Vol. 135, No. 2
JOURNAL OF BACTERIOLOGY, Aug. 1978, P. 517-520
0021-9193/78/0135-0517$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Galactose Catabolism in Caulobacter crescentus NURITH KURN, INES CONTRERAS, AND LUCILLE SHAPIRO* Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461 Received for publication 27 January 1978
Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Doudoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.
Galactose utilization proceeds by different pathways in enteric bacteria and in Pseudomonas. In enteric bacteria galactose catabolism is carried out by phosphorylation to galactose phosphate followed by conversion to the glucose derivative by UDP-galactose-4-epimerase (17). The catabolism of galactose, as well as other hexoses in Pseudomonas, on the other hand, is carried out by the enzymes of the Entner-Doudoroff pathway (1, 3): D-galactose NAD D-gal-
actono-y-lactone +H2-p D-galactonate H20 2-keto-3-deoxy-galactonate ATP 2-keto-3deoxy-6-phosphogalactonate -- pyruvate + Dglyceraldehyde-3-phosphate. In addition to the basic enzymatic differences in hexose catabolism exhibited by the enterics and Pseudomonas, the sole use of the EntnerDoudoroff enzymes requires independent pathways for utilization of different hexoses (1, 3). Moreover, regulation of gene expression for catabolic enzymes in Pseudomonas, as well as gene organization, also differs from that observed in the enteric bacteria (2, 4, 7, 10, 16). The genes for glucose catabolism in Pseudomonas putida are independently regulated although they map in a chromosomal cluster (2). Furthermore, other catabolic pathways in both P. putida (2, 7, 16) and P. aeruginosa (4, 10) appear to be similarly regulated. Caulobacter crescentus catabolizes glucose solely via the Entner-Doudoroff pathway (6, 9),
and the nicotinamide adenine dinucleotide (NAD)-linked glucose 6-phosphate dehydrogenase activity in this organism is inducible (9, 12). In the present report we demonstrate that galactose catabolism in C. crescentus is carried out by the Entner-Doudoroff pathway. Expression of three activities involved in galactose utilization, galactose dehydrogenase, 2-keto-3-deoxy-6phosphogalactonate (KDP-gal) aldolase, and galactose uptake, are inducible, and analysis of mutant strains revealed that these activities are independently regulated. It was previously observed that lactose uptake and ,-galactosidase activity were regulated independently in C. crescentus (6). It appears, therefore, that the genes for catabolic enzymes in C. crescentus are regulated in a manner similar to genes encoding catabolic functions in Pseudomonas (2, 4, 7, 10, 16).
MATERIALS AND METHODS Growth conditions and mutant isolation. C. crescentus CB13 and mutant strains AE22 and AE23
were grown with shaking at 300C in minimal medium (6), with glucose, lactose, mannose, or galactose as the sole carbon source, or in complex PYE medium (9). The parent strain, C. crescentus CB13, is able to grow on galactose as the sole carbon source only in the presence of 3 x 10-3 M dibutyryl cyclic AMP (6). Derivatives of cyclic GMP could not replace deriva-
tives of cyclic AMP. Mutant strains able to grow on galactose in the absence of the cyclic AMP derivative were selected by spreading 0.1 ml of a glucose-grown culture (OD = 0.8) of strain CB13 on agar plates containing galactose as 517
518
J. BACTERIOL.
KURN, CONTRERAS, AND SHAPIRO
the sole carbon source. Mutant colonies appeared on the galactose plates with a frequency of 10-8, and these colonies were then grown on liquid galactose (0.5%, wt/vol) medium, and the growth rate was determined (Table 1). Galactose uptake assay. The uptake of [ U'4C]galactose was measured as described previously (6). The initial rate of galactose uptake by the cells was determined at various substrate concentrations, and the kinetic constants for uptake were calculated from Lineweaver-Burk double-reciprocal plots. The initial rates of uptake were determined at galactose concentrations of 0.5 to 10 mM. Enzyme assays. Crude extracts of strains CB13, AE22, and AE23 were prepared by sonic disruption at 4°C (Sonifier Cell Disruptor, model W185, Heat-System Ultrasonics, Inc.), followed by centrifugation for 15 min at 15,000 rpm in a Sorvall SS-34 rotor at 4°C. Crude extracts were prepared in the buffers described below for the respective enzymes. All enzyme activities shown in Tables 2 and 3 were linearly dependent on protein concentration and time. D-Galactose dehydrogenase activity was measured spectrophotometrically by the method of Wallenfels and Kurz (14). Crude extracts were dialyzed against 0.03 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.0, prior to assay and contained between 0.3 and 0.5 mg of protein per ml per reaction. KDP-gal aldolase was measured spectrophotometrically by the method of Shuster (13). KDP-gal was a generous gift of H. P. Meloche. Crude extracts were dialyzed against 0.03 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.0, prior to assay and between 0.2 and 0.5 mg of protein per ml was used per reaction. UDP-galactose-4-epimerase was assayed by the method of Wilson and Hogness TABLE 1. Growth rate on various carbon sources Doubling time (h) Carbon source'
CB13
AE22
AE23
7 7 6 Glucose 11 10 10.5 Lactose 8 7 NGb Galactose 8 7 21 Galactose + dibutyryl cyclic AMP 32 37 37 Mannose a Cultures were grown at 30°C in minimal medium containing glucose (0.2%), mannose (0.2%), lactose (0.5%), or galactose (0.5%). When dibutyryl cyclic AMP was added, it was present at 3 x 10-3 M. 'NG, No growth observed.
