Arch Microbiol (1990) 154:489-495
Archives of
Hicrnbiolngy
9 Springer-Verlag1990
Catabolism of 3-hydroxybenzoate by the gentisate pathway in Klebsiella pneumoniae M5al David C. N. Jones and Ronald A. Cooper Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK Received October 2, 1989/Accepted June 12, 1990
Abstract. G r o w t h of Klebsiella pneumoniae M5al on 3-hydroxybenzoate leads to the induction of 3-hydroxybenzoate monooxygenase, 2,5-dihydroxybenzoate dioxygenase, maleylpyruvate isomerase and fumarylpyruvate hydrolase. G r o w t h in the presence of 2,5-dihydroxybenzoate also induces all of these enzymes including the 3-hydroxybenzoate monooxygenase which is not required for 2,5-dihydroxybenzoate catabolism. Mutants defective in 3-hydroxybenzoate monooxygenase fail to grow on 3-hydroxybenzoate but grow normally on 2,5dihydroxybenzoate. Mutants lacking maleylpyruvate isomerase fail to grow on 3-hydroxybenzoate and 2,5-dihydroxybenzoate. Both kinds of mutants grow normally on 3,4-dihydroxybenzoate. Mutants defective in maleylpyruvate isomerase accumulate maleylpyruvate when exposed to 3-hydroxybenzoate and growth is inhibited. Secondary mutants that have additionally lost 3-hydroxybenzoate monooxygenase are no longer inhibited by the presence of 3-hydroxybenzoate. The 3-hydroxybenzoate monooxygenase gene (mhbM) and the maleylpyruvate isomerase gene (mhbI) are 100% co-transducible by Pl phage.
pathway for homoprotocatechuate (3,4-dihydroxyphenylacetate) catabolism have been cloned and their organization and expression studied (Jenkins and Cooper 1988). However, the range of aromatic compounds degraded by E. coli is quite restricted (Cooper et al. 1985; Parrott et al. 1987) and to broaden the investigation we have turned our attention to the aromatic degradative ability of other enteric bacteria. The ability of certain Klebsiellae to degrade aromatic compounds was reported in 1969 (Grant and Patel 1969; Patel and Grant 1969) and has been further studied recently (Deschamps et al. 1983; Doten and Ornston 1987). We have been investigating the aromatic degradative abilities of Klebsiella pneumoniae M5al because this organism has been used extensively in molecular genetic studies of dinitrogen fixation (Dixon 1984) and its natural sensitivity to coliphage P1 (Streicher et al. 1971) makes it amenable to various kinds of genetic manipulation. K. pneumoniae M5al grows on various aromatic compounds including certain hydroxybenzoates and this paper describes the pathway for the catabolism of 3-hydroxybenzoate.
Key words: Klebsiella pneumoniae M5al - 3-Hydroxybenzoate degradation - Gentisate pathway - 3-Hydroxybenzoate monooxygenase mutants - Maleylpyrurate isomerase mutants
Materials and methods
In recent years we and others have studied the degradation of certain aromatic acids by Escherichia coli and established that the catabolic pathways for 4-hydroxyphenylacetate (Cooper and Skinner 1980) and 3-phenylpropionate (Burlinghame and Chapman 1983) are identical to those described earlier for various soil bacteria (Dagley et al. 1965; Sparnins et al. 1974). As a further part of this investigation the E. coli C genes encoding the Offprint requests to: R. A. Cooper
Bacterial strains and growth conditions. Klebstellapneumoniae M5al was kindly provided by M. J Merrick, ARC Nitrogen Fixation Unit, Brighton, England. The 3-hydroxybenzoate-negative, 2,5dihydroxybenzoate-positive mutant OH 101 (mhbM), the 3-hydroxybenzoate-negadve, 2,5-dihydroxybenzoate-negative mutant MI214 (mhbI) and the double mutant resistant to 3-hydroxybenzoate during growth on glycerol (M[214/l:mhblM) are described in the text. Cultures were grown aerobically at 30~ in minimal medium (Hareland et al. 1975). Carbon sources were sterilized separately and added aseptically to give a final concentration of 5 mM for aromatic acids, t5 mM for succmate and 20 mM for glycerol. To measure induction by 3-hydroxybenzoate and 2,5dihydroxybenzoate m mutants unable to grow on those compounds, a low catabolite repression carbon source (glycerol) was used as growth substrate with hydroxybenzoates included as appropriate. Likewise, induction by the chemically unstable dihydroxybenzoates was measured using glycerol as the main growth substrate. For this an overnight glycerol culture was inoculated into fresh medium to give 0D68o 0.05 and allowed to grow to 0D680 0.10. Then the
490 appropriate aromatic acid was added and growth continued until OD68o 0.50. Liquid media were solidified as required by the incorporation of 1.6% (w/v) Oxoid bacteriological agar. 2,5Dihydroxybenzoate and 3,4-dihydroxybenzoate plates contained 3 mM sodium dithionite to reduce their rate of spontaneous oxidation.
Substrate oxidation. Washed-cell suspensions were prepared, and oxygen consumption of cell suspensmns and cell-free extracts measured, as described previously (Cooper and Skinner 1980). Cell-free extracts. Bacteria from ~00 ml growth medium were harvested in the exponential growth phase (OD6s0 = 0.5), washed with 20 ml 0.1 M potassium phosphate buffer pH 7.1 and finally resuspended in 4 ml of the same buffer, The cells were broken at 0~C by ultrasonication in an MSE 100 W ultrasonic oscillator operating at 8 microns peak to peak amplitude for two 30 s bursts with 60 s cooling between. The crude extracts were centrifuged for 15 min at 20,000 x g and 4~ and the supernatant retained. When NADH was a substrate the extracts were ultracentrifuged for 90 min at 120,000 x g and 4~ to sediment NADH oxidase. Soluble protein was measured by the biuret method (Gornall et al. 1949) using crystalline bovine serum albumen as the standard. Enzyme assays. Assays of individual enzymes were carried out in 0.1 M potassium phosphate buffer pH 7.1 at 37~ 3-Hydroxybenzoate 6-monooxygenase (EC 1.14.13.-) activity was measured polarographically by following oxygen consumption concomitant with hydroxylation of the aromatic ring. Assays were performed with 0.25 mM 3-hydroxybenzoate and 0.25 mM electron donor. The rates were corrected for NADH oxidase activity as appropriate. The enzyme was also measured spectrophotometrieally at 340 nm as the 3-hydroxybenzoate-dependent oxidation of NADPH. The 1 mt reaction mixture contained 0.15 lamol NADPH and 0.2 lamol 3hydroxybenzoate. 2,5-Dihydroxybenzoate (gentisate) dioxygenase (EC 1.13.11.4) and 3,4-dihydroxybenzoate (protocatechuate) dioxygenase (EC 1.13.11.3) were measured polarographically by following oxygen consumption concomitant with ring cleavage of the aromatic substrate which was 0.25 mM concentration. The assays were started by addition of the enzyme. 2,5-Dihydroxybenzoate dioxygenase was also measured spectrophotometrically at 330 nm as the formation of maleylpyruvate. The 1 ml reaction mixture contained 0.1 gmol 2,5-dihydroxybenzoate. Maleytpyruvate isomerase (EC 5.2.1.4) was assayed spectrophotometrically at 330 nm by the method of Lack (1961) in the presence of an excess of fumarylpyruvate hydrolase. The latter was supplied by a crude extract from the maleylpyruvate isomerase-negative mutant MI214 grown on glycerol in the presence of 2,5-dihydroxybenzoate. The reaction mixture contained in 1 ml of buffer: GSH (0.1 gmol), maleylpyruvate (0.1 gmol), and MI214 crude extract (0.4 mg protein). The reaction was started by addition of the extract. The isomerization of maleylpyruvate (E330 13,000 M - 1 cm - 1 at pH 7.1) to fumarylpyruvate and the subsequent hydrolysis of the fumarylpyruvate results in a loss of absorbance at 330 nm. In this assay the rate of decrease was directly proportional to the amount of extract used. Fumarylpyruvate hydrolase (EC 3.7.1.-) was assayed spectrophotometrically at 335 nm by the method of Lack (1961). The reaction mixture contained 0.1 Ixmol of fumarylpyruvate in 1 ml buffer. The reaction was started by addition of extract and the decrease in absorbance measured. Anion exchange chromatography. Ultracentrifuged crude extract (10-12 mg protein) prepared in 20 mM Tris-HC1 buffer pH 7.0 was applied to a Pharmacia HR5/5 MonoQ anion exchange column attached to a Pharmacia fast protein liquid chromatography (FPLC) system. The column was washed with 5 ml of the 20 mM Tris-HC1 buffer pH 7.0 to elute those proteins not bound to the column. A 20 ml gradient o f 0 ~ l M NaC1 in 20 mM Tris-HC1 buffer pH 7.0, at a flow rate of 1 ml/mm, was used to elute bound proteins. The fractions (1 ml) obtained were assayed for gentisate pathway enzymes.
