chrysanthemi That Are Deficient in Polygalacturonate Catabolic

JOURNAL OF BACTERIOLOGY, May 1985, p. 708-714 0021-9193/85/050708-07$02.00/0 Copyright ©3 1985, American Society for Microbiology

Vol. 162, No. 2

Isolation and Characterization of Tn5 Insertion Mutants of Erwinia chrysanthemi That Are Deficient in Polygalacturonate Catabolic Enzymes Oligogalacturonate Lyase and 3-Deoxy-D-Glycero-2,5-Hexodiulosonate Dehydrogenaset ARUN K. CHATTERJEE,* KERRY K. THURN, AND DANA J. TYRELLt

Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506 Received 20 August 1984/Accepted 15 February 1985

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl1) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by TnS mutagenesis using the R plasmid pJB4JI. Ogl1 Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of TnS and that TnS (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl1 strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate Iyase than the KduD+ parents did. In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.

hexodiulosonate [ketodeoxyuronate] dehydrogenase) (KDUD), purified from Erwinia chrysanthemi (strain 3937j), was shown to catalyze the conversion of 3-deoxy-D-glycero2,5-hexodiulosonate (DGH) to KDG (Fig. 1, step 9) (14). Despite this biochemical knowledge, the organization and expression of the genes for the PGT catabolic enzymes (Fig. 1, steps 1, 2, 4, 8, and 9) have not been investigated. The genetics of D-galacturonate (hexuronate) catabolism (Fig. 1, steps 5, 6, and 7), however, have been extensively studied in Escherichia coli (2, 33, 36-38) and to some extent in E. chrysanthemi (45). E. chrysanthemi produces PL and PG and utilizes PGT as a carbon source. Genetic (5, 6) and biochemical (1, 18) studies have revealed that the PGT-depolymerizing enzyme, PL (Fig. 1, step 2), is required in the plant pathogenicity of this bacterium. Moreover, PL production in E. chrysanthemi, as in E. carotovora subsp. carotovora (44), appears to be regulated by PGT catabolic products (9, 11, 12). To assess the significance of PGT catabolic events in plant pathogenicity and to understand the regulation of the catabolic genes, we have initiated investigations in E. chrysanthemi EC16, for which various genetic tools (4, 7, 8) are available. In this communication we describe the isolation and properties of TnS insertion mutants defective in the genes for OGL (ogl) and KDUD (kduD). Our findings with these mutants indicate that the KDU pathway regulates the production of PL, PG, OGL, and KDUD. Preliminary reports of some of this work have appeared in abstracts (K. K. Thurn, D. J. Tyrell, and A. K. Chatterjee, Phytopathology 73:827, 1983; D. J. Tyrell, K. K. Thurn, and A. K. Chatterjee, Phytopathology 73:827, 1983).

Pectate (polygalacturonate [PGTJ), a major component of pectic substances, is present in the cell walls of higher plants. The primary chain of PGT is composed of ot-1,4linked D-galacturonate molecules with some rhamnose residues (17). Studies with cell extracts and partially purified enzymes of the soft-rot bacterium Erwinia carotovora subsp. carotovora (19, 23, 27, 28, 41) suggest four major catabolic events in the utilization of PGT: (i) the formation of dimers (oligomers) from the polymer by extracellular pectate lyase (PL) or polygalacturonase (PG) or both (Fig. 1, steps 1 and 2); (ii) the production of the monomer(s) from the dimers (oligomers) by oligogalacturonate lyase (OGL) (Fig. 1, step 4); (iii) the catabolism of monomers (D-galacturonate [Fig. 1, steps 5, 6, and 7] or 4-deoxy-L-threo-5-hexoseulose uronate [DTH] [Fig. 1, steps 8 and 9]) by two independent but converging pathways, ultimately producing the common intermediate 2-keto-3-deoxy-D-gluconate (KDG); and (iv) the assimilation of KDG with the formation of triose phosphate and pyruvate (Fig. 1, steps 10 and 11). In a Pseudomonas sp., the breakdown of PGT through the ketodeoxyuronate (KDU) and KDG pathways (Fig. 1) has been shown (34, 35). In addition, the enzyme 2-keto-3deoxygluconate oxidoreductase (3-deoxy-D-glycero-2,5* Corresponding author. t Contribution no. 85-72-J from the Department of Plant Pathology, Kansas Agricultural Experiment Station, Kansas State University, Manhattan. t Present address: Department of Laboratory Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

708

POLYGALACTURONATE CATABOLISM IN E. CHRYSANTHEMI

VOL. 162, 1985 POLYQALACTUROATEW

UN"TURATED OUGOGALACTUROIdATE

SATURATED TE

2

(UOG1

(5001

SATUITE/D ONAITE

/UNSATURTED DIGALACTURONATE 4

4

4

3

DGALA

(UJDG)