(17) in crude extracts dialyzed against 0.1 M glycine buffer, pH 8.5. Galactokinase activity was assayed by chromatographically identifying the product, galactose 6-PO4, on diethylaminoethyl paper (Whatman DE81) according to Wilson and Hogness (17). Materials. [U-_4C]galactose (95 mCi/mmol) was purchased from the Amersham/Searle Corp. The radioimmune assay kit for cyclic AMP was obtained from Collaborative Research. NfV,O2-dibutyryl cyclic 3',5'-AMP was purchased from Sigma Chemical Co.
RESULTS AND DISCUSSION Growth of C. crescentus wild type and mutant strains AE22 and AE23 on a variety of sugars as the sole carbon source. The parent strain CB13 is unable to grow on galactose as the sole carbon source (6). If dibutyryl cyclic AMP is added to the minimal medium in the presence of galactose, then growth can occur (Table 1). Butyrate did not replace dibutyryl cyclic AMP in the presence of galactose, nor did dibutyryl cyclic AMP alone permit growth in minimal media. Mutant strains AE22 and AE23, selected for their ability to grow on galactose in the absence of dibutyryl cyclic AMP, exhibited growth rates on lactose, glucose, and mannose similar to that of the wild-type strain CB13. Although wild-type cells were able to grow on galactose in the presence of dibutyryl cyclic AMP, the generation time was twice as long as that observed with mutant strains AE22 and AE23 grown in the absence of exogenous cyclic nucleotide (Table 1). TABLE 2. Specific activities of galactose dehydrogenase and KDP-gal aldolase in C. crescentus grown on various carbon sources Sp act with carbon source: Enzyme
Strain
Glucose Galactose Lactose CB13 8.0 26.0a 12.0b AE22 2.0 23.6 25.0 AE23 0.4 9.0 9.0 CB13 0.041 0.068a 0.032 AE22 0.032 0.156 0.085 AE23 0.202 0.219 0.180 a Strain CB13 was grown on galactose (0.5%, wt/vol) in the presence of dibutyryl cyclic AMP (3 x 10-3 M). b Reaction not linear with time.
D-Galactose dehydrogenase (nmol/mg per min) KDP-gal aldolase (,umol/mg per min)
TABLE 3. Kinetic constants for galactose uptake by C. crescentus CB13
AE23
AE22
Carbon source
Kma Vmaxb Km Glucose 1.28 1,056 1.21 Galactose 2.03c 1,421X 0.98 Lactose 960 1.28 1.50 a Units for Km are millimolar. b Units for Vm. are micromoles per milliliter per minute. c Galactose (0.5% [wt/vol]) + dibutyryl cyclic AMP (3 x 10-3 M).
Vm.
