Roles of Individual mgl Gene Products in the ,&Methylgalactoside Transport System of Escherichia coZi K12*

Previous findings showed that galactose-binding protein defective mutants (mgl B-,A+,C+) of Escherichia coli K12 are still capable of growth on methyl-P-o-galactopyranoside, while mgl A and mgl Cm 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-P-n-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 n-glyceryl-P-n-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 rnglm mutants. The kinetic parameters of transport in mgl B- mutants were compared to those of the isogenic mgl+ strain which accumulates methyl-fl-n-galactopyranoside against a lO,OOO-fold concentration gradient. The apparent K, of methyl-fi-n-galactopyranoside influx was 1,000 times greater in mgl B- than in mgl+ strains.

Mass. 02139. *The trivial name used is: @-methylgalactoside, methyl-P-r-galactopyranoside. S185 [his, str, kzc(Z,Y,A),,,, pts F, ara(C,O),,,, mgl+]. Then the mgl D,B, mgl D&C, and mg1 D lesions of S183-27, S183-27T, and S183-726, respectively, were introduced into S185 by transduction (5,6) to yield the corresponding derivatives of S185. Cells were grown at 37" in DM minimal medium . Blank values were obtained using formaldehyde-treated cells in the assay (9). The transport activity of mgl mutants was measured by a modification of the above assay. This modified test, referred to as the "60x assay, " was performed as follows: 0.08 ml of a washed cell suspension containing about lo9 cells was incubated with the radioactive substrate in a final volume of 0.1 ml which contained 2.77 nmol of ['%]methyl-@-n-galactopyranosidi (2 x 10' cpm) and 3.3 JL~ of chloramphenicol. The assay was terminated by adding 2.9 ml of growth medium containing 33 &ml of chloramphenicol, filtering the mixture through a Millipore membrane, and washing the membrane (at room temperature) with 3 ml of growth medium. The radioactivity of the membranes was determined and corrected for blank values as indicated above. Unless otherwise stated, cells were incubated with the substrate at 28". In measurements of initial rate of uptake, the cells accumulated less than 5%~ of the extracellular substrate. The characteristics of the 60x assay of Tra+ activity were investigated.

An
The intracellular concentration of P-methylgalactoside increased linearly with time from 0 to 5 min, reaching a steady state at about 20 min; the bulk of the intracellular radioactivity could be eliminated by a cold chase (Fig. 1). The steady state concentration of intracellular P-methylgalactoside was independent of the number of cells used in the assay over the range 4 x lo* to 1.2 x lo9 cells (Fig. 2). No evidence of chemical alteration of the intracellular substrate (at the detection level of 1.5%)) was found by chromatographic analyses of the accumulated radioactivity. The optimal temperature for Tra+ activity was 2%31", as compared to 18-21" for the /3-methylgalactoside permease in mgl+ cells.
Tra+ activity was also demonstrable using another substrate respectively. These assays were performed as described under "Materials and Methods" with the exceptions that in the 60x assay washing was omitted, and 5 mM unlabeled substrate was added, where indicated.
In both assays, accumulation was measured after 3-min incubation at 28". The results are corrected for blanks which did not differ significantly for Tra+ and Tra-cells. These blanks are 4 and 326 cpm for washed and unwashed cells, respectively.
The results are corrected for the indicated blanks which were prepared by adding the cells after dilution of the substrate.
The blanks were incubated 30 min at 28" before filtration. Thus these blanks are equivalent to results from routine permease assays in which the concentration of radioactive substrate is doubled. Blank values were inde-Tra+ mutant S185-27 was tested using the 60x assay at 28" with pendent of concentration of unlabeled substrate; for A, they ranged incubation times of 3 min. B, the isogenic mg1+ strain S185-726 was from 34 to 46 cpm/lOg cells, and for B, from 147 to 194 cpm/lOg cells. tested using the routine permease assay at 28" with incubation times of by guest on March 25, 2020 http://www.jbc.org/ Downloaded from by D-galactose inhibition of p-["C Jmethylgalactoside influx. This procedure was necessary since Escherichia coli possesses a number of transport systems with affinity for D-galactose (7). Our results indicate that D-galactose competitively inhibits influx of P-methylgalactoside with a Ki of 6.3 mM (Fig. 4). The apparent K, of D-glyceryl-P-D-galactopyranoside accumulation, measured directly at the steady state, was 20 mM. The accumulation of D-glyceryl-@-D-galactopyranoside was inhibited competitively by P-methylgalactoside with a Ki of 20 mM. A series of independent, isogenic mgl-mutants were com- pared with respect to their transport of P-methylgalactoside at 27.7 FM and 5 mM external substrate; these concentrations are near the K, values of influx of mgl+ and Tra+ cells, respectively. As shown in Fig. 5, at 27.7 ELM substrate all Tra+ mutants tested accumulated more than 0.1 nmol/109 cells; at 5 mM all transported more than 10 nmol/109 cells. In contrast, the accumulation in Tram mutants at 27 MM substrate was about 33% of the average Tra+ accumulation with the exception of one mgl A-mutant (S183-10) which accumulated to levels comparable to those attained by Tra+ cells. However, at 5 mM none of the Tra+ mutants (including S183-10) transported more than 2.4 nmol/lOg cells, only 15% of the accumulation observed in Tra+ cells.
Efflux of P-methylgalactoside down a concentration gradient was also compared in Tra+ and mgl+ cells. Cells were incubated with substrate until they had attained a steady state of accumulation, then diluted 50-fold into prewarmed medium, and samples were measured at intervals in order to determine the remaining intracellular substrate concentration. As shown in Fig. 6, the rates of efflux measured for Tra+ and mgl+ cells were comparable, loss of half the intracellular substrate occurring in 4.3 and 5.6 min, respectively. Recapture of substrate under the conditions of these experiments was shown to be negligible since accumulation was reduced by 95% upon the 50.fold dilution of extracellular substrate.

DISCUSSION
Isogenic mgl+ and Tra+ (mgl B-,A+,C+) cells, which differ only in the presence or absence of functional galactose-binding protein, accumulate P-methylgalactoside intracellularly to maxima of 10,000 and 6 times the concentration gradient, respectively. The lower level of accumulation achieved by cells which lack the binding protein is shown to result from a decrease in their affinity for extracellular substrate. This conclusion is based on our findings that while the apparent K, values of substrate influx in mgl+ and Tra+ cells differ l,OOO-fold, no significant differences are observed between I I !I c