PMID:4905670
| Citation |
Wong, PT, Kashket, ER and Wilson, TH (1970) Energy coupling in the lactose transport system of Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 65:63-9 |
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| Abstract |
A mutant (ML 308-22) was isolated from Escherichia coli ML 308, which had lost the normal capacity to accumulate lactose analogs despite an increase in the membrane carrier activity. The exit of thiomethylgalactoside was much faster than normal, accounting for the inability of the cell to maintain high intracellular concentrations of galactosides. Growth of the mutant on lactose was normal at high concentrations of sugar and impaired at low concentrations. This transport defect appeared to be limited to the lactose transport system as D-fucose and alpha-aminoisobutyric acid uptake and accumulation were normal. It is inferred from the data that the mutant possessed a defect in the coupling of metabolic energy to lactose transport. |
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| Keywords |
Aminoisobutyric Acids; Biological Transport, Active; Carbon Isotopes; Escherichia coli; Fucose; Genetics, Microbial; Glycosides; Lactose; Mutation |
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Main Points of the Paper
- Isolation of ML308-22, a mutant which fermented lactose, but failed to accumulate the galactoside T-ONPG
- Severe defect in the accumulation of thiogalactosides with a greater activity of membrane carriers
- An acceleration of sugar efflux accounted for this mutant's inability to maintain a high intracellular concentration of sugar
- Transport for D-fucose and AIB was equivalent to the parent, suggesting the exit pathway for these sugars is normal
Materials and Methods Used
- Parent Strain: ML308 (i-z+y+a+
- ONPG hydrolysis assay (measure of transport carriers)
- β-galactoside uptake assays
- Counterflow experiments
- M protein (LacY) assay
Phenotype Annotations
| Species | Taxon ID | Strain | Gene (if known) | OMP | Phenotype | Details | Evidence | Notes |
|---|---|---|---|---|---|---|---|---|
|
Escherichia coli |
NCBI:562 |
ML308-22 |
lacYUN |
severely reduced accumulation of beta-galactosides |
Other |
possesses 1/10 of the active transport capacity of the parent based on intracellular [14C]TMG concentration |
Other |
Figure 1- Filter assay |
|
Escherichia coli |
NCBI:562 |
ML308-22 |
lacYUN |
increased number of transporters |
Metabolic Activity |
The release of o-nitrophenol from ONPG by washed mutant was 153% of that f the parent |
Other |
Table 2- ONPG hydrolysis is a measure of membrane carriers for lactose |
|
Escherichia coli |
NCBI:562 |
ML308-22 |
lacYUN |
increased number of transporters |
Metabolic Activity |
The mutant showed a more rapid initial rate of entry |
Other |
Figure 2- TMG counterflow assay |
|
Escherichia coli |
NCBI:562 |
ML308-22 |
lacYUN |
increased beta-galactosidase activity |
Metabolic Activity |
The cell-free extract of the mutant contained 147% of the beta-galactosidase activity of that of the parent |
Other |
in text, pg. 66 |
|
Escherichia coli |
NCBI:562 |
ML308-22 |
lacYUN |
increased binding of substrate |
Other |
[3H]TDG binding is increased 163% compared to the parent |
Other |
Table 3- [3H]TDG binding assay |
|
Escherichia coli |
NCBI:562 |
ML308-22 |
lacYUN |
reduced growth rate at lower lactose concentrations |
Growth |
At an external lactose concentration of 0.5mM, the mutant had a longer doubling time and at 0.25mM lactose, the cells did not grow |
Other |
Table 4- evaluation of doubling times at varying lactose concentrations |
|
Escherichia coli |
NCBI:562 |
ML308-22 |
lacYUN |
increased rate of sugar efflux |
Other |
Other |
Figure 4- [3C]TMG filter assay | |
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