Km
Vmax
1,428 686 704
2.15 0.33 1.05
1,613 851 620
VOL. 135, 1978
GALACTOSE CATABOLISM IN C. CRESCENTUS
It was previously observed that when strain CB13 was shifted from growth on glucose to either lactose or galactose as the sole carbon source growth was blocked and the cells accumulated at the predivisional cell stage (6, 11). This cell cycle arrest was shown to occur before induction of the corresponding catabolic enzymes; when lactose was the sole carbon source, growth of the culture resumed after 20 h, or immediately upon the addition of cyclic AMP derivatives. Dibutyryl cyclic AMP was shown to stmulate catabolic enzyme induction and thus permit growth (6). Shift of mutants AE22 and AE23 from growth on glucose to either lactose or galactose similurly resulted in a transient arrest of growth and the accumulation of cells at the predivisional cell stage. It has been previously shown that the parent strain, C. crescentus CB13, contains both cyclic AMP and cyclic GMP (5, 6, 11). The intracellular concentration of cyclic AMP was identical in strains AE22, AE23, and wild-type strain CB13 and did not vary with carbon source (data not shown). Galactose utilization in C. crescntus. Crude extracts prepared from strain CB13 grown on galactose plus dibutyryl cyclic AMP or strains AE22 and AE23 grown on galactose were initially assayed for galactokinase activity. The enzyme assay was based on the retention of the charged phosphorylation product on diethylaminoethyl paper chromatograms, as described by Wilson and Hogness (17). Incubation of [14C]galactose with dialyzed crude extracts prepared from all three C. crescentus strains resulted in production of charged reaction products, none of which migrated as galactose 6phosphate when chromatographed on Whatman paper with the 1-propanol-formic acid-water system (1). In addition, the reaction was not dependent on ATP, and it was thus unlikely that a kinase activity was being measured. The formation of charged products, on the other hand, which are retained at the origin on diethylaminoethyl paper chromatography, may result from catabolism of galactose by enzymes of the Entner-Doudoroff pathway (1, 3). In this pathway oxidation of galactose by an NAD-dependent galactose dehydrogenase results in formation of galactono-y-lactone, which is spontaneously hydrolyzed at alkaline pH (pH 8.0). The reaction yields galactonate, which is then further converted to the corresponding a-keto acid (1). To test for the conversion of galactose -to galactonate and the a-keto acid, [14C]galactose was added to the crude extracts and incubated for 30 min at 37°C, and the reaction products were analyzed by paper chromatography (Whatman no. 1) using 1-propanol-formic acid-water (6:3:1), as de-
519
scribed by DeLey and Doudoroff (1). Only two reaction products were formed and these had Rf values of 0.45 and 0.57, corresponding to the migration of galactonic acid and the a-keto acid, respectively. The values agreed with previously reported Rf values (1). Although dialyzed crude extract exhibited enzyme activity, if the crude extract was treated with active charcoal before assaying, galactose dehydrogenase activity could not be measured. The two reaction products migrating as galactonic acid and keto acid were formed from ['4C]galactose, however, when NAD was added to crude extracts that had been treated with active charcoal. These results suggest that C. crescentus contains an NAD-dependent galactose dehydrogenase activity and that the untreated extracts likely contained tightly bound NAD. The specific activity of this galactose dehydrogenase in crude extracts was then determined spectrophotometrically (14; Table 2). In this assay lactose could not replace galactose as substrate. Further steps in the Entner-Doudoroff pathway are the formation of reduced and phosphorylated keto acid yielding KDP-gal (1) and its subsequent cleavage by KDP-gal aldolase. The substrate KDP-gal was used to assay KDP-gal aldolase (13) in extracts of strains CB13, AE22, and AE23 (Table 2). The specific activity of KDP-gal aldolase in all three strains grown on galactose or galactose plus dibutyryl cyclic AMP was similar to that reported for P. saccharophila (13). To determine whether C. crescentus can efficiently utilize galactose by its conversion to glucose, UDP-galactose-4-epimerase was assayed in crude extracts of mutant and wild-type strains grown on glucose, lactose, and galactose. The specific activity obtained in all cases, 0.28 to 0.33 umol of UDP-glucose produced per h per mg of protein, corresponded to less than 1% of that reported for Escherichia coli K-12 grown on galactose (17). This low level of activity was not increased by growth on galactose or lactose. It thus appears likely that galactose utilization in C. crescentus does not depend primarily on UDP-galactose-4-epimerase. The low level of activity may represent the route of galactose incorporation into cell wall. Effect of carbon source on galactose utilization enzymes in wild-type and mutant strains. Three activities involved in galactose