Isolation of mutants and genetic procedures. Cell suspensions were treated in minimal salts medium with ethylmethanesulphonate as described by Miller (1972). Survavors were allowed to grow overnight at 30~ on succinate minimal medium and 3-hydroxybenzoate-negative mutants were isolated after a penicillin-enrichment procedure (Miller 1972). Phage PI vir-mediated transduction was as described by Miller (1972). Chemicals and biochemicals. Ammonium maleylpyruvate was prepared enzymically from gentisate by the method of Lack (1959). Fumarylpyruvate was prepared freshly an solution by the acidcatalysed isomerization of maleylpyruvate (Lack 1959). 3-Hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, GSH, NADH and NADPH were from Sigma Chemical Co. Ltd., Poole, Dorset, England. Other chemicals were the best grades available commercially.
Results Growth and whole-cell oxidations G r o w t h on v a r i o u s h y d r o x y b e n z o a t e s was assessed b y p l a t e g r o w t h tests. Klebsiella pneumoniae M 5 a l grew well o n 3 - h y d r o x y b e n z o a t e , 2 , 5 - d i h y d r o x y b e n z o a t e (gentisate) a n d 3 , 4 - d i h y d r o x y b e n z o a t e ( p r o t o c a t e c h u a t e ) after 24 h i n c u b a t i o n at 30~ b u t d i d n o t g r o w on 4hydroxybenzoate. However, on continued incubation i n d i v i d u a l colonies a r o s e s p o n t a n e o u s l y o n the 4-hyd r o x y b e n z o a t e plate. Table 1 shows t h a t cells g r o w n o n 3 - h y d r o x y b e n z o a t e a n d glycerol + 2 , 5 - d i h y d r o x y b e n z o a t e r e a d i l y o x i d i z e d b o t h o f these a r o m a t i c c o m p o u n d s b u t n o t 3,4d i h y d r o x y b e n z o a t e . Conversely, cells g r o w n o n glycerol + 3,4-dihydroxybenzoate oxidised that aromatic comp o u n d b u t n o t 3 - h y d r o x y b e n z o a t e . Cells g r o w n on g l y c e r o l o x i d i s e d n e i t h e r 3 - h y d r o x y b e n z o a t e n o r 2,5dihydroxybenzoate.
Gentisate pathway enzymes in cell-free extracts Initial experiments with crude extracts of 3-hydroxyb e n z o a t e - g r o w n K. pneumoniae suggested the presence o f the gentisate p a t h w a y e n z y m e s a n d the p u t a t i v e r e a c t i o n s were s t u d i e d f u r t h e r u s i n g f r a c t i o n s after a n i o n e x c h a n g e c h r o m a t o g r a p h y o f a c r u d e extract. T h e 3 - h y d r o x y b e n z o a t e m o n o o x y g e n a s e was identified b y m e a s u r i n g the 3 - h y d r o x y b e n z o a t e - d e p e n d e n t o x i d a t i o n o f N A D P H . T h e f r a c t i o n w i t h this a c t i v i t y was d e v o i d o f 2 , 5 - d i h y d r o x y b e n z o a t e d i o x y g e n a s e activity. To identify the p r o d u c t o f the r e a c t i o n a 1 m l m i x t u r e c o n t a i n i n g 1 0 m M s o d i u m p h o s p h a t e buffer p H 7 . 1 , 0.15 m M N A D P H , 0.2 m M 3 - h y d r o x y b e n z o a t e a n d the e n z y m e was a l l o w e d to r e a c t until the N A D P H o x i d a t i o n ceased. A t this p o i n t the w a v e l e n g t h setting was c h a n g e d f r o m 340 n m to 330 n m a n d the 2 , 5 - d i h y d r o x y b e n z o a t e dioxygenase fraction added. The formation of a comp o u n d w i t h a n a b s o r b a n c e m a x i m u m a t 330 nm, w h i c h was lost o n a c i d i f i c a t i o n , was o b s e r v e d . These p r o p e r t i e s are c h a r a c t e r i s t i c o f m a l e y l p y r u v a t e ( L a c k 1959) suggesting t h a t 2 , 5 - d i h y d r o x y b e n z o a t e h a d b e e n p r e s e n t in the r e a c t i o n m i x t u r e . In a s e p a r a t e m o n o o x y g e n a s e reac-
491 Table 1. Patterns of substrate oxidation by washed cells of Klebswlla pneumoniae M5al Growth substrate
3-Hydroxybenzoate Glycerol + 2,5-dihydroxybenzoate Glycerol + 3,4-dihydroxybenzoate Glycerol
Oxidation rate [gmol 9min- 1 (mg dry wt.)- 1] of: 3-Hydroxybenzoate
2,5-Dihydroxybenzoate
3,4-Dihydroxybenzoate
0.15 0.13 < 0.001 < 0.001
0.23 0.15 NT < 0.001
0.002 0.002 0.07 NT
Oxygen consumption was measured polarographicallyat 37~ in 0.1 M potassium phosphate buffer pH 7.1. NT = not tested. The rates were measured on at least 3 occasionsand representativevalues are presented tion H2SO4 was added to 0.01 M concentration when the NADPH oxidation ceased. The acidified solution was extracted three times with 1-ml portions of diethyl ether, the pooled ether extracts dried over anhydrous sodium sulphate and the ether removed by evaporation. The residue was dissolved in 300 gl ethyl acetate and a 100 gl sample analysed by paper chromatography using the 2propanol-ammonia-H20 solvent (Smith et al. 1969). A UV-fluorescent spot corresponding to authentic 2,5dihydroxybenzoate was observed. The 2,5-dihydroxybenzoate dioxygenase obtained from the column had very low activity but addition of ferrous sulphate to 1 mM final concentration readily reactivated the enzyme. The enzyme was incubated with 0.1 mM 2,5-dihydroxybenzoate in 10 mM sodium phosphate buffer pH 7.1 and the absorbance at 330 nm monitored until the increase ceased. That this was due to reaction of all the 2,5-dihydroxybenzoate rather than to inactivation of the dioxygenase was confirmed when addition of more enzyme caused no further increase in absorbance at 330 nm. The product of the reaction had an absorbance maximum at 330 nm at pH 7.1 and this absorbance was lost on addition of H2SO4 to 0.01 M concentration, indicating that the compound formed was maleylpyruvate (Lack 1959). Fractionation of a crude extract on the Mono Q column served to remove GSH as well as to separate the various enzymes. There was no loss of absorbance at 330 nm when dialysed crude extract from 3-hydroxybenzoate-grown cells was added to the reaction mixture containing maleylpyruvate, suggesting that no maleylpyruvate hydrolase, such as that reported by Bayley et al. (1980), was present. However, addition of GSH to the reaction led to a very rapid loss of absorbance. The fractions containing the proteins that did not bind to the Mono Q column at pH 7.0 catalysed the GSHdependent decrease in absorbance of maleylpyruvate at 330nm. When the decrease ceased the absorbance maximum had shifted to 335 nm and was unaffected by the addition of H2SO4 to 0.01 M, This is characteristic of fumarylpyruvate (Lack 1959). The fraction from the anion exchange chromatography containing the highest fumarylpyruvate hydrolase activity was identified using fumarylpyruvate as substrate and monitoring the absorbance decrease at 335 nm. At the end of the reaction the wavelength was changed to 340 nm and N A D H added to 0.15 mM. Addition of lactate dehydrogenase caused a very rapid oxidation of
