Correction: in the Article

7844 Corrections: Proc. Natl. Acad. Sci. USA 78 (1981)

Correction. In the article "X-ray diffraction of strained muscle Correction. In the article "Purified lexA protein is a repressor fibers in rigor" by G. R. S. Naylor and R. J. Podolsky, which of the recA and lexA genes" by John W. Little, David W. appeared in the September 1981 issue ofProc. NatL Acad. Sci. Mount, and Celeste R. Yanisch-Perron, which appeared in the USA (78, 5559-5563), two printer's errors occurred. In the July 1981 issue of Proc. NatL Acad. Sci. USA (78, 4199-4203), Abstract, line 5 should read: printer's errors deleted some lines of text. On p. 4202, the last sentence of the first full paragraph should read "A given pro- "the intensity ratio, I(1O/1(11). Because the intensity ratio de- moter gives rise to a run-offtranscript ofa particular size, which pends .. . " forms a band in the autoradiogram." On p. 4203, the last sentence before the acknowledgements should read "We conclude Also, on p. 5563, in the last paragraph of the Discussion, the that this model, originally based largely on genetic evidence, first-word in line 3 should be "and." is also completely consistent with the known biochemical prop- erties of the lexA and recA proteins. "

Correction: In the article "N-(6-Aminohexyl)-5-chloro-1-naph- thalenesulfonamide, a calmodulin antagonist, inhibits cell pro- liferation" by Hiroyoshi Hidaka, Yasuharu Sasaki, Toshio Tan- Correction. In the article "Impaired induction and self-catab- aka, Toyoshi Endo, Shinichi Ohno, Yasuhisa Fujii, and Tetsuji olite repression of extracellular pectate lyase in Erwinia chry- Nagata, which appeared in the July 1981 issue of Proc. Nati santhemi mutants deficient in oligogalacturonide lyase" by Alan Acad. Sci. USA (78, 4354-4357), an undetected printer's error Collmer and Durward F. Bateman, which appeared in the June resulted in the omission of Table 1. The table is reproduced 1981 issue of Proc. Nati Acad. Sci. USA (78, 3920-3924), an here. editorial error and an undetected printer's error occurred in Table 2 on p. 3922. The heading for the first column of data should be (GalUA)2 (the saturated digalacturonic acid). The Table 1. Affinity (IC50) of W-7 and W-5 for calmodulin heading for the third column of data should-be u(GalUA)2 (the W-7 W-5 unsaturated digalacturonic acid). Inhibition of phosphodiesterase activity 28 240 Inhibition of myosin light chain kinase activity 51 230 Displacement of [3H]W-7 from calmodulin 31 210 The IC50 value is defined as the concentration of drug required to produce 50% inhibition of enzyme activity or of labeled W-7 binding to purified calmodulin. These values were determined graphically and all experiments were run in triplicate.

Correction. In the article "Tumor-promoting phorbol-esters stimulate myelopoiesis and suppress erythropoiesis in cultures ofmouse bone marrow cells" by Fritz Sieber, Robert K. Stuart, and Jerry L. Spivak, which appeared in the July 1981 issue of Proc. NatL Acad. Sci. USA (78, 4402-4406), several printer's errors occurred in Table 2 on p. 4405. The corrected version ofTable 2 is printed here. Table 2. Colony formation by mixed cultures of TPA-treated and untreated marrow cells TPA treatment Colonies* No. of cells juM min BFU-E CFU-E CFU-GM 1 x 105 None - 53.0 ± 3.9 507 ± 36 15.3 ± 2.7 1 x 105 10 45 0 99± 9 74.7 ± 2.2 1 x 105 1 45 3.5 ± 0.5 ND ND 1 x 105 0.1 45 52.3 ± 2.1 ND ND

5 x 0 104 None 4 0 305 ± 18 100.3 ± 4.8 (Expected) (26.5) (303) (45)

5 x 104 None1 10.8 ± 1.7 ND ND (Expected) (28.3)

5 x 104 None - 51.8 ± 2.9 ND ND with5 x 104 0.1 45 (Expected) (52.7) B6D2F1 marrow cells were incubated in TPA as outlined in Table!1, washed, and cultured either sep- arately or mixed with untreated cells. ND, not done. * Mean of quadruplicate cultures ± SEM. Downloaded by guest on September 25, 2021 Proc. Natl. Acad. Sci. USA Vol. 78, No. 6, pp. 3920-3924, June 1981 Microbiology

