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
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.
Links	PubMed
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	Decreased active transport of small molecule Decreased ability to sustain tranport gradient?	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 Increased transport of small molecules	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 Increased transport of small molecules	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 Increased utilization of carbon souce (lactose) Increased lactose catabolic process	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 affinity for substrate Increased substrate binding Increased transport of small molecules	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	Concentration-dependent growth (lactose) Decreased growth rate Decreased transporter activity Decreased active transport?	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 efflux of substrate Decreased active transport Decreased energy-coupled transport?	Other		Other	Figure 4- [3C]TMG filter assay
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