the NADH, suggesting that pyruvate had been formed from fumarylpyruvate. The overall conversion of 2,5-dihydroxybenzoate to equimolar amounts of pyruvate and fumarate by the combined action of the dioxygenase, isomerase and hydrolase was demonstrated using the anion exchange chromatography fractions with the peak activities of these enzymes. The 50-ml reaction mixture contained 10 mM sodium phosphate buffer pH 7.1, 0.1 mM 2,5dihydroxybenzoate, the isomerase and hydrolase fractions and the dioxygenase was added last to initiate the reaction. A sample (1 ml) of the mixture was transferred to a cuvette so that the reaction could be followed spectrophotometrically. When all the 2,5-dihydroxybenzoate had reacted, as indicated by the cessation of absorbance increase at 330 nm, GSH was added and the reaction allowed to proceed until the absorbance decrease ceased. A sample (5 ml) of the reaction mixture was removed for subsequent enzymic analysis of pyruvate and fumarate. The remainder was acidified by addition of H2SO4 to 0.01 M and extracted three times with an equal volume of diethyl ether. The ether extracts were pooled, dried over anhydrous sodium sulphate, evaporated to dryness under vacuo at 30~C and the residue dissolved in ethyl acetate (1 ml). Samples (50 btl) were analysed by paper chromatography using butanol-acetic acid-water and ethanol-ammonia-water solvents (Nordmann and Nordmann 1969). A UV-absorbing spot that corresponded to authentic fumaric acid was observed. Likewise, when analysed with the 2,4-dinitrophenylhydrazine reagent (Nordmann and Nordmann 1969) spots corresponding to those given by authentic pyruvic acid were seen. Pyruvate in the reaction mixture was measured using N A D H and lactate dehydrogenase (Czok and Lamprecht 1974) and corresponded to 0.10raM. Fumarate was measured by the action of fumarase to convert it to malate. Malate dehydrogenase in the presence of hydrazine at pH 9.0 was then used to convert the malate to oxaloacetate with the concomitant formation of N A D H (Gutmann and Wahlefeld 1974). The fumarate content of the reaction mixture was 0.085 raM. The various gentisate pathway enzymes were measured individually in crude extracts of K. pneumoniae. As can be seen from Table 2 all the enzymes were present at high activity after growth on 3-hydroxybenzoate or glycerol + 2,5-dihydroxybenzoate but essentially absent from glycerol-grown cells. The 3-hydroxybenzoategrown cell extract showed no 3,4-dihydroxybenzoate
492 Table 2. Activities of enzymesassociated with 3-hydroxybenzoatecatabolism m K. pneumoniae M5al Growth substrate
Specificactivity [pmol 9min- 1 (rag protein)- 1] 3-Hydroxybenzoate monooxygenase
3-Hydroxybenzoate Glycerol + 2,5-dihydroxybenzoate Glycerol + 3,4-dihydroxybenzoate Glycerol
(NADH)
(NADPH)
0.91 0.36 NT < 0.001
0.76 0.31 NT NT
2,5-Dihydroxy- MaleylF u m a r y l - 3,4-Dihydroxybenzoate pyruvate p y r u v a t e benzoate dioxygenase lsomerase hydrolase dioxygenase 1.04 0.78 NT < 0.001
8.8 4.1 NT < 0.001
0.40 0.25 NT < 0.001
< 0.001 NT 0.07 NT
Details of the various assays are given in Methods. The figures are representativevalues. NT = not tested
dioxygenase activity but this enzyme was detected in extracts prepared from glycerol + 3,4-dihydroxybenzoategrown cells, Table 2.
Properties of 3-hydroxybenzoate-negative mutants Mutants unable to grow in 3-hydroxybenzoate (gene symbol: m-hydroxybenzoate; mhb) but capable of normal growth on fumarate and pyruvate were isolated. When these mutants were tested for growth on 2,5-dihydroxybenzoate some grew but others did not. The 3hydroxybenzoate-negative, 2,5-dihydroxybenzoate-positive class could be defective in 3-hydroxybenzoate uptake or hydroxylation and the 3-hydroxybenzoate-negative, 2,5-dihydroxybenzoate-negative class defective in 2,5dihydroxybenzoate dioxygenase, maleylpyruvate isomerase or fumarylpyruvate hydrolase. A 3-hydroxybenzoate-negative, 2,5-dihydroxybenzoate-positive mutant (designated OH101) was grown in glycerol + 2,5-dihydroxybenzoate and extracts analysed. Table 3 shows that the activity of 2,5-dihydroxybenzoate dioxygenase, maleylpyruvate isomerase and fumarylpyruvate hydrolase in K. pneumoniae M5al and mutant OH101 were essentially equivalent but that OH101 had no detectable 3-hydroxybenzoate monooxygenase activity with either N A D H or NADPH. Unlike the situation for K. pneumoniae M5al none of the enzymes were present in significant amounts after growth of OH10a on glycerol + 3-hydroxybenzoate. Thus OH 101 was defective in 3-hydroxybenzoate monooxygenase (gene symbol
mhbM). A 3-hydroxybenzoate-negative, 2,5-dihydroxybenzoate-negative mutant designated MI214 was also analysed. When either 3-hydroxybenzoate or 2,5-dihydroxybenzoate was added to cultures of MI214 growing on glycerol severe inhibition of growth was seen after 2 - 3 cell doublings. Extracts prepared from such inhibited cells showed 3-hydroxybenzoate monooxygenase, 2,5dihydroxybenzoate dioxygenase and fumarylpyruvate hydrolase activities that were about 25% of those from similarly grown, but not inhibited, K. pneumoniae M5al, Table 3. However, maleylpyruvate isomerase was undetectable in MI214 extracts suggesting that the mutant was defective in this enzyme (gene symbol mhbI).
Skinner and Cooper (1982) reported that the growth of an Escherichia coli 4-hydroxyphenylacetate-negative mutant on glycerol was severely inhibited by addition of 4-hydroxyphenylacetate due to the accumulation of the substrate of the blocked reaction. So the failure of mutant MI214 to grow normally in the presence of 3-hydroxybenzoate seemed likely to be caused by accumulation of maleylpyruvate. To see whether maleylpyruvate was accumulated, the UV spectra of culture supernatants from wild-type and mutant cells during growth on glycerol + 3-hydroxybenzoate were measured. At pH 7.1 3-hydroxybenzoate itself shows an absorbance peak at 290 nm. During growth of K. pneumoniae this peak was gradually reduced as the 3-hydroxybenzoate was utilized. The UV spectrum ( 2 5 0 - 410 nm) of the culture medium showed no other significant change. In contrast, the 290 nm absorbance of the culture medium did not fall in cultures of mutant MI214. Rather a strong absorbance at 330 nm appeared and there was a slight increase in the absorbance at 290 nm due, presumably, to a contribution from the compound with the absorbance maximum at 330 nm. The 330-nm absorbance was lost when the pH was reduced to pH 1.5. An absorbance maximum of 330 nm which is lost on acidification is characteristic of maleylpyruvate and not shown by fumarylpyruvate (Lack 1959).
Double mutants of 3-hydroxybenzoate catabolism When mutant MI214 (mhbI) was spread onto succinate + 3-hydroxybenzoate plates and incubated at 30~ for 24 h there was little or no growth. However, after 4 8 72 h incubation strongly growing individual colonies were seen on the weak background lawn of growth. If the initial inhibition of growth was due to the accumulation of maleylpyruvate to a toxic concentration such single colonies could have arisen by the following means: (1) by reversion of the original mutation; (2) by a further mutation which allowed the cell to metabolize the maleylpyruvate by an alternative route; (3) by a mutation which made the cell resistant to the toxic compound; (4) by a further mutation in the 3-hydroxybenzoate catabolic pathway which then prevented formation of the inhibitory compound. When a selection of the colonies were
493 Table 3. Gentisate pathway enzymes in various mutants
Enzyme
Strain M5al
3-Hydroxybenzoate monooxygenase 2,5-Dihydroxybenzoate dioxygenase Maleylpyruvate isomerase Fumarylpyruvate hydrolase
OH101
a
b
0.10 0.67 3.2 0.43
0.12 0.35 4.1 0.25
MI214
a
b
< 0.001 < 0.00i 0.005 0.015
< 0.001 0.78 4.1 0.60
a
MI214/1 b
0.03 0.14 < 0.001 0.10
a
0.03 0.10 < 0.001 0.07
< 0.001 0.01 < 0.001 0.002
b < 0.001 0.35 < 0.001 0.14
Details of the assays are given in Methods. a, b = specific activities [gmol 9rain 1 (mg protein)-1] for cells grown on (a) glycerol + 3-hydroxybenzoate and (b) glycerol + 2,5-dihydroxybenzoate. Representative values are presented
VII COOH I
C=O
COOH
02
COOH
COOH
~
%/, I
ooo.