(600) A

~

13 ~~~~

D-RONATE

4-DEOXY-L-thro-5HEXOSEULOSE URO4ATE (DT1H

PTU)

D-TAGATURONATE (TGT) 3-PE0XY,D-GLYCERO-

D-ALTRONATR,

2,5-HEXODIULOSONATE

(ALT)

(DGH)

24ETO0-34-EOXY-D-GLUCONATE

OM) 110

2-KETO-3-DEOXY-6-PHOSPHLUCONATE (KDPG) 111

PYRUVATE+TRICE- 3-PHOSPHATE

FIG. 1. Major pathways of PGT catabolism in bacteria. Enzymes and genes for the catabolic steps are: (1) PG (peh), (2) PL (pel), (3) a-galacturonidase or oligogalacturonate hydrolase (ogh), (4) OGL (ogi), (5) uronate isomerase (uxaC), (6) altronate oxidoreductase (uxaB), (7) altronate hydrolyase (uxaA), (8) 4-deoxy-L-threo-5hexoseulose uronate isomerase (kduI), (9) KDUD (kduD), (10) 2-keto-3-deoxygluconate kinase (kdgK), (11) 2-keto-3-deoxy-6phosphogluconate aldolase (kdgA). The D-galacturonate pathway consists of steps 5, 6, and 7; the KDU pathway consists of steps 8 and 9; and the KDG pathway consists of steps 10 and 11. (See the text and references 17 and 20 for the occurrence of various enzymes.)

MATERIALS AND METHODS Organisms. The strains of E. chrysanthemi, described in Table 1 and below, were maintained at 4°C on yeast-glucoseCaCO3 agar slants. Strains carrying F'ts114 lac+ zzf21::TnJO (10) were kept at 4°C on minimal lactose agar medium supplemented with tetracycline and the required amino acids. E. coli HB101(pRZ102) (47) and AC8001 (8) were stored at 4°C on L agar containing the appropriate antibiotics, and E. coli LA1016 (24) was maintained at room temperature in an L-agar stab. Saccharomycesfragilis C-351 was obtained from H. Phaff (University of California, Davis) and kept at 4°C on malt agar slants (32). TnS insertion mutants were obtained as described previously (8; also see Results). Media. L agar, yeast-glucose-CaCO3 agar, PGT-yeast extract agar, and minimal media were described previously (3, 42). PGT-salts-Casamino Acids medium was prepared as follows. PGT (0.2%, wt/vol) was suspended in water, and NaOH was added with stirring until the pH was approximately 5.5. Casamino acids (1%, wt/vol) was added, and the pH was adjusted to 7. After the mixture was autoclaved, stock solutions of salts were added to the same final concen trations as in the minimal medium (3). Minimal Casamino

709

Acids medium was identical to PGT-salts-Casamino Acids medium except that the PGT was omitted. PGT-salts-calcium medium, modified from Moran et al. (27), contained (in grams per liter): PGT, 2; MgSO4 *7H20, 0.197; (NH4)2SO4, 2.45; KH2PO4, 2.45; Na2HPO4, 0.89; and CaCI2, 0.074. Modified minimal salts medium was identical to PGT-salts-calcium medium except that the PGT was omitted. For solid media, agar (15 g/liter) was added. When desired, media were supplemented with amino acids or uracil (50 ,ug/ml), tetracycline (10 Fg/ml), or drugs as indicated in the footnotes to Table 3. Carbon utilization. Mutants and their parents were grown overnight on modified minimal salts-glycerol (0.1% wt/vol) agar, and the cells were suspended in 55 mM potassium phosphate (pH 7.4). The suspension was spotted on modified minimal salts agar containing the desired carbon source (0.1%, wt/vol) and incubated at 30°C. Growth was scored after 24 h. Construction of donor strains, bacterial crosses, and plant tissue maceration. The procedures used are detailed in previous publications (3, 5, 42). Preparation of culture samples for enzyme assays. Bacterial cultures (4 to 5 ml), at a Klett value of 100 to 150, were harvested (12,000 x g, 4°C, 10 min), and the sdlpernatants were stored at 4°C or frozen. The cells were suspended in 1 ml of 10 mM Tris-hydrochloride (pH 7) and frozen. Cell extracts were prepared by sonication with a Braunsonic sonicator 1510 at full power with a 3/8-in. probe for two 30-s pulses. Assay procedures. PL was assayed according to the method of Starr et al. (42) in a 0.6-ml reaction mixture with a Gilford recording spectrophotometer. One unit of PL activity is defined as the amount of enzyme that produced 1 FImol of unsaturated digalacturonate (UDG) per min at 30°C. PG activity was determined (13) in a 1- or 2-ml reaction mixture. One unit of activity is defined as the amount of enzyme that produced 1 pumol of galacturonic acid per min at 300C. OGL activity was assayed according to the method of Moran et al. (28) in a 0.1-ml reaction mixture by using a modified periodate-thiobarbituric acid procedure (31). One unit of OGL activity is defined as the amount of enzyme that produced 1 nmol of 3-formyl pyruvate per min at 30°C. KDUD activity was determined by measuring the decrease in absorbance at 340 nm by using a modified method TABLE 1. E. chrysanthemi strains Strain

