Difference between revisions of "PMID:4577939"

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(Main Points of the Paper)
(Table edited by Azweifel via TableEdit)
Line 77: Line 77:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
inability to grow on lactose  
+
*Abolished lactose catabolic process
 +
*Absent growth on minimal salts medium with lactose
 +
*Abolished utilization of carbon source (lactose)
 +
 
 +
 
 
|
 
|
 
Growth
 
Growth
 
|
 
|
in a minimal salts medium
+
inability to grow on lactose in a minimal salts medium
 
|
 
|
 
Other
 
Other
Line 96: Line 100:
 
''lacY<sup>-</sup>''
 
''lacY<sup>-</sup>''
 
|
 
|
no induction of alpha-galactosidase
+
*Decreased rate of alpha-galactosidase expression
 +
*Abolished induction of alpha-galactosidase expression
 +
 
 
|
 
|
 
Metabolic Activity
 
Metabolic Activity
Line 115: Line 121:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
White colonies on MacConkey melibiose indicator plates
+
*Abolished utilization of carbon source (melibiose)
 +
*Abolished melibiose catabolic process
 +
*White colonies on MacConkey melibiose indicator plates
 
|
 
|
 
Growth
 
Growth
Line 135: Line 143:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
reduced intracellular IPTG accumulation
+
*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
 
Other
 
|
 
|
mutant reached a maximum intracellular concentration of 0.1 mM compared to 0.4 mM IPTG for the energy-coupled parent
+
*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
 
Other
Line 154: Line 167:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
reduced rate of beta-galactosidase induction
+
*Decreased induction of beta-galactosidase
 +
*Decreased rate of expression of beta-galactosidase
 +
*Decreased lactose catabolic process
 +
 
 
|
 
|
 
Metabolic Activity
 
Metabolic Activity
 
|
 
|
 
+
*reduced rate of beta-galactosidase induction
 
|
 
|
 
Biochemical Assay
 
Biochemical Assay
Line 174: Line 190:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
reduced growth rate on melibiose
+
*Decreased utilization of carbon source (melibiose)
 +
*Decreased melibiose catabolic process
 +
*Decreased growth rate on minimal salts medium with melibiose
 
|
 
|
 
Growth
 
Growth
 
|
 
|
in a minimal salts medium
+
*reduced growth rate on melibiose in a minimal salts medium
 
|
 
|
 
Plating Assay
 
Plating Assay
Line 193: Line 211:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
reduced rate of alpha-galactosidase induction  
+
*Decreased rate of alpha-galactosidase expression
 +
*Decreased induction of alpha-galactosidase
 
|
 
|
 
Metabolic Activity
 
Metabolic Activity
 
|
 
|
 
+
reduced rate of alpha-galactosidase induction
 
|
 
|
 
Biochemical Assay
 
Biochemical Assay
Line 212: Line 231:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
decreased affinity for lactose
+
*Decreased affinity for lactose binding
 +
*Decreased substrate binding
 +
*Decreased transport of small molecules
 
|
 
|
 
Other
 
Other
 
|
 
|
 
+
decreased affinity for lactose
 
|
 
|
 
Other
 
Other
Line 231: Line 252:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
Light pink colonies on MacConkey lactose indicator plates
+
*Decreased utilization of carbon source (lactose)
 +
*Decreased lactose catabolic process
 +
*Light pink colonies on MacConkey lactose indicator plates
 
|
 
|
 
Growth
 
Growth
Line 271: Line 294:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
Light pink colonies on MacConkey melibiose indicator plates
+
*Decreased utilization of carbon source (melibiose)
 +
*Decreased melibiose catabolic activity
 +
*Light pink colonies on MacConkey melibiose indicator plate
 
|
 
|
 
Growth
 
Growth
Line 291: Line 316:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
no growth on lactose  
+
*Abolished lactose catabolic process
 +
*Absent growth on minimal salts medium with lactose
 +
*Abolished utilization of carbon source (lactose)
 +
 
 +
 
 
|
 
|
 
Growth
 
Growth
 
|
 
|
in liquid culture with lactose concentrations b/t 5M and 50mM  
+
no growth on lactose in liquid culture with lactose concentrations b/t 5M and 50mM  
  
 
|
 
|
Line 311: Line 340:
 
''lacY<sup>UN</sup>''
 
''lacY<sup>UN</sup>''
 
|
 
|
increased affinity for melibiose  
+
*Increased affinity for melibiose  
 +
*Increased small molecule transport
 +
*Increased melibiose binding
 
|
 
|
 
Other
 
Other

Revision as of 16:35, 26 January 2011

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

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+yUNa-)- 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+yUNa-)
  • 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

<protect>

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

</protect>

Notes