utilization, galactose dehydrogenase, KDP-gal aldolase, and galactose uptake, were assayed in
cultures grown with different carbon sources (Table 2 and 3). The wild-type strain had threefold-higher galactose dehydrogenase activity when grown on galactose plus dibutyryl cyclic AMP than when grown on glucose as the sole
520
KURN, CONTRERAS, AND SHAPIRO
carbon source. Neither the KDP-gal aldolase activity nor galactose uptake varied with carbon source (Tables 2 and 3). Since galactose dehydrogenase was induced in the presence of galactose and dibutyryl cyclic AMP, but neither galactose uptake nor KDP-gal aldolase activity was simultaneously induced, it appears that these activities are independently regulated in the wild-type strain. Further indication that the activities of the enzymes involved in galactose catabolism are regulated independently of one another was obtained from analysis of mutant strains AE22 and AE23. Both galactose dehydrogenase and KDPgal aldolase were induced in mutant AE22 grown on galactose, although the rate of galactose uptake was the same whether the cells were grown on glucose or galactose. Specifically, the levels of galactose dehydrogenase were over 10-fold higher in cultures grown on galactose than when grown on glucose. The activity of KDP-gal aldolase in this mutant strain was increased fivefold in the presence of galactose as compared to the activity when glucose was the sole carbon source, and twofold when compared to lactosegrown cells. Galactose uptake was unaffected by carbon source in mutant strain AE22. In mutant AE23, on the other hand, KDP-gal aldolase was constitutively expressed, and galactose uptake and galactose dehydrogenase were induced by growth on galactose. These results suggest that these activities are independently regulated, as has been found to be the case for catabolic gene expression in Pseudomonas (10, 16). We have previously demonstrated that the regulation of lactose uptake in C. crescentus is independent of the regulation of,-galactosidase; lactose uptake in mutant strains constitutive for the expression of,-galactosidase was shown to be induced by growth on lactose (6). ACKNOWLEDGMENIS This work was supported by gant no. PCM 77-06714 from the National Science Foundation and by Public Health Service grant no. GM 11301 from the National Institute of General Medical Sciences. L S. is a recipient of a Hirschl Trust Award and N. K. is a recipient of Public Health Service Fellowship
J. BACTERIOL. HD05087-02 from the National Institute of Child Health and Human Development.
LITERATURE CITED 1. DeLey, J., and M. Doudoroff. 1957. The metabolism of D-galactose in Pseudomonas saccharophila. J. Biol. Chem. 227:745-757. 2. de Torrontegui, G., R. Diaz, M. L Wheelis, and J. L Conova. 1976. Supra-operonic clustering of genes specifying glucose dissimilation in Pseudomonas putida. Mol. Gen. Genet. 144:407411. 3. Entner, N., and M. Doudoroff. 1952. Glucose and gluconic acid oxidation in Pseudomonas aeruginosa. J. Biol. Chem. 196:853862. 4. Kemp, M. B., and G. D. Hegeman. 1968. Genetic control of the ,B-ketoadipate pathway in Pseudomonas aeruginosa. J. Bacteriol. 96:1488-1499. 5. Kurn, N., and L Shapiro. 1976. Effect of 3',5'-cyclic GMP derivatives on the formation of Caulobacter surface structures. Proc. Natl. Acad. Sci. U.S.A. 73:3303-3307. 6. Kurn, N., L Shapiro, and N. Agabian. 1977. Effect of carbon source and the role of cyclic adenosine 3',5'monophosphate on the Caulobacter cell cycle. J. Bacteriol. 131:951-959. 7. Leidigh, B. J., and M. L Wheelis. 1973. The clustering on the P. putida chromosome of genes specifying dissimilatory functions. J. Mol. Evol. 2:235-242. 8. OrnsWton, L N. 1971. Regulation of catabolic pathways in Pseudomonas. Bacteriol. Rev. 35:87-116. 9. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. 10. Rosenberg, S. C., and G. D. Hegeman. 1969. Clustering of functionally related genes in Pseudomonas aeruginosa. J. Bacteriol. 99:353-355. 11. Shapiro, L, N. Agabian-Keshishian, A. Hirsch, and 0. M. Rosen. 1972. Effect of dibutyryladenosine 3',5'cyclic monophosphate on growth and differentiation in Caulobacter crescentus. Proc. Natl. Acad. Sci. U.S.A. 69:1225-1229. 12. Shedlarsi, J. G. 1974. Glucose 6-phosphate dehydrogenase from Caulobacter crescentus. Biochim. Biophys. Acta 358:33-43. 13. Shuster, C. W. 1966. 2-Keto-3-deoxy-6-phosphogalactonic acid aldolase. Methods Enzymol. 9:524-528. 14. Wallenfels, K., and G. Kurz. 1966. ,8-D-Galactose dehydrogenase from Pseudomonas saccharophila. Methods Enzymol. 9:112-115. 15. Wheelis, M. L 1975. The genetics of dissimilatory pathways in Pseudomonas. Annu. Rev. Microbiol. 29:505-524. 16. Wheelis, M. L, and R. Y. Stanier. 1966. The genetic control of dissimilatory pathways in Pseudomonas putida. Genetics 66:245-266. 17. Wilson, D. B., and D. S. Hogness. 1966. Galactokinase and uridine diphosphogalactose-4-epimerase from E. coli. Methods Enzymol. 8:229-240.