EC16 AC4074 AC4095

Description"

Wild type, prototrophic thr-l his-i trp-J Nalr Strr kduD2 exu4: :TnS(Kmr) Nair

AC4110 ogl-2::Tn5, derivative of AC4074 AC4149 thr-J exu-3 Strr Nalr AC4150 AC4156

Nalr derivative of EC16 thr-I exu-3 car-I Strr Nalr Rifr

AC4194

kduDI :Tn5(Kmr) zae-J :Tn5(Kmr), derivative of AC4149 ogl-::TnS(Kmr), derivative of AC4150 kduDl::TnS(Kmr), recombinant of AC4156

AC4197 AC4961

Reference

or

source

5

3 Laboratory collection This paper Laboratory collection 8

Laboratory collection This paper This paper This paper

a Uncommon markers: ogl+, production of OGL; kduD', production of KDUD; exu+, utilization of D-galacturonate.

710

CHATTERJEE, THURN, AND TYRELL

of Moran et al. (28). The reaction mixture (0.3 ml) contained 0.17 mM substrate (DGH) prepared as described below, 0.33 mM NADH, 50 mM phosphate buffer (pH 7.2), and cell

lysate. One unit of activity is defined as the amount of enzyme that oxidized 1 ,umol of NADH per min at 30°C. Protein was estimated by the method of Lowry et al. (25) with bovine serum albumin as the standard. DNA techniques. Total DNA was extracted as described previously (39), except that all steps after the ethanol precipitation were omitted. DNA was digested with restriction endonuclease EcoRI, KpnI, or SalI (Bethesda Research Laboratories, Gaithersburg, Md.) as described in the supplier's instructions. The isolation of Mu DNA and the TnS-containing plasmid pRZ102 (22) and the electrophoresis of DNA fragments were carried out as described previously (47). Southern blot hybridizations (40) were done according to the method of Zink et al. (47), except that sonicated and denatured salmon DNA (0.1 to 0.2 mg/ml) was included in the prehybridization and hybridization steps. Preparation of UDG. UDG was prepared by using E. chrysanthemi EC16 PL. The enzyme was obtained from the supernatant of a 1-liter PGT-salts-Casamino Acids culture which was grown to a Klett value of 300 and concentrated 10-fold by ultrafiltration. The reaction mixture (1000 ml), which contained 1% (wt/vol) PGT, 0.33 mM CaCl2, 0.1 M Tris-hydrochloride (pH 8.5) and 3.9 x 107 U of PL, was incubated at 30°C for 16 to 24 h. The oligogalacturonates were precipitated as barium salts. UDG was purified from a 2-g sample of barium salts by the method of Dave et al. (15), with the following modifications. The barium salts were precipitated with 60% (vol/vol) ethanol and air dried after the diethyl ether wash but were not converted to the acid form before being loaded on the ion-exchange column (2.6 by 40 cm). Elution was carried out with the following volumes and concentrations of sodium formate (pH 4.7): 500 ml, 0.3 M; 500 ml, 0.4 M; 1,000 ml, 0.5 M; 500 ml, 0.6 M; and 500 ml, 0.7 M. The column fractions were assayed for the presence of oligogalacturonates by the arsenomolybdate assay (30). Peak fractions were converted to the acid form with AG 50W-X8 or Dowex 50W-X4 cation-exchange resin (Bio-Rad Laboratories, Richmond, Calif.), lyophilized, and stored at -20°C. Thin-layer or paper chromatography (15, 21) revealed that fractions containing UDG were free of other oligogalacturonates. UDG was eluted in 0.5 M sodium formate and had a final yield of 0.4 to 0.5 g. Preparation of SDG. Saturated digalacturonate (SDG) was produced by using S. fragilis C-351 PG prepared according to the method of Phaff (32), except that the ammonium sulfate precipitation step was omitted. PGT was digested with the yeast PG, and the saturated oligogalacturonates were precipitated as strontium salts (26). SDG was purified by the method of Nagel and Wilson (29), with the following modifications. The column dimensions were 2.6 by 40 cm, and the resin was AG-1-X8 (Bio-Rad) 200-400 mesh. The sample was applied as the strontium salt (2 g dissolved in 50 mM sodium formate, pH 4.7) and eluted with a 2-liter linear gradient (0.3 to 0.6 M) of sodium formate (pH 4.7). The column fractions were treated as described above for UDG. SDG eluted in ca. 0.39 M sodium formate, and the final yield was 0.3 to 0.4 g. Preparation of DGH. DGH was prepared by using an enzyme preparation from an E. chrysanthemi mutant (AC4095 [Table 1]) that did not degrade DGH. The mutant was grown for 16 h in PGT-salts-Casamino Acids medium at 28°C. The cells were collected by centrifugation (17,600 x g, 20 min, 4°C), suspended in 10 mM Tris hydrochloride (pH 7),

