R. Hughes ◽

Michael T. Rennie ◽

Michael P. Barrett ◽

Andrew P. Waters

Keyword(s):

Anticancer Agents ◽

Drug Targets ◽

Reverse Genetics ◽

Enzyme Inhibitors ◽

Artemisinin Resistance ◽

Drug Repurposing ◽

Antimalarial Drugs ◽

Malaria Parasites

AbstractCurrent malaria control efforts rely significantly on artemisinin combinational therapies which have played massive roles in alleviating the global burden of the disease. Emergence of resistance to artemisinins is therefore, not just alarming but requires immediate intervention points such as development of new antimalarial drugs or improvement of the current drugs through adjuvant or combination therapies. Artemisinin resistance is primarily conferred by Kelch13 propeller mutations which are phenotypically characterised by generalised growth quiescence, altered haemoglobin trafficking and downstream enhanced activity of the parasite stress pathways through the ubiquitin proteasome system (UPS). Previous work on artemisinin resistance selection in a rodent model of malaria, which we and others have recently validated using reverse genetics, has also shown that mutations in deubiquitinating enzymes, DUBs (upstream UPS component) modulates susceptibility of malaria parasites to both artemisinin and chloroquine. The UPS or upstream protein trafficking pathways have, therefore, been proposed to be not just potential drug targets, but also possible intervention points to overcome artemisinin resistance. Here we report the activity of small molecule inhibitors targeting mammalian DUBs in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasites in vitro and possess antiparasitic activity in vivo and can be used in combination with additive effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisinin in vitro as well as in vivo to the extent that ART resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream 20s proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed into in vivo antimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.Graphical abstract

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Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice

Journal of Virology ◽

10.1128/jvi.00364-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9490-9501 ◽

Cited By ~ 18

Author(s):

Christine D. Krempl ◽

Anna Wnekowicz ◽

Elaine W. Lamirande ◽

Giw Nayebagha ◽

Peter L. Collins ◽

...

Keyword(s):

Virulence Factor ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Vaccine Development ◽

Virulent Strain ◽

Consensus Sequence ◽

Cytoplasmic Tail ◽

Syncytial Virus

ABSTRACT Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (ΔG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the ΔG mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

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Influenza B Virus NS1-Truncated Mutants: Live-Attenuated Vaccine Approach

Journal of Virology ◽

10.1128/jvi.01213-08 ◽

2008 ◽

Vol 82 (21) ◽

pp. 10580-10590 ◽

Cited By ~ 73

Author(s):

Rong Hai ◽

Luis Martínez-Sobrido ◽

Kathryn A. Fraser ◽

Juan Ayllon ◽

Adolfo García-Sastre ◽

...

Keyword(s):

Reverse Genetics ◽

Influenza Viruses ◽

Influenza B Virus ◽

Influenza B ◽

Type I ◽

Live Attenuated Vaccine ◽

Live Virus ◽

B Virus

ABSTRACT Type B influenza viruses can cause substantial morbidity and mortality in the population, and vaccination remains by far the best means of protection against infections with these viruses. Here, we report the construction of mutant influenza B viruses for potential use as improved live-virus vaccine candidates. Employing reverse genetics, we altered the NS1 gene, which encodes a type I interferon (IFN) antagonist. The resulting NS1 mutant viruses induced IFN and, as a consequence, were found to be attenuated in vitro and in vivo. The absence of pathogenicity of the NS1 mutants in both BALB/c and C57BL/6 PKR−/− mice was confirmed. We also provide evidence that influenza B virus NS1 mutants induce a self-adjuvanted immune response and confer effective protection against challenge with both homologous and heterologous B virus strains in mice.

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Refined experimental design may increase the value of murine models for estimation of bluetongue virus virulence

Laboratory Animals ◽

10.1177/0023677220930056 ◽

2020 ◽

pp. 002367722093005

Author(s):

Maria Stokstad ◽

Peter Coetzee ◽

Mette Myrmel ◽

Paidamwoyo Mutowembwa ◽

Estelle H Venter ◽

...