JOURNAL OF BACTERIOLOGY, Aug. 1978, P. 517-520
0021-9193/78/0135-0517$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Galactose Catabolism in Caulobacter crescentus NURITH KURN, INES CONTRERAS, AND LUCILLE SHAPIRO* Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461 Received for publication 27 January 1978
Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Doudoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.
Galactose utilization proceeds by different pathways in enteric bacteria and in Pseudomonas. In enteric bacteria galactose catabolism is carried out by phosphorylation to galactose phosphate followed by conversion to the glucose derivative by UDP-galactose-4-epimerase (17). The catabolism of galactose, as well as other hexoses in Pseudomonas, on the other hand, is carried out by the enzymes of the Entner-Doudoroff pathway (1, 3): D-galactose NAD D-gal-
actono-y-lactone +H2-p D-galactonate H20 2-keto-3-deoxy-galactonate ATP 2-keto-3deoxy-6-phosphogalactonate -- pyruvate + Dglyceraldehyde-3-phosphate. In addition to the basic enzymatic differences in hexose catabolism exhibited by the enterics and Pseudomonas, the sole use of the EntnerDoudoroff enzymes requires independent pathways for utilization of different hexoses (1, 3). Moreover, regulation of gene expression for catabolic enzymes in Pseudomonas, as well as gene organization, also differs from that observed in the enteric bacteria (2, 4, 7, 10, 16). The genes for glucose catabolism in Pseudomonas putida are independently regulated although they map in a chromosomal cluster (2). Furthermore, other catabolic pathways in both P. putida (2, 7, 16) and P. aeruginosa (4, 10) appear to be similarly regulated. Caulobacter crescentus catabolizes glucose solely via the Entner-Doudoroff pathway (6, 9),
and the nicotinamide adenine dinucleotide (NAD)-linked glucose 6-phosphate dehydrogenase activity in this organism is inducible (9, 12). In the present report we demonstrate that galactose catabolism in C. crescentus is carried out by the Entner-Doudoroff pathway. Expression of three activities involved in galactose utilization, galactose dehydrogenase, 2-keto-3-deoxy-6phosphogalactonate (KDP-gal) aldolase, and galactose uptake, are inducible, and analysis of mutant strains revealed that these activities are independently regulated. It was previously observed that lactose uptake and ,-galactosidase activity were regulated independently in C. crescentus (6). It appears, therefore, that the genes for catabolic enzymes in C. crescentus are regulated in a manner similar to genes encoding catabolic functions in Pseudomonas (2, 4, 7, 10, 16).
MATERIALS AND METHODS Growth conditions and mutant isolation. C. crescentus CB13 and mutant strains AE22 and AE23
were grown with shaking at 300C in minimal medium (6), with glucose, lactose, mannose, or galactose as the sole carbon source, or in complex PYE medium (9). The parent strain, C. crescentus CB13, is able to grow on galactose as the sole carbon source only in the presence of 3 x 10-3 M dibutyryl cyclic AMP (6). Derivatives of cyclic GMP could not replace deriva-
tives of cyclic AMP. Mutant strains able to grow on galactose in the absence of the cyclic AMP derivative were selected by spreading 0.1 ml of a glucose-grown culture (OD = 0.8) of strain CB13 on agar plates containing galactose as 517
518
J. BACTERIOL.
KURN, CONTRERAS, AND SHAPIRO
the sole carbon source. Mutant colonies appeared on the galactose plates with a frequency of 10-8, and these colonies were then grown on liquid galactose (0.5%, wt/vol) medium, and the growth rate was determined (Table 1). Galactose uptake assay. The uptake of [ U'4C]galactose was measured as described previously (6). The initial rate of galactose uptake by the cells was determined at various substrate concentrations, and the kinetic constants for uptake were calculated from Lineweaver-Burk double-reciprocal plots. The initial rates of uptake were determined at galactose concentrations of 0.5 to 10 mM. Enzyme assays. Crude extracts of strains CB13, AE22, and AE23 were prepared by sonic disruption at 4°C (Sonifier Cell Disruptor, model W185, Heat-System Ultrasonics, Inc.), followed by centrifugation for 15 min at 15,000 rpm in a Sorvall SS-34 rotor at 4°C. Crude extracts were prepared in the buffers described below for the respective enzymes. All enzyme activities shown in Tables 2 and 3 were linearly dependent on protein concentration and time. D-Galactose dehydrogenase activity was measured spectrophotometrically by the method of Wallenfels and Kurz (14). Crude extracts were dialyzed against 0.03 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.0, prior to assay and contained between 0.3 and 0.5 mg of protein per ml per reaction. KDP-gal aldolase was measured spectrophotometrically by the method of Shuster (13). KDP-gal was a generous gift of H. P. Meloche. Crude extracts were dialyzed against 0.03 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.0, prior to assay and between 0.2 and 0.5 mg of protein per ml was used per reaction. UDP-galactose-4-epimerase was assayed by the method of Wilson and Hogness TABLE 1. Growth rate on various carbon sources Doubling time (h) Carbon source'
CB13
AE22
AE23
7 7 6 Glucose 11 10 10.5 Lactose 8 7 NGb Galactose 8 7 21 Galactose + dibutyryl cyclic AMP 32 37 37 Mannose a Cultures were grown at 30°C in minimal medium containing glucose (0.2%), mannose (0.2%), lactose (0.5%), or galactose (0.5%). When dibutyryl cyclic AMP was added, it was present at 3 x 10-3 M. 'NG, No growth observed.