J. BACTERIOL.

and disrupted with a French pressure cell (43). Unbroken cells were removed by centrifugation (12,000 x g, 15 min, 4°C), and the lysate was ultracentrifuged at 360,000 x g for 1 h. The supernatant was used as described below. DGH was produced by incubating 5 mM UDG, previously titrated to pH 8 with NaOH, for 2 h at 30°C with the enzymatic preparation described above, containing 5 U of OGL. The products were converted to the acid form by using AG 5OW-X8 cation-exchange resin (Bio-Rad) and separated by descending paper chromatography on Whatman 3MM paper previously washed with water. The chromatogram was developed at 4°C for 23 to 33 h with ethyl acetate-acetic acid-water-formic acid (18:3:4:1, vol/vol) (11), and the reaction products were visualized (46). Two periodate-thiobarbituric acid-positive products were produced by the reaction: a faster-moving major component, DGH, and a second component, present in trace amounts, DTH. The relative migrations on Whatman no. 1 paper with galacturonic acid as the reference were DGH, 1.86; DTH, 1.07; and UDG, 0.73; these are similar to published values (11). DGH was eluted from the chromatogram by descending chromatography with water. The eluate, which contained approximately 16 to 20 ,umol of ,B-formyl pyruvate equivalent groups (31), was stored at -80°C. Digestion products of UDG. Digestion products of UDG were prepared from 5 mM UDG as described above for DGH, except that the cell lysate was not ultracentrifuged and the chromatographic fractionation steps were omitted. Protein was removed (16), and the resulting preparation was used in the induction experiments. The conversion of UDG to the digestion products, DTH and DGH, was almost complete as determined by the disappearance of UDG (decrease in absorbance at 235 nm) and the concomitant increase in periodate-thiobarbituric acid-reactive products

(31). RESULTS Isolation of TnS insertion mutants. E. coli AC8001 carrying the R plasmid pJB4JI was crossed with appropriate E. chrysanthemi recipients, and Kmr Gms transconjugants were obtained as previously described (8). For the isolation of OGL-deficient mutants, we used E. chrysanthemi AC4150 and AC4074, both of which utilized PGT, presumably by the steps shown in Fig. 1. Since OGL activity is required in PL induction with PGT (11, 12; see below), we looked for colonies that produced little or no clearing on PGT-yeast extract agar (5), which indicated a deficiency in extracellular PL activity. A group of such mutants, later identified as Oglby enzymatic assays, failed to utilize PGT or digalacturonates (Table 2). The Ogl- mutants, however, macerated potato tuber tissue and utilized glycerol, glucose, gluconate, and galacturonate (Table 2). To isolate mutants defective in the KDU pathway (Fig. 1, steps 8 and 9), we used a strain of E. chrysanthemi (AC4149) blocked in the galacturonate pathway. In this strain, PGT catabolism should occur through the KDU pathway if the scheme shown in Fig. 1 is operative. Based upon this assumption, we replicated Kmr Gms colonies of AC4149 on PGT-salts-calcium agar. The colonies that failed to grow on this medium but were able to utilize gluconate and produced about normal levels of PL on PGT-yeast extract agar were presumed to have a defect in the KDU pathway. One such mutant (AC4194) and its genetic recombinant (AC4961), both deficient in KDUD, macerated potato tuber tissue and utilized glycerol and glucose as carbon sources, but failed to utilize PGT and digalacturonates (Table 2).