,,,,j NAD(P)
',
COOH
COOH
I CH3
v
II
r
"~ .,
|1
,v
C
+
HOOC /
H20 Fig. 1. The gentisate pathway for 3-hydroxybenzoate catabolism. 3Hydroxybenzoate (I) ~s converted to pyruvate (VII) and fumarate (VI) via 2.5-dihydroxybenzoate [gentisate] (II), maleylpyruvate [enol] (ITt), fumarylpyruvate [enol] (IV) and fumarylpyruvate (V)
tested for growth on 3-hydroxybenzoate very few grew i.e. were of categories I and 2 above. Many colonies that grew in succinate + 3-hydroxybenzoate were still unable to grow on succinate + 2,5-dihydroxybenzoate. It was thus unlikely that in these cases growth was due to developing a tolerance o f the inhibitor. The most likely explanation was that formation of the inhibitor from 3hydroxybenzoate, but not from 2,5-dihydroxybenzoate, had been blocked. One such secondary mutant, designated MI214/1, was grown on glycerol + 3-hydroxybenzoate and glycerol + 2,5-dihydroxybenzoate and cell extracts assayed. Table 3 shows that MI214/1 had undergone a mutation in 3-hydroxybenzoate monooxygenase and so was doubly defective in 3-hydroxybenzoate catabolic enzymes.
Trans&tctional analysis of mhbM and mhbI genes To see if the mhbM and mhbI mutations were closely linked a phage P1 vir lysate prepared on wild-type K. pneumoniae M5al was used to transduce MI214/1 to growth on 2,5-dihydroxybenzoate which selects only for repair ofmhbL When the 79 transductants obtained were
~'H
Vl by the sequential action of 3-hydroxybenzoate monooxygenase, 2.5dihydroxybenzoate &oxygenase, maleylpyruvate isomerase, a nonenzymic (NE) keto-enol tautomerization and fumarylpyruvate hydrolase
tested for growth on 3-hydroxybenzoate all were positive indicating 100% linkage between the mhbI and mhbM genes.
Growth of 3-hydroxybenzoate-negative mutants on 3,4-dihydroxybenzoate When the two 3-hydroxybenzoate-negative mutants OH101 and MI214 were tested on 3,4-dihydroxybenzoate plates both grew as well as K. pneumoniae M5al.
Discussion
Two principal pathways for the bacterial aerobic catabolism of 3-hydroxybenzoate have been described (Yano and Arima 1958) and the utilization of a particular pathway may be of taxonomic significance (Wheelis et al. 1967). 2,5-Dihydroxybenzoate or 3,4-dihydroxybenzoate are the ring-fission intermediates in these two routes for 3hydroxybenzoate breakdown but which is used by enteric bacteria was not known.
494
The results presented here for whole cells, cell-free extracts and mutants are all consistent with 3-hydroxybenzoate being catabolised via 2,5-dihydroxybenzoate, maleylpyruvate and fumarylpyruvate in Klebsiella pneumoniae M5al. This reaction sequence (Fig. 1) is known as the gentisate pathway (Dagley 1975). All the enzymes involved, 3-hydroxybenzoate monooxygenase, 2,5-dihydroxybenzoate dioxygenase, maleylpyruvate isomerase and fumarylpyruvate hydrolase were present at high activities in 3-hydroxybenzoate-grown cells and low or undetectable in extracts from glycerol-grown cells. Growth in the presence of 2,5-dihydroxybenzoate led to induction of all the enzymes of the gentisate pathway even though the 3-hydroxybenzoate monooxygenase is not needed for 2,5-dihydroxybenzoate catabolism. However, none of these enzymes were induced during growth of 3-hydroxybenzoate monooxygenase-negative mutants in the presence of 3-hydroxybenzoate, so this compound itself was not an inducer. Since fumarylpyruvate hydrolase was still induced in the maleylpyruvate isomerasenegative mutant that could form Z5-dihydroxybenzoate and maleylpyruvate but not fumarylpyruvate, it seems likely that either 2,5-dihydroxybenzoate or maleylpyruvate is the inducer of all the gentisate pathway enzymes. The 3-hydroxybenzoate monooxygenase of crude extracts worked almost equally well with NADH or NADPH. It was thus possible that this was due to one enzyme with the ability to utilise both coenzymes or to two enzymes each specific for a particular coenzyme. However, the ratio of activity with the two electron donors remained constant as the amount of the 3hydroxybenzoate monooxygenase activity varied and both activities were missing in mutant OH101. This suggested that it was a single enzyme that could utilize either coenzyme, similar to the enzyme from Pseudomonas aeruginosa (Groseclose and Ribbons 1973). The general growth inhibition seen for the maleylpyruvate isomerase-negative mutant in the presence of 3-hydroxybenzoate and its abolition by a mutation removing 3-hydroxybenzoate monooxygenase activity suggested that accumulated maleylpyruvate was responsible. Thus, like succinic semialdehyde in 4-hydroxyphenylacetate catabolism (Skinner and Cooper 1982) an excess of a normal pathway intermediate can cause severe growth inhibition. The ability of the 3-hydroxybenzoate-negative mutants to grow normally on 3,4-dihydroxybenzoate was further support for the view that this compound was not an intermediate in 3-hydroxybenzoate catabolism in K. pneumoniae. T h e small n u m b e r o f steps in the g e n t i s a t e p a t h w a y a n d the evidence f r o m c o t r a n s d u c t i o n studies o f clustering f o r at least two o f the genes i n v o l v e d m a k e s it likely t h a t all the p a t h w a y genes c o u l d be c l o n e d on a single s m a l l piece o f D N A to facilitate their fine genetic analysis. This p o s s i b i l i t y is c u r r e n t l y u n d e r i n v e s t i g a t i o n . Acknowledgements. DCNJ was supported by a research studentship from the SERC. We thank Mrs. U. Gervind-Richards for typing the manuscript.
References Bayly RC, Chapman PJ, Dagley S, Di Berardino D (1980) Purification and some properties of maleylpyruvate hydrolase and fumarylpyruvate hydrolase from Pseudomonas alcaligenes. J Bacteriol 143 : 7 0 - 77 Burhnghame R, Chapman PJ (1983) Catabolism ofphenylpropionic acid and its 3-hydroxy derivative by Escherichia coli. J Bacteriol 155:113-121 Cooper RA, Skinner MA (1980) Catabolism of 3- and 4-hydroxyphenylacetate by the 3,4-dihydroxyphenylacetate pathway in Escherichia coli. J Bacteriol 143 : 302 - 306 Cooper RA, Jones DCN, Parrott S (1985) Isolation and mapping of Escherichia coli Kt2 mutants defective in phenylacetate degradation. J Gen Microbiol 131:2753-2757 Czok R, Lamprecht W (1974) Pyruvate, phosphoenolpyruvate and D-glycerate 2-phosphate. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vot 3, 2nd english edn. Verlag Chemie, Weinheim; Academic Press, New York London~ pp 1446 - 1451 Dagley S (1975) A biochemical approach to some problems of environmental pollution. In: Campbell PN, Aldridge WN (eds) Essays in biochemistry, vol 11. Academic Press, London New York San Francisco, pp 8J -138 Dagley S, Chapman PJ, Gibson DT (1965) The metabolism of fiphenylpropionic acid by an Achromobacter. Biochem J 97: 643 - 650 Deschamps AM, Richard C, Lebeault J-M (1983) Bacteriology and nutrition of environmental strains of Klebsiella pneumoniae involved in wood and bark decay. Ann Microbiol (Paris) 134A:189-- 196 Dixon RA (1984) The genetic complexity of nitrogen fixation. J Gen Microbiol 130 : 2745 - 2755 Doten RC, Ornston LN (1987) Protocatechuate is not metabolized via catechol in Enterobacter aerogenes. J Bacteriol 169:58275830 Gornall AC, Bardawill CS, David MM (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751-766 Grant DJW, Patel JC (1969) The non-oxidative decarboxylation of p-hydroxybenzoic acid, gentisic acid, protocatechuic acid and gallic acid by Klebsiella aerogenes (Aerobacter aerogenes) Antonie van Leuwenhoek 35: 325-- 343 Groseclose EE, Ribbons DW (1973) 3-Hydroxybenzoate 6-hydroxylase from Pseudomonas aeruginosa. Biochem Biophys Res Commun 55:897-903 Gutmann I, Wahlefeld AW (1976) L-(--)-Malate. Determination with malate dehydrogenase and NAD. In: Bergmeyer HU (ed) Methods of enzymatic anaiysis, vol 3, 2nd english edn. Verlag Chemie, Weinheim; Academic Press, New York London, pp 1585-1589 Hareland WA, Crawford RL, Chapman PJ, Dagley S (1975) Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. J Bacteriol 121:272-285 Jenkins JR, Cooper RA (1988) Molecular cloning, expression, and analysis of the genes of the homoprotocatechuate catabolic pathway of Escherichia coli C. J Bacteriol 170: 5317 - 5324 Lack L (1959) The enzymatic oxidation of gentisic acid Biochim Biophys Acta 34:117-123 Lack L (1961) Enzymatic cis-trans isomerization of maleylpyruvate. J Biol Chem 236:2835--2840 Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Nordmann J, Nordmann R (1969) Organic acids. In: Smith I (ed) Chromatographic and electrophoretic techniques, 3rd edn. William Heinemann, London, pp 342--363 Parrott S, Jones S, Cooper RA (1987) 2-Phenylethylamine catabolism by Escherichia coli K12. J Gen Microbiol 133:347--351 Patel JC, Grant DJW (1969) The formation of phenol in the degradation of p-hydroxybenzoic acid by Klebsiella aerogenes (Aerobacter aerogenes). Antonie van Leuwenhoek 35 : 53 - 64
495 Skinner MA, Cooper RA (1982) An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase. Arch Microblol 132 :270 - 275 Smith I, Seakins JWT, Dayman J (1969) Phenolic acids. In: Smith I (ed) Chromatographic and electrophoretic techniques. 3rd edn. William Heinemann, London, pp 3 6 4 - 389 Sparnins VL, Chapman PJ, Dagley S (1974) Bacterial degradation of 4-hydroxyphenylacetic acid and homoprotocatechuic acid. J Bacteriol 120 : 1 5 9 - - 1 6 7 Strelcher S, Gurney E, Valentine RC (1971) Transduction of the nitrogen-fixation genes in Klebsiella pneumoniae. Proc Natl Acad Sci USA 68:1174-1177
Wheelis ML, Palleroni NJ, Stanier RY (1967) The metabohsm of aromatic compounds by Pseudomonas testosteroni and P. acidovorans. Arch Mikrobiol 59: 302 - 3] 4 Yano K, Arima K (1958) Metabolism of aromatic compounds by bacteria. II. m-Hydroxybenzoic acid hydroxylase A and B; 5dehydroshikimic acid, a precursor of protocatechuic acid, a new pathway from salicylic acid to gentislc acid. J Gen Appl Microbiol 4 :241 - 258
Archives of
Hicrnbiolngy
9 Springer-Verlag1990
Catabolism of 3-hydroxybenzoate by the gentisate pathway in Klebsiella pneumoniae M5al David C. N. Jones and Ronald A. Cooper Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK Received October 2, 1989/Accepted June 12, 1990
Abstract. G r o w t h of Klebsiella pneumoniae M5al on 3-hydroxybenzoate leads to the induction of 3-hydroxybenzoate monooxygenase, 2,5-dihydroxybenzoate dioxygenase, maleylpyruvate isomerase and fumarylpyruvate hydrolase. G r o w t h in the presence of 2,5-dihydroxybenzoate also induces all of these enzymes including the 3-hydroxybenzoate monooxygenase which is not required for 2,5-dihydroxybenzoate catabolism. Mutants defective in 3-hydroxybenzoate monooxygenase fail to grow on 3-hydroxybenzoate but grow normally on 2,5dihydroxybenzoate. Mutants lacking maleylpyruvate isomerase fail to grow on 3-hydroxybenzoate and 2,5-dihydroxybenzoate. Both kinds of mutants grow normally on 3,4-dihydroxybenzoate. Mutants defective in maleylpyruvate isomerase accumulate maleylpyruvate when exposed to 3-hydroxybenzoate and growth is inhibited. Secondary mutants that have additionally lost 3-hydroxybenzoate monooxygenase are no longer inhibited by the presence of 3-hydroxybenzoate. The 3-hydroxybenzoate monooxygenase gene (mhbM) and the maleylpyruvate isomerase gene (mhbI) are 100% co-transducible by Pl phage.
pathway for homoprotocatechuate (3,4-dihydroxyphenylacetate) catabolism have been cloned and their organization and expression studied (Jenkins and Cooper 1988). However, the range of aromatic compounds degraded by E. coli is quite restricted (Cooper et al. 1985; Parrott et al. 1987) and to broaden the investigation we have turned our attention to the aromatic degradative ability of other enteric bacteria. The ability of certain Klebsiellae to degrade aromatic compounds was reported in 1969 (Grant and Patel 1969; Patel and Grant 1969) and has been further studied recently (Deschamps et al. 1983; Doten and Ornston 1987). We have been investigating the aromatic degradative abilities of Klebsiella pneumoniae M5al because this organism has been used extensively in molecular genetic studies of dinitrogen fixation (Dixon 1984) and its natural sensitivity to coliphage P1 (Streicher et al. 1971) makes it amenable to various kinds of genetic manipulation. K. pneumoniae M5al grows on various aromatic compounds including certain hydroxybenzoates and this paper describes the pathway for the catabolism of 3-hydroxybenzoate.
Key words: Klebsiella pneumoniae M5al - 3-Hydroxybenzoate degradation - Gentisate pathway - 3-Hydroxybenzoate monooxygenase mutants - Maleylpyrurate isomerase mutants
Materials and methods
In recent years we and others have studied the degradation of certain aromatic acids by Escherichia coli and established that the catabolic pathways for 4-hydroxyphenylacetate (Cooper and Skinner 1980) and 3-phenylpropionate (Burlinghame and Chapman 1983) are identical to those described earlier for various soil bacteria (Dagley et al. 1965; Sparnins et al. 1974). As a further part of this investigation the E. coli C genes encoding the Offprint requests to: R. A. Cooper
Bacterial strains and growth conditions. Klebstellapneumoniae M5al was kindly provided by M. J Merrick, ARC Nitrogen Fixation Unit, Brighton, England. The 3-hydroxybenzoate-negative, 2,5dihydroxybenzoate-positive mutant OH 101 (mhbM), the 3-hydroxybenzoate-negadve, 2,5-dihydroxybenzoate-negative mutant MI214 (mhbI) and the double mutant resistant to 3-hydroxybenzoate during growth on glycerol (M[214/l:mhblM) are described in the text. Cultures were grown aerobically at 30~ in minimal medium (Hareland et al. 1975). Carbon sources were sterilized separately and added aseptically to give a final concentration of 5 mM for aromatic acids, t5 mM for succmate and 20 mM for glycerol. To measure induction by 3-hydroxybenzoate and 2,5dihydroxybenzoate m mutants unable to grow on those compounds, a low catabolite repression carbon source (glycerol) was used as growth substrate with hydroxybenzoates included as appropriate. Likewise, induction by the chemically unstable dihydroxybenzoates was measured using glycerol as the main growth substrate. For this an overnight glycerol culture was inoculated into fresh medium to give 0D68o 0.05 and allowed to grow to 0D680 0.10. Then the
490 appropriate aromatic acid was added and growth continued until OD68o 0.50. Liquid media were solidified as required by the incorporation of 1.6% (w/v) Oxoid bacteriological agar. 2,5Dihydroxybenzoate and 3,4-dihydroxybenzoate plates contained 3 mM sodium dithionite to reduce their rate of spontaneous oxidation.