(17) in crude extracts dialyzed against 0.1 M glycine buffer, pH 8.5. Galactokinase activity was assayed by chromatographically identifying the product, galactose 6-PO4, on diethylaminoethyl paper (Whatman DE81) according to Wilson and Hogness (17). Materials. [U-_4C]galactose (95 mCi/mmol) was purchased from the Amersham/Searle Corp. The radioimmune assay kit for cyclic AMP was obtained from Collaborative Research. NfV,O2-dibutyryl cyclic 3',5'-AMP was purchased from Sigma Chemical Co.
RESULTS AND DISCUSSION Growth of C. crescentus wild type and mutant strains AE22 and AE23 on a variety of sugars as the sole carbon source. The parent strain CB13 is unable to grow on galactose as the sole carbon source (6). If dibutyryl cyclic AMP is added to the minimal medium in the presence of galactose, then growth can occur (Table 1). Butyrate did not replace dibutyryl cyclic AMP in the presence of galactose, nor did dibutyryl cyclic AMP alone permit growth in minimal media. Mutant strains AE22 and AE23, selected for their ability to grow on galactose in the absence of dibutyryl cyclic AMP, exhibited growth rates on lactose, glucose, and mannose similar to that of the wild-type strain CB13. Although wild-type cells were able to grow on galactose in the presence of dibutyryl cyclic AMP, the generation time was twice as long as that observed with mutant strains AE22 and AE23 grown in the absence of exogenous cyclic nucleotide (Table 1). TABLE 2. Specific activities of galactose dehydrogenase and KDP-gal aldolase in C. crescentus grown on various carbon sources Sp act with carbon source: Enzyme
Strain
Glucose Galactose Lactose CB13 8.0 26.0a 12.0b AE22 2.0 23.6 25.0 AE23 0.4 9.0 9.0 CB13 0.041 0.068a 0.032 AE22 0.032 0.156 0.085 AE23 0.202 0.219 0.180 a Strain CB13 was grown on galactose (0.5%, wt/vol) in the presence of dibutyryl cyclic AMP (3 x 10-3 M). b Reaction not linear with time.
D-Galactose dehydrogenase (nmol/mg per min) KDP-gal aldolase (,umol/mg per min)
TABLE 3. Kinetic constants for galactose uptake by C. crescentus CB13
AE23
AE22
Carbon source
Kma Vmaxb Km Glucose 1.28 1,056 1.21 Galactose 2.03c 1,421X 0.98 Lactose 960 1.28 1.50 a Units for Km are millimolar. b Units for Vm. are micromoles per milliliter per minute. c Galactose (0.5% [wt/vol]) + dibutyryl cyclic AMP (3 x 10-3 M).
Vm.