711

POLYGALACTURONATE CATABOLISM IN E. CHRYSANTHEMI

VOL. 162, 1985

TABLE 2. Utilization of various carbon sources and maceration of potato tuber tissue by E. chrvysanthlemni strains" Maceration

Growth with the following carbon source":

Strain

AC4150 AC4197 AC4074 AC4110 AC4149 AC4194 AC4156 AC4961

Relevant

Ogl+ OglOgl+ OglOgl+ Ogl+ Ogl+ Ogl+

phenotype'

KduD+ KduD+ KduD+ KduD+ KduD+ KduD KduD+ KduD-

Exu+ Exu+ Exu+ Exu+ ExuExuExuExu-

of potato

tuber

GLY

GLU

GTU

GLC

PGT

UDG

SDG

+ + + + + + + +

+ + + +

+ + + + -

+ + + + + + + +

+ -

+ -

+ -

+ +

+ + + -

+ + + -

+ + -" + -"

+ + + + + +

+ + + +

tissue

aSee Materials and Methods for the details of experimental conditions. bSee Table 1, footnote a, for definition of these phenotypes. The media were supplemented with uracil or amino acids or both as required. GLY, Glycerol; GLU, glucose; GTU, galacturonate; GLC, gluconate. ' Extended incubation periods (48 h) resulted in leaky growth of these strains.

Physical and genetic characterization of TnS insertion mutations. Southern blot hybridizations were performed to determine the presence of Mu DNA. The lack of hybridization signals between the Mu probe and EcoRI chromosomal fragments of the Tn5 mutants (AC4110, AC4194, and AC4197) indicated the absence of the bacteriophage DNA (data not shown). To determine if more than one TnS copy was present in the chromosome of Ogl- mutants (AC4110 and AC4197), total DNA was restricted with EcoRI or KpnI, which do not cleave TnS, or with Sall, which cleaves TnS in one site (22), and hybridized with pRZ102(Tn5). The autoradiogram revealed the presence of a single hybridization band with EcoRI or KpnI chromosomal fragments (Fig. 2, lanes 3, 4, 9, and 10) and two bands with Sall fragments (data not shown), indicating the presence of Tn5 at a single site on the chromosomes of these mutants. Linkage between the Ogl- phenotype and Kmr (TnS), was estimated by crossing Ogl- (Kmr) donor strains with the Ogl+ Exu- strain AC4156, selecting for Kmr colonies, and testing for the inability to utilize PGT. The data (Table 3) show that between 93 and 96% of the Kmr recombinants were concomitantly Pgt-, indicating the acquisition of the defective ogl gene. Southern blot hybridization with the TnS probe (Fig. 2, lanes 3 through 12) strongly suggested that Tn5 was present in the same chromosomal fragment in the Ogl- parent strains and three of their recombinants (AC4951, AC4952, and AC4953). Thus, based upon both physical and

1 2 3 4 5 6 7 8 9 10111213141516

daa de FIG. 2. Autoradiogram of a Southern blot of total cellular DNA digested with EcoRI or KpnI and hybridized with pRZ102(Tn5) DNA. Lanes 1 and 2, AC4156 (Ogl+ parent); lanes 3 and 4, AC4197 (ogl-J::Tn5 parent); lanes 5 and 6, AC4951 (ogl-l::Tn5 recombinant); lanes 7 and 8, AC4952 (ogl-l::TnS recombinant); lanes 9 and 10, AC4110 (ogl-2::Tn5 parent); lanes 11 and 12, AC4953 (ogl-2::TnS recombinant); lanes 13 and 14, AC4194 (kduDl::TnS and zae-l::Tn5 parent); lanes 15 and 16, AC4961 (kduDl::TnS recombinant). Oddnumbered lanes are KpnI digests; even-numbered lanes are EcoRI digests. See the text and Table 3 for the construction of the recombinants.