Substrate oxidation. Washed-cell suspensions were prepared, and oxygen consumption of cell suspensmns and cell-free extracts measured, as described previously (Cooper and Skinner 1980). Cell-free extracts. Bacteria from ~00 ml growth medium were harvested in the exponential growth phase (OD6s0 = 0.5), washed with 20 ml 0.1 M potassium phosphate buffer pH 7.1 and finally resuspended in 4 ml of the same buffer, The cells were broken at 0~C by ultrasonication in an MSE 100 W ultrasonic oscillator operating at 8 microns peak to peak amplitude for two 30 s bursts with 60 s cooling between. The crude extracts were centrifuged for 15 min at 20,000 x g and 4~ and the supernatant retained. When NADH was a substrate the extracts were ultracentrifuged for 90 min at 120,000 x g and 4~ to sediment NADH oxidase. Soluble protein was measured by the biuret method (Gornall et al. 1949) using crystalline bovine serum albumen as the standard. Enzyme assays. Assays of individual enzymes were carried out in 0.1 M potassium phosphate buffer pH 7.1 at 37~ 3-Hydroxybenzoate 6-monooxygenase (EC 1.14.13.-) activity was measured polarographically by following oxygen consumption concomitant with hydroxylation of the aromatic ring. Assays were performed with 0.25 mM 3-hydroxybenzoate and 0.25 mM electron donor. The rates were corrected for NADH oxidase activity as appropriate. The enzyme was also measured spectrophotometrieally at 340 nm as the 3-hydroxybenzoate-dependent oxidation of NADPH. The 1 mt reaction mixture contained 0.15 lamol NADPH and 0.2 lamol 3hydroxybenzoate. 2,5-Dihydroxybenzoate (gentisate) dioxygenase (EC 1.13.11.4) and 3,4-dihydroxybenzoate (protocatechuate) dioxygenase (EC 1.13.11.3) were measured polarographically by following oxygen consumption concomitant with ring cleavage of the aromatic substrate which was 0.25 mM concentration. The assays were started by addition of the enzyme. 2,5-Dihydroxybenzoate dioxygenase was also measured spectrophotometrically at 330 nm as the formation of maleylpyruvate. The 1 ml reaction mixture contained 0.1 gmol 2,5-dihydroxybenzoate. Maleytpyruvate isomerase (EC 5.2.1.4) was assayed spectrophotometrically at 330 nm by the method of Lack (1961) in the presence of an excess of fumarylpyruvate hydrolase. The latter was supplied by a crude extract from the maleylpyruvate isomerase-negative mutant MI214 grown on glycerol in the presence of 2,5-dihydroxybenzoate. The reaction mixture contained in 1 ml of buffer: GSH (0.1 gmol), maleylpyruvate (0.1 gmol), and MI214 crude extract (0.4 mg protein). The reaction was started by addition of the extract. The isomerization of maleylpyruvate (E330 13,000 M - 1 cm - 1 at pH 7.1) to fumarylpyruvate and the subsequent hydrolysis of the fumarylpyruvate results in a loss of absorbance at 330 nm. In this assay the rate of decrease was directly proportional to the amount of extract used. Fumarylpyruvate hydrolase (EC 3.7.1.-) was assayed spectrophotometrically at 335 nm by the method of Lack (1961). The reaction mixture contained 0.1 Ixmol of fumarylpyruvate in 1 ml buffer. The reaction was started by addition of extract and the decrease in absorbance measured. Anion exchange chromatography. Ultracentrifuged crude extract (10-12 mg protein) prepared in 20 mM Tris-HC1 buffer pH 7.0 was applied to a Pharmacia HR5/5 MonoQ anion exchange column attached to a Pharmacia fast protein liquid chromatography (FPLC) system. The column was washed with 5 ml of the 20 mM Tris-HC1 buffer pH 7.0 to elute those proteins not bound to the column. A 20 ml gradient o f 0 ~ l M NaC1 in 20 mM Tris-HC1 buffer pH 7.0, at a flow rate of 1 ml/mm, was used to elute bound proteins. The fractions (1 ml) obtained were assayed for gentisate pathway enzymes.
Isolation of mutants and genetic procedures. Cell suspensions were treated in minimal salts medium with ethylmethanesulphonate as described by Miller (1972). Survavors were allowed to grow overnight at 30~ on succinate minimal medium and 3-hydroxybenzoate-negative mutants were isolated after a penicillin-enrichment procedure (Miller 1972). Phage PI vir-mediated transduction was as described by Miller (1972). Chemicals and biochemicals. Ammonium maleylpyruvate was prepared enzymically from gentisate by the method of Lack (1959). Fumarylpyruvate was prepared freshly an solution by the acidcatalysed isomerization of maleylpyruvate (Lack 1959). 3-Hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, GSH, NADH and NADPH were from Sigma Chemical Co. Ltd., Poole, Dorset, England. Other chemicals were the best grades available commercially.
Results Growth and whole-cell oxidations G r o w t h on v a r i o u s h y d r o x y b e n z o a t e s was assessed b y p l a t e g r o w t h tests. Klebsiella pneumoniae M 5 a l grew well o n 3 - h y d r o x y b e n z o a t e , 2 , 5 - d i h y d r o x y b e n z o a t e (gentisate) a n d 3 , 4 - d i h y d r o x y b e n z o a t e ( p r o t o c a t e c h u a t e ) after 24 h i n c u b a t i o n at 30~ b u t d i d n o t g r o w on 4hydroxybenzoate. However, on continued incubation i n d i v i d u a l colonies a r o s e s p o n t a n e o u s l y o n the 4-hyd r o x y b e n z o a t e plate. Table 1 shows t h a t cells g r o w n o n 3 - h y d r o x y b e n z o a t e a n d glycerol + 2 , 5 - d i h y d r o x y b e n z o a t e r e a d i l y o x i d i z e d b o t h o f these a r o m a t i c c o m p o u n d s b u t n o t 3,4d i h y d r o x y b e n z o a t e . Conversely, cells g r o w n o n glycerol + 3,4-dihydroxybenzoate oxidised that aromatic comp o u n d b u t n o t 3 - h y d r o x y b e n z o a t e . Cells g r o w n on g l y c e r o l o x i d i s e d n e i t h e r 3 - h y d r o x y b e n z o a t e n o r 2,5dihydroxybenzoate.
Gentisate pathway enzymes in cell-free extracts Initial experiments with crude extracts of 3-hydroxyb e n z o a t e - g r o w n K. pneumoniae suggested the presence o f the gentisate p a t h w a y e n z y m e s a n d the p u t a t i v e r e a c t i o n s were s t u d i e d f u r t h e r u s i n g f r a c t i o n s after a n i o n e x c h a n g e c h r o m a t o g r a p h y o f a c r u d e extract. T h e 3 - h y d r o x y b e n z o a t e m o n o o x y g e n a s e was identified b y m e a s u r i n g the 3 - h y d r o x y b e n z o a t e - d e p e n d e n t o x i d a t i o n o f N A D P H . T h e f r a c t i o n w i t h this a c t i v i t y was d e v o i d o f 2 , 5 - d i h y d r o x y b e n z o a t e d i o x y g e n a s e activity. To identify the p r o d u c t o f the r e a c t i o n a 1 m l m i x t u r e c o n t a i n i n g 1 0 m M s o d i u m p h o s p h a t e buffer p H 7 . 1 , 0.15 m M N A D P H , 0.2 m M 3 - h y d r o x y b e n z o a t e a n d the e n z y m e was a l l o w e d to r e a c t until the N A D P H o x i d a t i o n ceased. A t this p o i n t the w a v e l e n g t h setting was c h a n g e d f r o m 340 n m to 330 n m a n d the 2 , 5 - d i h y d r o x y b e n z o a t e dioxygenase fraction added. The formation of a comp o u n d w i t h a n a b s o r b a n c e m a x i m u m a t 330 nm, w h i c h was lost o n a c i d i f i c a t i o n , was o b s e r v e d . These p r o p e r t i e s are c h a r a c t e r i s t i c o f m a l e y l p y r u v a t e ( L a c k 1959) suggesting t h a t 2 , 5 - d i h y d r o x y b e n z o a t e h a d b e e n p r e s e n t in the r e a c t i o n m i x t u r e . In a s e p a r a t e m o n o o x y g e n a s e reac-
491 Table 1. Patterns of substrate oxidation by washed cells of Klebswlla pneumoniae M5al Growth substrate
3-Hydroxybenzoate Glycerol + 2,5-dihydroxybenzoate Glycerol + 3,4-dihydroxybenzoate Glycerol
Oxidation rate [gmol 9min- 1 (mg dry wt.)- 1] of: 3-Hydroxybenzoate
2,5-Dihydroxybenzoate
3,4-Dihydroxybenzoate
0.15 0.13 < 0.001 < 0.001
0.23 0.15 NT < 0.001
0.002 0.002 0.07 NT