Km
Vmax
1,428 686 704
2.15 0.33 1.05
1,613 851 620
VOL. 135, 1978
GALACTOSE CATABOLISM IN C. CRESCENTUS
It was previously observed that when strain CB13 was shifted from growth on glucose to either lactose or galactose as the sole carbon source growth was blocked and the cells accumulated at the predivisional cell stage (6, 11). This cell cycle arrest was shown to occur before induction of the corresponding catabolic enzymes; when lactose was the sole carbon source, growth of the culture resumed after 20 h, or immediately upon the addition of cyclic AMP derivatives. Dibutyryl cyclic AMP was shown to stmulate catabolic enzyme induction and thus permit growth (6). Shift of mutants AE22 and AE23 from growth on glucose to either lactose or galactose similurly resulted in a transient arrest of growth and the accumulation of cells at the predivisional cell stage. It has been previously shown that the parent strain, C. crescentus CB13, contains both cyclic AMP and cyclic GMP (5, 6, 11). The intracellular concentration of cyclic AMP was identical in strains AE22, AE23, and wild-type strain CB13 and did not vary with carbon source (data not shown). Galactose utilization in C. crescntus. Crude extracts prepared from strain CB13 grown on galactose plus dibutyryl cyclic AMP or strains AE22 and AE23 grown on galactose were initially assayed for galactokinase activity. The enzyme assay was based on the retention of the charged phosphorylation product on diethylaminoethyl paper chromatograms, as described by Wilson and Hogness (17). Incubation of [14C]galactose with dialyzed crude extracts prepared from all three C. crescentus strains resulted in production of charged reaction products, none of which migrated as galactose 6phosphate when chromatographed on Whatman paper with the 1-propanol-formic acid-water system (1). In addition, the reaction was not dependent on ATP, and it was thus unlikely that a kinase activity was being measured. The formation of charged products, on the other hand, which are retained at the origin on diethylaminoethyl paper chromatography, may result from catabolism of galactose by enzymes of the Entner-Doudoroff pathway (1, 3). In this pathway oxidation of galactose by an NAD-dependent galactose dehydrogenase results in formation of galactono-y-lactone, which is spontaneously hydrolyzed at alkaline pH (pH 8.0). The reaction yields galactonate, which is then further converted to the corresponding a-keto acid (1). To test for the conversion of galactose -to galactonate and the a-keto acid, [14C]galactose was added to the crude extracts and incubated for 30 min at 37°C, and the reaction products were analyzed by paper chromatography (Whatman no. 1) using 1-propanol-formic acid-water (6:3:1), as de-
519
scribed by DeLey and Doudoroff (1). Only two reaction products were formed and these had Rf values of 0.45 and 0.57, corresponding to the migration of galactonic acid and the a-keto acid, respectively. The values agreed with previously reported Rf values (1). Although dialyzed crude extract exhibited enzyme activity, if the crude extract was treated with active charcoal before assaying, galactose dehydrogenase activity could not be measured. The two reaction products migrating as galactonic acid and keto acid were formed from ['4C]galactose, however, when NAD was added to crude extracts that had been treated with active charcoal. These results suggest that C. crescentus contains an NAD-dependent galactose dehydrogenase activity and that the untreated extracts likely contained tightly bound NAD. The specific activity of this galactose dehydrogenase in crude extracts was then determined spectrophotometrically (14; Table 2). In this assay lactose could not replace galactose as substrate. Further steps in the Entner-Doudoroff pathway are the formation of reduced and phosphorylated keto acid yielding KDP-gal (1) and its subsequent cleavage by KDP-gal aldolase. The substrate KDP-gal was used to assay KDP-gal aldolase (13) in extracts of strains CB13, AE22, and AE23 (Table 2). The specific activity of KDP-gal aldolase in all three strains grown on galactose or galactose plus dibutyryl cyclic AMP was similar to that reported for P. saccharophila (13). To determine whether C. crescentus can efficiently utilize galactose by its conversion to glucose, UDP-galactose-4-epimerase was assayed in crude extracts of mutant and wild-type strains grown on glucose, lactose, and galactose. The specific activity obtained in all cases, 0.28 to 0.33 umol of UDP-glucose produced per h per mg of protein, corresponded to less than 1% of that reported for Escherichia coli K-12 grown on galactose (17). This low level of activity was not increased by growth on galactose or lactose. It thus appears likely that galactose utilization in C. crescentus does not depend primarily on UDP-galactose-4-epimerase. The low level of activity may represent the route of galactose incorporation into cell wall. Effect of carbon source on galactose utilization enzymes in wild-type and mutant strains. Three activities involved in galactose