Keyword(s):

Experimental Design ◽

Response Surface ◽

Bluetongue Virus ◽

Reverse Genetics ◽

Lethal Dose ◽

Murine Models ◽

Negative Consequences ◽

Infectious Dose

Bluetongue is a serious non-contagious vector-borne viral disease in ruminants, causing poor animal welfare and economic consequences globally. Concern has been raised about the development of novel bluetongue virus (BTV) strains and their possibly altered virulence through the process of viral reassortment. Virulence is traditionally estimated in lethal dose 50 (LD50) studies in murine models, but agreement with both in vitro and virulence in ruminants is questionable, and a refined experimental design is needed. Specific reassortants between wild-type and vaccine strains of BTV-1, -6 and -8 have previously been developed by reverse genetics. The aim of the present study was to rank the in vivo virulence of these parental and reassortant BTV strains by calculating LD50 in a murine model by using an experimental design that is new to virology: a between-patient optimised three-level response surface pathway design. The inoculation procedure was intracranial. Fifteen suckling mice were used to establish LD50 for each strain. Three parental and five reassortant virus strains were included. The LD50s varied from of 0.1 (95% confidence interval (CI) 0–0.20) to 3.3 (95% CI 2.96–3.72) tissue culture infectious dose 50/ml. The results support the hypothesis that reassortment in BTV may lead to increased virulence in mice with potential negative consequences for the natural ruminant host. The ranking showed low agreement with in vitro properties and virulence in ruminants according to existing literature. Refined design such as response surface pathway design was found suitable for use in virology, and it introduces significant ethical and scientific improvements.

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Inositol 1,4-bisphosphate is an allosteric activator of muscle-type 6-phosphofructo-1-kinase

Biochemical Journal ◽

10.1042/bj2590463 ◽

1989 ◽

Vol 259 (2) ◽

pp. 463-470 ◽

Cited By ~ 9

Author(s):

G W Mayr

Keyword(s):

Inositol Phosphates ◽

Head Group ◽

Muscle Type ◽

Allosteric Effectors ◽

Fructose 1,6 Bisphosphate ◽

Regulatory Properties ◽

Allosteric Activator

The allosteric effects of various inositol biphosphate (InsP2) isomers and other inositol phosphates, of glycerophosphoinositol phosphates (GroPInsPx) and of phosphoinositides (PtdInsPx) on muscle-type 6-phosphofructo-1-kinase (PFK) were investigated. The binding of these substances to PFK was indirectly estimated by their ability to stabilize the tetrameric enzyme. At near-physiological concentrations of other allosteric effectors, muscle PFK was activated AMP-dependently by Ins(1,4)P2 (Ka = 43 microM), Ins(2,4)P2 (Ka = 70 microM) and GroPIns4P (Ka = 20 microM). These compounds activated PFK by a mechanism similar to that established for activating hexose bisphosphates. Indirect binding experiments indicated minimal Kd,app. values of about 5 microM for the binding of Ins(1,4)P2 in the presence of 0.1 mM-AMP at pH 7.4. This apparent affinity was comparable with that of fructose 1,6-bisphosphate and glucose 1,6-bisphosphate at identical conditions. The enzyme was also found to interact specifically with PtdIns4P (Kd,app. = 37 microM), the inositol phospholipid carrying Ins(1,4)P2 as its head group. The regulatory behaviour of muscle-type PFK in vitro and the concentrations of Ins(1,4)P2 in vivo (between 4 and greater than 50 nmol/g wet wt. of tissue) are consistent with the hypothesis that there is a functional interaction in vivo. Furthermore, a role of PtdIns4P in membrane compartmentation of PFK is suggested. Comparative experiments with liver PFK indicate that these regulatory properties may be relatively specific for the muscle isoform. Unlike muscle PFK, the liver isoform was slightly activated by sub-micromolar concentrations of Ins(1,4,5)P3.

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