CO2 production from galactose in galactose-1-phosphate uridyl transferase-deficient Escherichia coli

CO2 Production from Galactose in Galactose-1-Phosphate Uridyl Transferase-Deficient Escherichia coli Robert J. LaPolla, Mark R. Geier, Thomas B. Friedman and Carl R. Merril J. Bacteriol. 1975, 124(1):558.

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JOURNAL OF BACTERIOLOGY, Oct. 1975, p. 558-561 Copyright © 1975 American Society for Microbiology

Vol. 124, No. 1 Printed in U.S.A.

NOTES CO2 Production from Galactose in Galactose-1-Phosphate Uridyl Transferase-Deficient Escherichia coli

Received for publication 25 June 1975

Escherichia coli K-12 deficient in galactose-1-phosphate uridyl transferase is capable of converting significant amounts of D- [1- "C ]galactose to "CO,, whereas strains deficient in other enzymes of the Leloir pathway cannot do so. The LeLoir pathway is a major metabolic undertaken to determine whether gal-transferroute for the metabolism of galactose in eukary- ase-deficient E. coli, as has been found in otes and in many prokaryotes (4). This pathway human gal- transferase- deficient fibroblasts, utilizes three sequential reactions that are cata- might be capable of converting galactose to CO2 lyzed by galactokinase (EC 2.7.1.6), galactose- despite their similar inability to grow on galac1-phosphate uridyl transferase (gal-transferase; tose. The ability of the bacterial strains to (EC 2.7.7.12), and 5'-uridine diphosphate- convert D- [1- "C ]galactose to 14CO, is shown in galactose-4-epimerase (EC 5.1.3.2). Human de- Fig. 1 and Table 1. The galactokinase-deficient, ficiencies in gal-transferase result in a clinical the epimerase-deficient, and the galactose opdisorder known as galactosemia (9). The absence eron deletion strains make almost no "CO2 of galactokinase results in another metabolic from D- [1-14C ]galactose, whereas the galdisorder characterized by juvenile cataracts transferase-deficient strain was able to metabo(8). Human fibroblasts that lack detectable gal- lize significant amounts of galactose. The galtransferase activity are known to be capable of converting significant amounts of D- [1+ 2,000000 _ "IC ]galactose to "4CO2 despite their inability to S grow on galactose (3, 7). Galactokinase-deficient fibroblasts, on the other hand, have little or no ability to metabolize galactose (3, 7). The 1.500.000additional pathway(s) that the gal-transferasedeficient human cells utilizes must, therefore, require phosphorylation of galactose for further metabolism (3). Neither galactokinase- nor galtransferase-deficient human fibroblasts will 0. 1,000.000 grow on galactose (3, 5). Bacterial mutants of Escherichia coli K-12 that lack similar enzymes have been isolated 500.000 and characterized extensively (1). These strains do not grow in minimal galactose medium and are not fermenters, thus forming white colonies ^A GAL 2 = 200,000 M/ on MacConkey galactose indicator plates. Ga100.000 lactose fermenters produce red colonies on this 2 6 10 14 18 22 26 30 34 indicator plate. TIME IN HOURS In this study, the ability of each of these bacterial mutants to convert D- [1- 4C ]galactose FIG. 1. Galactose metabolism as reflected by the to "4CO2 was explored. This project was also amount of "4CO2 produced by gal-deficient E. coli K-12 from D- [1-14C ]galactose. Techniques employed Present address: Department of Biological Sciences, here are the same as those described in footnote I

a of Table 1.

Oakland Universitv. Rochester, Mich. 48063.

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ROBERT J. LAPOLLA,* MARK R. GEIER, THOMAS B. FRIEDMAN,' AND CARL R. MERRIL Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, Bethesda, Maryland 20014

VOL. 124, 1975

NOTES

559

TABLE 1. Ability of E. coli K-12 strains to metabolize D- [1- 4C]galactosea Strain

Growth Color on minimal MacConkey galactose m indicator galactose plates plates

Galactose phenotype

Wild type Transferase deficient Kinase deficient

+

N14

SA758

SA814 SA622 N99

Rate of galactose utilizationc

Final % utilization of input galactose L SDc

106,300 + 9,000 80,700 ± 3.300

19.3 ± 2.7 14.0 ± 0.6

6,700

3.2 ± 0.4

HrfH thi str+ FgalT151 (amber) his str+ FgalK2str-

on

Mating type type

Genotype

-

Red White

-

White

Epimerase

-

Dies

2,900 4 300

0.5 ± 0.2

HfrH

deficient Gal deletion

thi str+ galE PL2

-

White

5,300 + 2,700

1.4 ± 0.4

F+

Agal str- his

14,300

+

TABLE 2. Specific activities of galactokinase, gal-transferase, and uridine 5'-diphosphategalactose-4-epimerase (epimerase) in strains employeda Strain

SA814 SA622 N99 N14 SA758

Galactose phenotype

+ TKE-

Agal

Galactokinase activity

Gal-transferase activity

Epimerase

75.8 97.8 0.7 18.5 8.9

32.7