Oxygen consumption was measured polarographicallyat 37~ in 0.1 M potassium phosphate buffer pH 7.1. NT = not tested. The rates were measured on at least 3 occasionsand representativevalues are presented tion H2SO4 was added to 0.01 M concentration when the NADPH oxidation ceased. The acidified solution was extracted three times with 1-ml portions of diethyl ether, the pooled ether extracts dried over anhydrous sodium sulphate and the ether removed by evaporation. The residue was dissolved in 300 gl ethyl acetate and a 100 gl sample analysed by paper chromatography using the 2propanol-ammonia-H20 solvent (Smith et al. 1969). A UV-fluorescent spot corresponding to authentic 2,5dihydroxybenzoate was observed. The 2,5-dihydroxybenzoate dioxygenase obtained from the column had very low activity but addition of ferrous sulphate to 1 mM final concentration readily reactivated the enzyme. The enzyme was incubated with 0.1 mM 2,5-dihydroxybenzoate in 10 mM sodium phosphate buffer pH 7.1 and the absorbance at 330 nm monitored until the increase ceased. That this was due to reaction of all the 2,5-dihydroxybenzoate rather than to inactivation of the dioxygenase was confirmed when addition of more enzyme caused no further increase in absorbance at 330 nm. The product of the reaction had an absorbance maximum at 330 nm at pH 7.1 and this absorbance was lost on addition of H2SO4 to 0.01 M concentration, indicating that the compound formed was maleylpyruvate (Lack 1959). Fractionation of a crude extract on the Mono Q column served to remove GSH as well as to separate the various enzymes. There was no loss of absorbance at 330 nm when dialysed crude extract from 3-hydroxybenzoate-grown cells was added to the reaction mixture containing maleylpyruvate, suggesting that no maleylpyruvate hydrolase, such as that reported by Bayley et al. (1980), was present. However, addition of GSH to the reaction led to a very rapid loss of absorbance. The fractions containing the proteins that did not bind to the Mono Q column at pH 7.0 catalysed the GSHdependent decrease in absorbance of maleylpyruvate at 330nm. When the decrease ceased the absorbance maximum had shifted to 335 nm and was unaffected by the addition of H2SO4 to 0.01 M, This is characteristic of fumarylpyruvate (Lack 1959). The fraction from the anion exchange chromatography containing the highest fumarylpyruvate hydrolase activity was identified using fumarylpyruvate as substrate and monitoring the absorbance decrease at 335 nm. At the end of the reaction the wavelength was changed to 340 nm and N A D H added to 0.15 mM. Addition of lactate dehydrogenase caused a very rapid oxidation of
the NADH, suggesting that pyruvate had been formed from fumarylpyruvate. The overall conversion of 2,5-dihydroxybenzoate to equimolar amounts of pyruvate and fumarate by the combined action of the dioxygenase, isomerase and hydrolase was demonstrated using the anion exchange chromatography fractions with the peak activities of these enzymes. The 50-ml reaction mixture contained 10 mM sodium phosphate buffer pH 7.1, 0.1 mM 2,5dihydroxybenzoate, the isomerase and hydrolase fractions and the dioxygenase was added last to initiate the reaction. A sample (1 ml) of the mixture was transferred to a cuvette so that the reaction could be followed spectrophotometrically. When all the 2,5-dihydroxybenzoate had reacted, as indicated by the cessation of absorbance increase at 330 nm, GSH was added and the reaction allowed to proceed until the absorbance decrease ceased. A sample (5 ml) of the reaction mixture was removed for subsequent enzymic analysis of pyruvate and fumarate. The remainder was acidified by addition of H2SO4 to 0.01 M and extracted three times with an equal volume of diethyl ether. The ether extracts were pooled, dried over anhydrous sodium sulphate, evaporated to dryness under vacuo at 30~C and the residue dissolved in ethyl acetate (1 ml). Samples (50 btl) were analysed by paper chromatography using butanol-acetic acid-water and ethanol-ammonia-water solvents (Nordmann and Nordmann 1969). A UV-absorbing spot that corresponded to authentic fumaric acid was observed. Likewise, when analysed with the 2,4-dinitrophenylhydrazine reagent (Nordmann and Nordmann 1969) spots corresponding to those given by authentic pyruvic acid were seen. Pyruvate in the reaction mixture was measured using N A D H and lactate dehydrogenase (Czok and Lamprecht 1974) and corresponded to 0.10raM. Fumarate was measured by the action of fumarase to convert it to malate. Malate dehydrogenase in the presence of hydrazine at pH 9.0 was then used to convert the malate to oxaloacetate with the concomitant formation of N A D H (Gutmann and Wahlefeld 1974). The fumarate content of the reaction mixture was 0.085 raM. The various gentisate pathway enzymes were measured individually in crude extracts of K. pneumoniae. As can be seen from Table 2 all the enzymes were present at high activity after growth on 3-hydroxybenzoate or glycerol + 2,5-dihydroxybenzoate but essentially absent from glycerol-grown cells. The 3-hydroxybenzoategrown cell extract showed no 3,4-dihydroxybenzoate
492 Table 2. Activities of enzymesassociated with 3-hydroxybenzoatecatabolism m K. pneumoniae M5al Growth substrate
Specificactivity [pmol 9min- 1 (rag protein)- 1] 3-Hydroxybenzoate monooxygenase
3-Hydroxybenzoate Glycerol + 2,5-dihydroxybenzoate Glycerol + 3,4-dihydroxybenzoate Glycerol
(NADH)
(NADPH)
0.91 0.36 NT < 0.001
0.76 0.31 NT NT
2,5-Dihydroxy- MaleylF u m a r y l - 3,4-Dihydroxybenzoate pyruvate p y r u v a t e benzoate dioxygenase lsomerase hydrolase dioxygenase 1.04 0.78 NT < 0.001
8.8 4.1 NT < 0.001
0.40 0.25 NT < 0.001
< 0.001 NT 0.07 NT
Details of the various assays are given in Methods. The figures are representativevalues. NT = not tested
dioxygenase activity but this enzyme was detected in extracts prepared from glycerol + 3,4-dihydroxybenzoategrown cells, Table 2.
Properties of 3-hydroxybenzoate-negative mutants Mutants unable to grow in 3-hydroxybenzoate (gene symbol: m-hydroxybenzoate; mhb) but capable of normal growth on fumarate and pyruvate were isolated. When these mutants were tested for growth on 2,5-dihydroxybenzoate some grew but others did not. The 3hydroxybenzoate-negative, 2,5-dihydroxybenzoate-positive class could be defective in 3-hydroxybenzoate uptake or hydroxylation and the 3-hydroxybenzoate-negative, 2,5-dihydroxybenzoate-negative class defective in 2,5dihydroxybenzoate dioxygenase, maleylpyruvate isomerase or fumarylpyruvate hydrolase. A 3-hydroxybenzoate-negative, 2,5-dihydroxybenzoate-positive mutant (designated OH101) was grown in glycerol + 2,5-dihydroxybenzoate and extracts analysed. Table 3 shows that the activity of 2,5-dihydroxybenzoate dioxygenase, maleylpyruvate isomerase and fumarylpyruvate hydrolase in K. pneumoniae M5al and mutant OH101 were essentially equivalent but that OH101 had no detectable 3-hydroxybenzoate monooxygenase activity with either N A D H or NADPH. Unlike the situation for K. pneumoniae M5al none of the enzymes were present in significant amounts after growth of OH10a on glycerol + 3-hydroxybenzoate. Thus OH 101 was defective in 3-hydroxybenzoate monooxygenase (gene symbol
mhbM). A 3-hydroxybenzoate-negative, 2,5-dihydroxybenzoate-negative mutant designated MI214 was also analysed. When either 3-hydroxybenzoate or 2,5-dihydroxybenzoate was added to cultures of MI214 growing on glycerol severe inhibition of growth was seen after 2 - 3 cell doublings. Extracts prepared from such inhibited cells showed 3-hydroxybenzoate monooxygenase, 2,5dihydroxybenzoate dioxygenase and fumarylpyruvate hydrolase activities that were about 25% of those from similarly grown, but not inhibited, K. pneumoniae M5al, Table 3. However, maleylpyruvate isomerase was undetectable in MI214 extracts suggesting that the mutant was defective in this enzyme (gene symbol mhbI).