Impaired induction and self-catabolite repression of extracellular pectate lyase in Erwinia chrysanthemi mutants deficient in oligogalacturonide lyase (phytopathogenic bacteria/exoenzyme regulation/regulatory mutants/product induction/deoxyketuronic acid) ALAN COLLMER* AND DURWARD F. BATEMANt Department of Plant Pathology, Cornell University, Ithaca, New York 14853 Communicated by Ellis B. Cowling, March 4, 1981 ABSTRACT The pectate lyase (PL; EC 4.2.2.2) secreted by Bacteria secrete a variety of pectic enzymes differing in re- the plant pathogenErwinia chrysanthemi is induced and catabolite action mechanism (hydrolysis vs. /3 elimination) and action pat- repressed by different concentrations ofits own product, digalac- tern (exo vs. endo) (7). Regulatory studies thus far have focused turonic acid 4,5-unsaturated at the nonreducing end [u(GalUA)2]. on PL, which cleaves internal linkages in D-galacturonan by (3 Both activities ofu(GalUA)2 depend on its cleavage by oligogalac- elimination, generating a series of oligomers with a 4,5-unsat- turonide Iyase (OGL; EC 4.2.2.6). This intracellular enzyme con- urated bond at the nonreducing end. Several features of PL verts u(GalUA)2 to the deoxyketuronic acid 4-deoxy-L-threo-5-hex- regulation in E. carotovora have recently been described. In- osulose uronic acid, which is then isomerized to 3-deoxy-D-glycero- duction on D-galacturonan is mediated by PL reaction products 2,5-hexodiulosonic acid. An OGL-deficient mutant unable to grow (presumably generated by a basal level of PL): the adaptive lag on u(GalUA)2 was poorly induced by u(GalUA)2 or by D-galactu- digalacturonic acid ronan but produced wild-type levels of PL when supplied with 3- is shortened by adding 4,5-unsaturated deoxy-D-glycero-2,5-hexodiulosonic acid. PL synthesis. in the mu- [u(GalUA)2] and lengthened by adding EDTA, -which chelates tant could also be stimulated by 4,5-unsaturated trigalacturonic divalent cations essential for PL activity (8). PL synthesis is sub- acid, from which deoxyketuronic acid is released by another in- ject to cyclic AMP-controlled catabolite repression (9), and a tracellular enzyme. An OGL-deficient mutant that grew slowly on mutant deficient in cyclic AMP was similarly deficient in PL u(GalUA)2 in comparison with the wild-type parent was hyperin- (10). Finally, u(GalUA)2 at high concentrations exerts cyclic duced by u(GalUA)2 unless catabolite repression was relieved by AMP-reversible self-catabolite repression on PL (11). cyclic AMP or imposed by logarithmic growth on glycerol. PL syn- Our studies on the regulation ofthe PL produced by E. chry- thesis is also stimulated by saturated digalacturonic acid, which santhemi (12) suggested that saturated digalacturonic acid is released from D-galacturonan by another extracellular enzyme, [(GalUA)2], released by basal levels ofan extracellular exo-poly- exo-poly-a-D-galacturonosidase (EC 3.2.1.82). Because these di- a-D-galacturonosidase [PG; poly(1,4-a-D-galactosiduronate) mers stimulate PL synthesis at concentrations (wt/vol) 1/1000th digalacturonohydrolase, EC 3.2.1.82], could mediate induction ofthe concentration required by D-galacturonan, and because an on D-galacturonan even when u(GalUA)2 formation was blocked OGL-deficient mutantuninducible by dimers was also uninducible by EDTA (13). by D-galacturonan, we postulate that PL induction by pectic poly- Although the intracellular enzymes that degrade oligogalac- mers entails extracellular formation ofdimers and subsequent in- turonic acids have long been known (14-16), their involvement tracellular conversion to deoxyketuronic acids, the apparent in- in the regulation of extracellular PL has not been addressed. ducers of PL. To gain a deeper understanding of the induction and self-catabolite repression of PL in E. chrysanthemi we identified the Several bacterial plant pathogens in the genus Erwinia are char- pectic intermediates formed during the conversion ofextracel- acterized by their ability to produce pectolytic enzymes and to