genetic evidence, we concluded that the Tn.5 DNA in the Ogl- strains was located either in or very close to the structural or regulatory gene causing its inactivation. After restriction of the chromosomal DNA of the KduDstrain, AC4194, and subsequent Southern blot hybridization, two KpnI (Fig. 2, lane 13) and four Sall (data not shown) chromosomal fragments hybridized with the TnS probe. In contrast, only one EcoRI fragment hybridized with the Tn5 DNA (Fig. 2, lane 14). These results indicated that two copies of Tn5 were present on the chromosome of strain AC4194 and that the chromosomal DNA between the two TnS copies lacked an EcoRI site but contained KpnI and Sall sites. To construct a strain carrying only the kduD::TnS mutation, crosses were performed between the KduD- donor and the recipient strain AC4156 (KduD+ Exu-). The Kmr recombinants were tested for the inability to utilize PGT, which would indicate the acquisition of the defective kduiD gene from the donor. About 27% of the Kmr recombinants were concomitantly Pgt- (Table 3). A Kmr Pgt- recombinant (AC4961) carrying a single TnS copy (Fig. 2, lane 15) linked to the KduD- phenotype (Table 3) was used in our subsequent physiological investigations. Levels of PGT catabolic enzymes in Ogl- and KduDmutants. The data (Table 4) show that PL, PG, OGL, and KDUD were induced by SDG in the parent Ogl+ strains. In the Ogl- mutants, however, PL, PG, and KDUD activities remained at about the basal level. OGL activity was not detected in the mutants grown either with glycerol or with SDG. Analogous results were obtained when these bacteria were grown with UDG (Table 5) or PGT (data not shown). To determine if a catabolic product of UDG induced PL, PG, and KDUD, cells of Ogl+ and Ogl- strains were incubated with glycerol, UDG, enzymatic products of UDG, or DGH. The data (Table 5) reveal that in the Ogl- mutant, AC4197, the enzymes were induced by DGH or the enzymatic products of UDG but not by the digalacturonate. Moreover, OGL activity was not detected in Ogl- mutants grown in the presence of any of the substrates (Tables 4 and 5). All of the enzymes, including OGL, were induced in the Ogl+ strain by DGH, UDG, or the enzymatic products of UDG. These results suggested that a product of the digalacturonate induced PL, PG, OGL, and KDUD of E. chrysanthemi EC16. Further support for this hypothesis was the pattern of enzyme induction in the KduD- strains AC4194 and AC4961. KDUD activity was not detected in these strains when the cells were grown in the presence of SDG or glycerol (Table

712

CHATTERJEE, THURN, AND TYRELL

J. BACTERIOL.

TABLE 3. Linkage between kanamycin resistance and OGL deficiency (Ogl-) or KDUD deficiency (KduD-) in genetic recombinants of E. chrysanthemia Inheritance of unselected character Donor Recipient Selection' % with No. tested Phenotype' phentyp phenotype AC4110(F'tsl4) lac+ zzf-21::TnlO Kmr EC16 44 93 PgtAC4197(F'tsll4) lac+ zzf-21::TnlO AC4156 Kmr 73 Pgt96 AC4194(F'ts114) lac+ zzf-21::TnlO Kmr AC4156 100 Pgt27 EC16(F'tsll4) lac+ zzf-21::TnlO AC4961 Pgt+ 99 Kms 97 a See Materials and Methods for the construction of the donor strains and Table 1 for the phenotypes of the bacterial strains. Crosses were done for 16 h at 30°C, except for AC4110(F'ts114) lac+ zzf-21::TnIO x EC16, which was for 6 h (3). Utilization of PGT as a carbon source was the criterion for the presence of the ogl+ or kduD+ gene (see Table 1 and the text). b AC4110(F'ts114) lac' zzf-21::TnIO was counterselected by auxotrophy, AC4197(F'ts114) lac+ zzf-21::TnlO by rifampicin (50 ,ug/ml) and streptomycin (100 ,ug/ ml), AC4194(F'ts114) lac' zzf-21::TnIO by rifampicin (20 ,g/mI), and EC16(F'ts114) lac+ zzf-21::TnIO by nalidixic acid (50 ,ug/ml). The recipients were counterselected by kanamycin (50 ,ug/ml) or the inability to utilize PGT. ' Kmr recombinants were scored for the Pgt- phenotype on PGT-salts-calcium agar containing uracil and the appropriate amino acids, while Pgt+ recombinants were scored for Kms on L agar supplemented with kanamycin (50 pg/ml) and nalidixic acid (50 ,ug/ml).

4). However, the levels of PL, PG, and OGL in the mutants induced with SDG were considerably higher than those in the parents (Table 4); yet, the basal levels of these enzymes were similar. These observations suggested that the kduD::TnS mutation caused the accumulation of an intermediate(s) of the KDU pathway and that this intermediate induced the enzymes in a "gratuitous" manner. If this is true, low concentrations of digalacturonates should induce high levels of the enzymes in the KduD- strain. Indeed, the activities of PL and OGL were about 10-fold higher in the KduD- strain AC4961 than in the KduD+ strain AC4156 at a UDG concentration of 1 jig/ml (Fig. 3). The OGL activity of strain AC4961 increased dramatically with increasing UDG concentrations up to 25 ,ug/ml and then decreased (about 30%) at 50 ,ug of UDG per ml. Likewise, an increase in the PL activity was observed with UDG concentrations up to 5 jig/ml. However, the PL activity was about 22% lower with UDG at 10 ,ug/ml, and about 72% lower with 50 ,ug/ml, than with UDG at 5 ,ug/ml. DISCUSSION The genetic and physical data presented above support the conclusion that the Ogl- and KduD- phenotypes resulted from the insertion of TnS into the chromosome of E. chrysanthemi. The following findings with these mutants, taken together with earlier observations (9, 11-14, 45), strongly suggest that PGT is catabolized by E. chrysanthemi

