Abstract
Previous findings showed that galactose-binding protein defective mutants (mgl B-,A+,C+) of Escherichia coli K12 are still capable of growth on methyl-beta-D-galactopyranoside, while mgl A- and mgl C- mutants are not. When assayed by previous methods, none of these mutants exhibited methylgalactoside transport system activity. In this study, we present a modified assay developed for measuring low levels of transport. Using this assay, we found that mgl B-,A+,C+ mutants defective in galactose-binding protein accumulate methyl-beta-D-galactopyranoside up to six times the concentration gradient while mgl A- and mgl C- mutants failed to accumulate this substrate. Similar results were obtained using D-glyceryl-beta-D-galactopyranoside, another substrate of the methylgalactoside transport system. In contrast, all sugars tested which are not substrates of this system were transported equally by all mgl- mutants. The kinetic parameters of transport in mgl B- mutants were compared to those of the isogenic mgl+ strain which accumulates methyl-beta-D-galactopyranoside against a 10,000-fold concentration gradient. The apparent Km of methyl-beta-D-galactopyranoside influx was 1,000 times greater in mgl B- than in mgl+ strains. In contrast, there was no significant difference between these strains in either the Vmax of substrate influx or the rate of substrate exit. D-Galactose competitively inhibited methyl-beta-D-galactopyranoside influx into both mgl B- and mgl+ strains; the Ki of inhibition in mgl B- cells was 2,000-fold greater than that in mgl+ cells.
Highlights
We found that mgl B,A +,C+ mutants defective in galactose-binding protein accumulate methyl-P-n-galactopyranoside up to six times the concentration gradient while mgl
There was no significant difference between these strains in either the V, of substrate influx or the rate of substrate exit. o-GaIactose competitively topyranoside influx into both mgl B- and mgl+ strains; the K, of inhibition inhibited methyl-P-n-galacin mgl B- cells was 2,000-fold greater than that in mgl’ cells
I.e. growth of bacteria on P-methylgalactoside, was previously used to demonstrate a transport function in mgl mutants which exhibit no transport activity in routine measurements of intracellular substrate accumulation. This partial transport function was found to depend on two of the three genes required for p-methylgalactoside permease activity; no dependence on galactosebinding protein was observed [5]
Summary
When assayed by previous methods, none of these mutants exhibited methylgalactoside transport system activity. We present a modified assay developed for measuring low levels of transport. Using this assay, we found that mgl B-,A +,C+ mutants defective in galactose-binding protein accumulate methyl-P-n-galactopyranoside up to six times the concentration gradient while mgl. A- and mgl C.- mutants failed to accumulate this substrate. All sugars tested which are not substrates of this system were transported by all rnglm mutants. There was no significant difference between these strains in either the V,,, of substrate influx or the rate of substrate exit. A galactose-binding protein independent activity mgl A and mgl C has been demonstrated; observed to grow on P-methylgalactoside’
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