lular D-galacturonan to an intracellular inducer of PL. and sup- macerate parenchymatous plant tissues. Our understanding of plied them to strains deficient in oligogalacturonide lyase (OGL; the relationship between these two capacities has become EC 4.2.2.6].The subsequent PL production of these mutants clearer in the past decade (1). Pectic polymers [chains of 1,4- indicates the importance ofthis intracellular enzyme in the reg- linked a-D-galacturonic acid (GalUA) and methoxylated deriv- ulation of extracellular PL. atives] are structural components of the middle lamellae and primary cell walls ofhigher plants (2, 3). Highly purified pectate MATERIALS AND METHODS lyase [PL; poly(1,4-a-D-galacturonide) lyase, EC 4.22.2], by its Origin and Growth of E. chrysanthemi Strains. CU 1, a action on this substrate, appears sufficient to account for both spontaneous mutant resistant to rifampicin (200 ,g/ml) and the maceration and the cell killing characteristics ofsoft rot dis- streptomycin (10 ,g/ml) was derived from E. chrysanthemi eases (4, 5). Evidence that PL production is necessary for maceration has been reported for E. chrysanthemi; the capacity Abbreviations: PL, pectate lyase; GalUA, galacturonic acid; u(GalUA)2, for maceration and PL activity transfer together during conju- u(GalUA)3, and u(GalUA)4, di-, tri-, and tetragalacturonic acid 4,5-un- gation, suggesting that they are controlled by the same gene saturated at the nonreducing end; (GalUA)2, saturated digalacturonic (6). The apparent importance of pectic enzymes in the biology acid; PG, exo-poly-a-D-galacturonosidase; OGL, oligogalacturonide of their lyase; TBA, thiobarbituric acid; exoPL, exopolygalacturonate lyase; DK of these pathogens invites a thorough exploration I, 4-deoxy-L-threo-5-hexosulose uronic acid; DK II, 3-deoxy-D-glycero- regulation. 2,5-hexodiulosonic acid; KDG, 2-keto-3-deoxy-D-gluconic acid. * Present address: Section ofBiochemistry, Molecularand Cell Biology, The publication costs ofthis article were defrayed in part by page charge Cornell University, Ithaca, NY 14853. payment. This article must therefore be hereby marked "advertise- t Present address: North Carolina Agricultural Research Service, North ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Carolina State University, Raleigh, NC 27650. 3920 Microbiology: Collmer and Bateman Proc. Natl. Acad. Sci. USA 78 (1981) 3921 CUCPBt 630 [= strain 307 of Garibaldi (12)] which was origi- Preparation of Pectic Intermediates. u(GalUA)2, 4,5-unsat- nally isolated from carnation. CUCPB numbers of the strains urated trigalacturonic acid [u(GalUA)3], and 4,5-unsaturated studied are: CU 1, 1237; CU 2, 1238; CU 3, 1239; CU 4, 1240. tetragalacturonic acid [u(GalUA)4] were prepared by incubating Cultures were maintained at 80C on nutrient agar slants and 4% D-galacturonan in 50 mM Tris-HCl, pH 8.75/0.1 mM grown in 0.5% glycerol and minimal salts (17) prior to all ex- CaCl2, and partially purified PL. After 18 hr the mixture was periments. Except where noted, 250C, minimal salts, and shake treated with Dowex-50 in the H' form until the pH was reduced cultures were used throughout. to 2.1, then immediately removed from the resin by decanta- Selection of Mutants Deficient in OGL. CU 1 was mutation, and titrated to pH 5.5 with NaOH. The mixture of unsat- genized with ethylmethane sulfonate (18) in separate batches urated oligomers was then eluted in several 10-ml portions yielding 45% survival (CU 3) and 5% survival (CU 2). Muta- through a 2.5 x 90 cm column of Bio-Gel P-2 (Bio-Rad), -400 genized cultures (incubated for the rest of the procedure at mesh, at 25 mi/hr in 50 mM NaCl. u(GalUA)4 was further re- 350C) were grown to early stationary phase on 0.5% glucose, solved from larger oligomers by descending preparative paper harvested by centrifugation, and incubated at an OD6w of0. 