utilization, galactose dehydrogenase, KDP-gal aldolase, and galactose uptake, were assayed in
cultures grown with different carbon sources (Table 2 and 3). The wild-type strain had threefold-higher galactose dehydrogenase activity when grown on galactose plus dibutyryl cyclic AMP than when grown on glucose as the sole
520
KURN, CONTRERAS, AND SHAPIRO
carbon source. Neither the KDP-gal aldolase activity nor galactose uptake varied with carbon source (Tables 2 and 3). Since galactose dehydrogenase was induced in the presence of galactose and dibutyryl cyclic AMP, but neither galactose uptake nor KDP-gal aldolase activity was simultaneously induced, it appears that these activities are independently regulated in the wild-type strain. Further indication that the activities of the enzymes involved in galactose catabolism are regulated independently of one another was obtained from analysis of mutant strains AE22 and AE23. Both galactose dehydrogenase and KDPgal aldolase were induced in mutant AE22 grown on galactose, although the rate of galactose uptake was the same whether the cells were grown on glucose or galactose. Specifically, the levels of galactose dehydrogenase were over 10-fold higher in cultures grown on galactose than when grown on glucose. The activity of KDP-gal aldolase in this mutant strain was increased fivefold in the presence of galactose as compared to the activity when glucose was the sole carbon source, and twofold when compared to lactosegrown cells. Galactose uptake was unaffected by carbon source in mutant strain AE22. In mutant AE23, on the other hand, KDP-gal aldolase was constitutively expressed, and galactose uptake and galactose dehydrogenase were induced by growth on galactose. These results suggest that these activities are independently regulated, as has been found to be the case for catabolic gene expression in Pseudomonas (10, 16). We have previously demonstrated that the regulation of lactose uptake in C. crescentus is independent of the regulation of,-galactosidase; lactose uptake in mutant strains constitutive for the expression of,-galactosidase was shown to be induced by growth on lactose (6). ACKNOWLEDGMENIS This work was supported by gant no. PCM 77-06714 from the National Science Foundation and by Public Health Service grant no. GM 11301 from the National Institute of General Medical Sciences. L S. is a recipient of a Hirschl Trust Award and N. K. is a recipient of Public Health Service Fellowship
J. BACTERIOL. HD05087-02 from the National Institute of Child Health and Human Development.
LITERATURE CITED 1. DeLey, J., and M. Doudoroff. 1957. The metabolism of D-galactose in Pseudomonas saccharophila. J. Biol. Chem. 227:745-757. 2. de Torrontegui, G., R. Diaz, M. L Wheelis, and J. L Conova. 1976. Supra-operonic clustering of genes specifying glucose dissimilation in Pseudomonas putida. Mol. Gen. Genet. 144:407411. 3. Entner, N., and M. Doudoroff. 1952. Glucose and gluconic acid oxidation in Pseudomonas aeruginosa. J. Biol. Chem. 196:853862. 4. Kemp, M. B., and G. D. Hegeman. 1968. Genetic control of the ,B-ketoadipate pathway in Pseudomonas aeruginosa. J. Bacteriol. 96:1488-1499. 5. Kurn, N., and L Shapiro. 1976. Effect of 3',5'-cyclic GMP derivatives on the formation of Caulobacter surface structures. Proc. Natl. Acad. Sci. U.S.A. 73:3303-3307. 6. Kurn, N., L Shapiro, and N. Agabian. 1977. Effect of carbon source and the role of cyclic adenosine 3',5'monophosphate on the Caulobacter cell cycle. J. Bacteriol. 131:951-959. 7. Leidigh, B. J., and M. L Wheelis. 1973. The clustering on the P. putida chromosome of genes specifying dissimilatory functions. J. Mol. Evol. 2:235-242. 8. OrnsWton, L N. 1971. Regulation of catabolic pathways in Pseudomonas. Bacteriol. Rev. 35:87-116. 9. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. 10. Rosenberg, S. C., and G. D. Hegeman. 1969. Clustering of functionally related genes in Pseudomonas aeruginosa. J. Bacteriol. 99:353-355. 11. Shapiro, L, N. Agabian-Keshishian, A. Hirsch, and 0. M. Rosen. 1972. Effect of dibutyryladenosine 3',5'cyclic monophosphate on growth and differentiation in Caulobacter crescentus. Proc. Natl. Acad. Sci. U.S.A. 69:1225-1229. 12. Shedlarsi, J. G. 1974. Glucose 6-phosphate dehydrogenase from Caulobacter crescentus. Biochim. Biophys. Acta 358:33-43. 13. Shuster, C. W. 1966. 2-Keto-3-deoxy-6-phosphogalactonic acid aldolase. Methods Enzymol. 9:524-528. 14. Wallenfels, K., and G. Kurz. 1966. ,8-D-Galactose dehydrogenase from Pseudomonas saccharophila. Methods Enzymol. 9:112-115. 15. Wheelis, M. L 1975. The genetics of dissimilatory pathways in Pseudomonas. Annu. Rev. Microbiol. 29:505-524. 16. Wheelis, M. L, and R. Y. Stanier. 1966. The genetic control of dissimilatory pathways in Pseudomonas putida. Genetics 66:245-266. 17. Wilson, D. B., and D. S. Hogness. 1966. Galactokinase and uridine diphosphogalactose-4-epimerase from E. coli. Methods Enzymol. 8:229-240.