according to the scheme presented in Fig. 1. (i) The Oglmutants failed to grow with either PGT or digalacturonates as a sole carbon source (Table 2), suggesting that OGL plays a central role in the conversion of digalacturonates or oligogalacturonates to monomers, i.e., galacturonate or DTH (Fig. 1, step 4). (ii) Mutants blocked in the galacturonate pathway (Fig. 1, step 5, 6, or 7) can still utilize PGT and digalacturonates but not galacturonate (Table 2), indicating the occurrence of an alternate pathway. (iii) KduD- Exumutants, in which both the galacturonate (Fig. 1, step 5, 6, or 7) and KDU (Fig. 1, step 9) pathways are blocked, failed to utilize PGT or digalacturonate (Table 2). Furthermore, while the enzymes and the genes of the KDG pathway have not been studied in strain EC16, evidence presented by Van Gijsegem and Toussaint (45) indicates the occurrence of the KDG pathway in E. chrysanthemi. The data obtained with the Ogl- and KduD- strains confirm and extend previous observations (9, 11, 12, 44) on the regulation of PL and provide new information on the induction of other PGT catabolic enzymes (PG, OGL, and KDUD). We have observed the lack of induction of PL, PG, and KDUD in Ogl- mutants by UDG or SDG, the induction of these enzymes in the presence of DGH or the enzymatic products of UDG, and the hyperinduction of PL, PG, and OGL in the KduD- strains by SDG or UDG. These findings lead to the conclusion that DTH or DGH (or both) induces all of these enzymes. The differential role of DTH and DGH in the induction of these enzymes can only be clarified by the

TABLE 4. Production of PL, PG, OGL, and KDUD in E. chrysanthemi strains grown in glycerol (GLY) or SDG' Enzymatic activityb Strain

AC4150 AC4197 AC4074 AC4110 AC4149 AC4194 AC4156 AC4961

Relevant Ogl+ OglOgl+ Og1KduD+ KduDKduD+ KduD-

PL

PG

OGL

KDUD

GLY

SDG

GLY

SDG

GLY

SDG

GLY

SDG

0.14 0.15 0.10 0.13 0.13 0.15 0.22 0.40

2.0 0.3 2.5 0.6 2.4 18.5 4.2 20.8

0.04 0.03 0.03 0.04 0.02 0.02 0.05 0.04

0.19 0.04 0.22 0.05 0.13 0.42 0.21 0.40

1.5

12.7 ND 13.5 ND 13.8 58.6 4.9 30.2

0.03 0.02 0.02 0.02 0.01 ND 0.03 ND

0.28 0.03 0.23 0.02 0.32 ND 0.42 ND

ND' 1.1 ND 1.0 0.6 2.0 1.4

a Bacterial cultures at early exponential growth (about 30 to 40 Klett units) in minimal Casamino acids medium at 28°C were divided and supplemented either with glycerol (0.2%) or with SDG (50 jig/ml). Cultures were then incubated for an additional 4 h. PL and PG were assayed in the supernatants. Cell extracts were prepared as described in Materials and Methods and assayed for PL, PG, OGL, KDUD, and protein. b See Materials and Methods for assay conditions. PL and PG activities are expressed as the units of activity in cells and supernatants per milligram of cell protein per milliliter of culture. OGL and KDUD activities are expressed as units of activity per milligram of cell protein per milliliter of culture. ' ND, Not detected.

POLYGALACTURONATE CATABOLISM IN E. CHRYSANTHEMI

VOL. 162, 1985

713

TABLE 5. Induction of PL, PG, OGL, and KDUD by UDG, enzymatic products of UDG (UDGP), and DGH in E. (hrvsanthemi strains" Enzymatic activity' Strain

AC4150 AC4197

Relevant

phenotype

Ogl+ OgI-

GLY

UDG

0.07 0.03

0.23 0.06

PL UDGP' DGH'