Skinner and Cooper (1982) reported that the growth of an Escherichia coli 4-hydroxyphenylacetate-negative mutant on glycerol was severely inhibited by addition of 4-hydroxyphenylacetate due to the accumulation of the substrate of the blocked reaction. So the failure of mutant MI214 to grow normally in the presence of 3-hydroxybenzoate seemed likely to be caused by accumulation of maleylpyruvate. To see whether maleylpyruvate was accumulated, the UV spectra of culture supernatants from wild-type and mutant cells during growth on glycerol + 3-hydroxybenzoate were measured. At pH 7.1 3-hydroxybenzoate itself shows an absorbance peak at 290 nm. During growth of K. pneumoniae this peak was gradually reduced as the 3-hydroxybenzoate was utilized. The UV spectrum ( 2 5 0 - 410 nm) of the culture medium showed no other significant change. In contrast, the 290 nm absorbance of the culture medium did not fall in cultures of mutant MI214. Rather a strong absorbance at 330 nm appeared and there was a slight increase in the absorbance at 290 nm due, presumably, to a contribution from the compound with the absorbance maximum at 330 nm. The 330-nm absorbance was lost when the pH was reduced to pH 1.5. An absorbance maximum of 330 nm which is lost on acidification is characteristic of maleylpyruvate and not shown by fumarylpyruvate (Lack 1959).
Double mutants of 3-hydroxybenzoate catabolism When mutant MI214 (mhbI) was spread onto succinate + 3-hydroxybenzoate plates and incubated at 30~ for 24 h there was little or no growth. However, after 4 8 72 h incubation strongly growing individual colonies were seen on the weak background lawn of growth. If the initial inhibition of growth was due to the accumulation of maleylpyruvate to a toxic concentration such single colonies could have arisen by the following means: (1) by reversion of the original mutation; (2) by a further mutation which allowed the cell to metabolize the maleylpyruvate by an alternative route; (3) by a mutation which made the cell resistant to the toxic compound; (4) by a further mutation in the 3-hydroxybenzoate catabolic pathway which then prevented formation of the inhibitory compound. When a selection of the colonies were
493 Table 3. Gentisate pathway enzymes in various mutants
Enzyme
Strain M5al
3-Hydroxybenzoate monooxygenase 2,5-Dihydroxybenzoate dioxygenase Maleylpyruvate isomerase Fumarylpyruvate hydrolase
OH101
a
b
0.10 0.67 3.2 0.43
0.12 0.35 4.1 0.25
MI214
a
b
< 0.001 < 0.00i 0.005 0.015
< 0.001 0.78 4.1 0.60
a
MI214/1 b
0.03 0.14 < 0.001 0.10
a
0.03 0.10 < 0.001 0.07
< 0.001 0.01 < 0.001 0.002
b < 0.001 0.35 < 0.001 0.14
Details of the assays are given in Methods. a, b = specific activities [gmol 9rain 1 (mg protein)-1] for cells grown on (a) glycerol + 3-hydroxybenzoate and (b) glycerol + 2,5-dihydroxybenzoate. Representative values are presented
VII COOH I
C=O
COOH
02
COOH
COOH
~
%/, I
ooo.
,,,,j NAD(P)
',
COOH
COOH
I CH3
v
II
r
"~ .,
|1
,v
C
+
HOOC /
H20 Fig. 1. The gentisate pathway for 3-hydroxybenzoate catabolism. 3Hydroxybenzoate (I) ~s converted to pyruvate (VII) and fumarate (VI) via 2.5-dihydroxybenzoate [gentisate] (II), maleylpyruvate [enol] (ITt), fumarylpyruvate [enol] (IV) and fumarylpyruvate (V)
tested for growth on 3-hydroxybenzoate very few grew i.e. were of categories I and 2 above. Many colonies that grew in succinate + 3-hydroxybenzoate were still unable to grow on succinate + 2,5-dihydroxybenzoate. It was thus unlikely that in these cases growth was due to developing a tolerance o f the inhibitor. The most likely explanation was that formation of the inhibitor from 3hydroxybenzoate, but not from 2,5-dihydroxybenzoate, had been blocked. One such secondary mutant, designated MI214/1, was grown on glycerol + 3-hydroxybenzoate and glycerol + 2,5-dihydroxybenzoate and cell extracts assayed. Table 3 shows that MI214/1 had undergone a mutation in 3-hydroxybenzoate monooxygenase and so was doubly defective in 3-hydroxybenzoate catabolic enzymes.
Trans&tctional analysis of mhbM and mhbI genes To see if the mhbM and mhbI mutations were closely linked a phage P1 vir lysate prepared on wild-type K. pneumoniae M5al was used to transduce MI214/1 to growth on 2,5-dihydroxybenzoate which selects only for repair ofmhbL When the 79 transductants obtained were
~'H
Vl by the sequential action of 3-hydroxybenzoate monooxygenase, 2.5dihydroxybenzoate &oxygenase, maleylpyruvate isomerase, a nonenzymic (NE) keto-enol tautomerization and fumarylpyruvate hydrolase
tested for growth on 3-hydroxybenzoate all were positive indicating 100% linkage between the mhbI and mhbM genes.
Growth of 3-hydroxybenzoate-negative mutants on 3,4-dihydroxybenzoate When the two 3-hydroxybenzoate-negative mutants OH101 and MI214 were tested on 3,4-dihydroxybenzoate plates both grew as well as K. pneumoniae M5al.
Discussion
Two principal pathways for the bacterial aerobic catabolism of 3-hydroxybenzoate have been described (Yano and Arima 1958) and the utilization of a particular pathway may be of taxonomic significance (Wheelis et al. 1967). 2,5-Dihydroxybenzoate or 3,4-dihydroxybenzoate are the ring-fission intermediates in these two routes for 3hydroxybenzoate breakdown but which is used by enteric bacteria was not known.
494
The results presented here for whole cells, cell-free extracts and mutants are all consistent with 3-hydroxybenzoate being catabolised via 2,5-dihydroxybenzoate, maleylpyruvate and fumarylpyruvate in Klebsiella pneumoniae M5al. This reaction sequence (Fig. 1) is known as the gentisate pathway (Dagley 1975). All the enzymes involved, 3-hydroxybenzoate monooxygenase, 2,5-dihydroxybenzoate dioxygenase, maleylpyruvate isomerase and fumarylpyruvate hydrolase were present at high activities in 3-hydroxybenzoate-grown cells and low or undetectable in extracts from glycerol-grown cells. Growth in the presence of 2,5-dihydroxybenzoate led to induction of all the enzymes of the gentisate pathway even though the 3-hydroxybenzoate monooxygenase is not needed for 2,5-dihydroxybenzoate catabolism. However, none of these enzymes were induced during growth of 3-hydroxybenzoate monooxygenase-negative mutants in the presence of 3-hydroxybenzoate, so this compound itself was not an inducer. Since fumarylpyruvate hydrolase was still induced in the maleylpyruvate isomerasenegative mutant that could form Z5-dihydroxybenzoate and maleylpyruvate but not fumarylpyruvate, it seems likely that either 2,5-dihydroxybenzoate or maleylpyruvate is the inducer of all the gentisate pathway enzymes. The 3-hydroxybenzoate monooxygenase of crude extracts worked almost equally well with NADH or NADPH. It was thus possible that this was due to one enzyme with the ability to utilise both coenzymes or to two enzymes each specific for a particular coenzyme. However, the ratio of activity with the two electron donors remained constant as the amount of the 3hydroxybenzoate monooxygenase activity varied and both activities were missing in mutant OH101. This suggested that it was a single enzyme that could utilize either coenzyme, similar to the enzyme from Pseudomonas aeruginosa (Groseclose and Ribbons 1973). The general growth inhibition seen for the maleylpyruvate isomerase-negative mutant in the presence of 3-hydroxybenzoate and its abolition by a mutation removing 3-hydroxybenzoate monooxygenase activity suggested that accumulated maleylpyruvate was responsible. Thus, like succinic semialdehyde in 4-hydroxyphenylacetate catabolism (Skinner and Cooper 1982) an excess of a normal pathway intermediate can cause severe growth inhibition. The ability of the 3-hydroxybenzoate-negative mutants to grow normally on 3,4-dihydroxybenzoate was further support for the view that this compound was not an intermediate in 3-hydroxybenzoate catabolism in K. pneumoniae. T h e small n u m b e r o f steps in the g e n t i s a t e p a t h w a y a n d the evidence f r o m c o t r a n s d u c t i o n studies o f clustering f o r at least two o f the genes i n v o l v e d m a k e s it likely t h a t all the p a t h w a y genes c o u l d be c l o n e d on a single s m a l l piece o f D N A to facilitate their fine genetic analysis. This p o s s i b i l i t y is c u r r e n t l y u n d e r i n v e s t i g a t i o n . Acknowledgements. DCNJ was supported by a research studentship from the SERC. We thank Mrs. U. Gervind-Richards for typing the manuscript.
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