PMID:4577939

Citation	Kusch, M and Wilson, TH (1973) Defective lactose utilization by a mutant of Escherichia coli energy-uncoupled for lactose transport. The advantages of active transport versus facilitated diffusion.Biochim. Biophys. Acta 311:109-22
Abstract	No abstract in PubMed
Links	PubMed
Keywords	Biological Transport; Biological Transport, Active; Carbon Isotopes; Cell Division; Densitometry; Diffusion; Disaccharides; Enzyme Induction; Escherichia coli; Galactosidases; Kinetics; Lactose; Mutation; Transduction, Genetic
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Main Points of the Paper

• Mutants described in this paper were defective in energy-dependent accumulation of thiogalactosides, but showed no loss of carrier-mediated transport
• Inducible energy-uncoupled mutant (54-41) was unable to grow on lactose minimal medium. Inducer IPTG permitted normal growth in these cells
• In the energy-uncoupled mutant, the rate of induction of β-galactosidase by lactose and by low concentrations of IPTG was mucher slower than the parent
• X71-54 (constitutive mutant) grew poorly on melibiose, due to the inability of this sugar to induce α-galactosidase
• For normal growth of inducible cells on lactose and melibiose, carrier-mediated entry (facilitated diffusion) of substrates is inadequate; the membrane carriers must also be capable of accumulating the sugars

Materials and Methods Used

• Parent Strain: X71 (i^-z⁺y⁺a^-, proC^-, try^-, B₁^-)
• Additional Strain: X71-54 (i^-z⁺y^UNa^-)- energy uncoupled mutant
• Additional Strain: X71-15 (i⁺z⁺y⁺a^-)
• Additional Strain: X9003 (i⁺z^-y⁺a⁺, B₁^-)
• Additional Strain: 54-41 (i⁺z⁺y^UNa^-)

• Additional Strain: 316 (i^-z⁺y^-a^-)- y^- strain

• β-galactosidase assay
• α-galactosidase assay
• Carrier-mediated transport assay
• thiogalactoside transacetylase activity was determined

• P₁ Transduction

Phenotype Annotations

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Species	Taxon ID	Strain	Gene (if known)	OMP	Phenotype	Details	Evidence	Notes
Escherichia coli	NCBI:562	X71-54	lacYUN	Abolished lactose catabolic process Absent growth on minimal salts medium with lactose Abolished utilization of carbon source (lactose)	Growth	inability to grow on lactose in a minimal salts medium	Other	in text, pg. 113
Escherichia coli	NCBI:562	316	lacY-	Decreased rate of alpha-galactosidase expression Abolished induction of alpha-galactosidase expression	Metabolic Activity	alpha-gal activity	Biochemical Assay	in text- pg. 116
Escherichia coli	NCBI:562	54-41	lacYUN	Abolished utilization of carbon source (melibiose) Abolished melibiose catabolic process White colonies on MacConkey melibiose indicator plates	Growth	inability to ferment 0.2% melibiose	Plating Assay	Table 4
Escherichia coli	NCBI:562	54-41	lacYUN	Decreased intracellular accumulation of small molecule (IPTG) Decreased activity of LacY transporter Increased facilitative diffusion of LacY? Decreased active transport of LacY? Energy-uncoupled transport?	Other	mutant reached a maximum intracellular concentration of 0.1 mM compared to 0.4 mM IPTG for the energy-coupled parent reduced intracellular IPTG accumulation	Other	Figure 1- [14C]IPTG uptake
Escherichia coli	NCBI:562	54-41	lacYUN	Decreased induction of beta-galactosidase Decreased rate of expression of beta-galactosidase Decreased lactose catabolic process	Metabolic Activity	reduced rate of beta-galactosidase induction	Biochemical Assay	Figure 4- beta-galactosidase activity measured after lactose induction
Escherichia coli	NCBI:562	X71-54	lacYUN	Decreased utilization of carbon source (melibiose) Decreased melibiose catabolic process Decreased growth rate on minimal salts medium with melibiose	Growth	reduced growth rate on melibiose in a minimal salts medium	Plating Assay	Table 3
Escherichia coli	NCBI:562	X71-54	lacYUN	Decreased rate of alpha-galactosidase expression Decreased induction of alpha-galactosidase	Metabolic Activity	reduced rate of alpha-galactosidase induction	Biochemical Assay	Figure 5- alpha-galactosidase activity measured after melibiose induction
Escherichia coli	NCBI:562	54-41	lacYUN	Decreased affinity for lactose binding Decreased substrate binding Decreased transport of small molecules	Other	decreased affinity for lactose	Other	Table 5- kinetics of transporter for potential inducer molecules
Escherichia coli	NCBI:562	54-41	lacYUN	Decreased utilization of carbon source (lactose) Decreased lactose catabolic process Light pink colonies on MacConkey lactose indicator plates	Growth	reduced ability to ferment 0.2% lactose (fully restored when IPTG is present)	Plating Assay	Table 4
Escherichia coli	NCBI:562	X71-54	lacYUN	Light pink colonies on MacConkey melibiose indicator plates	Growth	inability to ferment 0.2% melibiose	Plating Assay	Table 4
Escherichia coli	NCBI:562	X71-54	lacYUN	Decreased utilization of carbon source (melibiose) Decreased melibiose catabolic activity Light pink colonies on MacConkey melibiose indicator plate	Growth	reduced ability to ferment 0.2% melibiose	Plating Assay	Table 4
Escherichia coli	NCBI:562	54-41	lacYUN	Abolished lactose catabolic process Absent growth on minimal salts medium with lactose Abolished utilization of carbon source (lactose)	Growth	no growth on lactose in liquid culture with lactose concentrations b/t 5M and 50mM	Plating Assay	in text, pg. 117- addition of 0.5mM IPTG added to lactose medium lead to growth comparable to parent with no constitutive cells
Escherichia coli	NCBI:562	54-41	lacYUN	Increased affinity for melibiose Increased small molecule transport Increased melibiose binding	Other		Other	Table 5- kinetics of transporter for potential inducer molecules
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