0.52 0.60

0.63 0.80

GLY

UDG

UDGP DGH

GLY

OGL UDG UDGP DGH

GLY

KDUD UDG UDGP DGH

0.05 0.05

0.07 0.05

0.09 0.09

1.20 ND"

14.0 ND

0.04 0.03

0.44 0.02

0.10 0.09

13.4 ND

17.6 ND

0.31 0.51

0.53 0.53

aBacterial cultures were grown at 28°C in modified minimal salts medium plus glycerol (GLY) (0.5%). At mid-exponential growth (about 80 to 100 Klett units), cells were collected by centrifugation (12,000 x g) at room temperature (ca. 25'C), suspended in 55 mM phosphate buffer (pH 7.4), and inoculated to modified minimal salts medium supplemented with glycerol (0.025%), UDG (0.5 mM), enzymatic products of UDG (1 mM), or DGH (1 mM). (The UDG concentration was determined from the absorbance at 235 nm [281. The concentrations of DGH and enzymatic products of UDG were calculated by using the periodate-thiobarbituric acid procedure [311 and expressed as 3-formyl pyruvate equivalent groups.) Cultures were then incubated for an additional 4 h. PL and PG were assayed in the supernatants and cell extracts. OGL, KDUD, and protein were assayed in the cell extracts. See Materials and Methods for the preparation of cell extracts and enzymatic assay conditions. bSee Table 4, footnote b, and Materials and Methods for the definition of enzymatic activities. ' DGH and enzymatic products of UDG were prepared as described in Materials and Methods. d ND, Not detected.

isolation of isomerase-deficient mutants. We currently are seeking such mutants by chemical as well as transpositional mutagenesis. An unexpected observation with the KduD- strains was the decrease in the PL and OGL specific activities when the bacteria were grown with higher concentrations (i.e., 50 ,ug/ml) of UDG (Fig. 3). Preliminary evidence indicates that 60 140

50

20

40

00

U~~~~~~~~~

.j.

10 20 30 40 UDG Concentration

5t0

(y/rml) FIG. 3. Effect of the UDG concentration on the production of PL ) and the KduD+ strain AC4156 (--- -). Bacterial cultures at early exponential growth (about 30 to 40 Klett units) in minimal Casamino Acids medium at 28°C were divided, supplemented with various concentrations of UDG, and incubated for an additional 4 h. PL was assayed in the supernatant and cell lysate, while OGL and protein were assayed in the cell lysate. Enzymatic activities were expressed as indicated in Table 4, footnote b.

(0) and OGL (0) in the KduD- strain AC4961 (

PG is influenced in a manner similar to that of PL. The higher UDG concentration may cause intermediates of the KDU pathway, especially DGH, to accumulate to nonphysiological levels in the KduD- strains, but not in the KduD+ strains. A second possibility is that an intracellular pool of DGH or DTH (or both) may regulate the production of the depolymerizing enzymes by a feedback mechanism. While we cannot at present rule out any of these possibilities, it is apparent that all the enzymes in the KduD- strains were induced at UDG concentrations considerably lower than those required by the parent. The maceration of potato tuber tissue by Ogl- and KduDmutants (Table 2) was somewhat unexpected and merits comment. The results with Ogl- mutants imply the presence in potato tuber tissue of monomers (DTH or DGH) or some other molecule of plant origin that may directly induce the production of PL. Tissue maceration by the Exu- KduDmutant, which no longer can catabolize PGT, digalacturonates, or galacturonate, suggests that in potato tuber tissue the mutant can sustain physiological activities by obtaining nutrients other than uronate derivatives from the host. Thus, a major implication of these findings is that production of extracellular PL may be the only PGT catabolic event of significance in the pathogenicity of E. chrysanthemi. We are currently testing the validity of this hypothesis by examining the population of E. chrysanthemi mutants in inoculated plant tissues and the quality and the quantity of PL produced in vivo. ACKNOWLEDGMENTS This investigation was supported by the National Science Foundation (grant PCM-8022003), the Science and Education Administration of the U.S. Department of Agriculture (grant 59-2201-1-1-686-0 from the Competitive Research Grants Office), and the Kansas Agricultural Experiment Station, Manhattan. We thank J. Roth and H. Phaff for the bacterial and yeast strains. LITERATURE CITED 1. Basham, H. G., and D. F. Bateman. 1975. Relationship of cell death in plant tissue treated with a homogeneous endopectate lyase to cell wall degradation. Physiol. Plant Pathol. 5:249-262. 2. Blanco, C., M. Mata-Gilsinger, and P. Ritzenthaler. 1983. Construction of hybrid plasmids containing the Escherichia coli uxaB gene: analysis of its regulation and direction of transcription. J. Bacteriol. 153:747-755. 3. Chatterjee, A. K. 1980. Acceptance by Erwinia spp. of R plasmid R68.45 and its ability to mobilize the chromosome of Erwinia chrysanthemi. J. Bacteriol. 142:111-119. 4. Chatterjee, A. K., M. A. Brown, J. S. Ziegle, and K. K. Thurn. 1981. Progress in chromosomal genetics of Erwinia chrysanth-

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