05 chromatography on Whatman no. 1, developed with 1-butanol/ (2 X 10' colony-forming units/ml) in 5 mM (GalUA)2 for 3 hr acetic acid/water, 2:1:1 (vol/vol). and with added ampicillin (20 ,ug/ml) for an additional 3 hr. Preparation of (GalUA)2 will be described in detail else- Washed survivors were grown on 0.5% glucose and incubated where. Briefly, D-galacturonan was digested with partially pu- with (GalUA)2 as before but with 2 mM D-cycloserine instead rified PG at pH 6.0 in the presence of1 mM EDTA. Undigested of ampicillin. Survivors of the second enrichment were again polymer was precipitated with an equal volume of redistilled recovered in glucose media and then replicate plated on 0.5% ethanol, then (GalUA)2 was precipitated from the supernatant D-galacturonan and 0.5% GalUA minimal salts agar. with 4 more volumes ofethanol at -20°C. (GalUA)2 used in all Clones growing on GalUA but not on D-galacturonan were experiments other than mutant selection was further purified screened for OGL activity as follows. The clones were trans- by gel filtration through Bio-Gel P-2, as described above. ferred to several nutrient agar plates (44 colonies per plate). The intracellular intermediates, tentatively identified as the After approximately 24 hr, a 9-cm-diameter circle ofWhatman deoxyketuronic acids, 4-deoxy-L-threo-5-hexosulose uronic no. 1 filter paper was pressed onto the surface and then placed acid (DK I) and 3-deoxy-D-glycero-2,5-hexodiulosonic acid (DK in 500 1d of50 mM (GalUA)2 in 100 mM potassium phosphate, II), were prepared by incubatinga mixture ofDowex-50-treated pH 7.0. After a 3-hr OGL induction period, the filter papers unsaturated oligomers in 50 mM Tris HCl, pH 7.7/1 mM CaCl2 were flooded with toluene for 2 min, then decanted and allowed with cell-free extracts (sonicated cells centrifuged at 10,000 X to incubate without cover for 90 min. The papers were then g for 30 min, then dialyzed). After 18 hr 5-ml portions of the stained with periodate/thiobarbituric acid (TBA) (19). Clones reaction mixture were passed through a Bio-Gel P-2 column as deficient in OGL (colonies not orange-red) were purified by described above. DK I, which eluted between residual u(GalUA)2 repeated streaking on nutrient agar plates supplemented with and DK II, was further purified by a second cycle of Bio-Gel rifampicin (100 pg/ml) and streptomcyin (10 ,ug/ml) then fur- P-2 gel filtration. DK I was independently generated by di- ther characterized in batch cultures. A spontaneous revertant gesting u(GalUA)3 with exoPL at pH 8.5 in 50 mM Tris/0. 1 mM ofCU 2 (CU 4) was selected by plating a glycerol-grown culture CaCl2, and by digesting u(GalUA)2 in 50 mM sodium acetate, on minimal salts agar containing 20 mM (GalUA)2, rifampicin pH 4.0, with the hydrolytic pectinase available from Sigma. (100 ,ug/ml), and streptomycin (10 ug/ml). All pectic intermediates were analyzed by descending paper Enzyme Assays. PL was assayed in the supernatants of cul- chromatography on Whatman no. 1 developed with ethyl ace- tures centrifuged at 6000 x g for 10 min. Reaction mixtures tate/acetic acid/water/formic acid, 18:3:4:1 (vol/vol), for 9.5 contained 0.07% D-galacturonan, 30 mM Tris at pH 8.5, 0.1 hr. Spots were detected by dipping papers in silver nitrate (20) mM CaC12, and 6.7% (vol/vol) supernatant sample. The reac- or phenylenediamine [36 mg/ml of70% (vol/vol) ethanol]. DK tion was monitored by the increase in absorbance at 230 nm I generated by cell-free extracts, pectinase, and partially pu- (4). OGL activity was determined by the production of TBA- rified OGL and exoPL (Table 1) migrated 12.5 cm and formed reactive material from (GalUA)2 or u(GalUA)2 (15). Centrifuged a purple spot after dipping in phenylenediamine. DK II mi- cells were resuspended in 1/20 the original volume of 100 mM grated 17.4 cm, formed a yellow spot with phenylenediamine, Tris-HCI, pH 7.7/1 mM CaCl2. After toluene had been added and was reduced to 2-keto-3-deoxy-D-gluconic acid (KDG) at 1 drop per ml, the suspension was Vortex-mixed repeatedly when incubated with cell-free extracts and NADH in 100 mM and incubated for 15 min at 25°C before addition of substrate potassium phosphate, pH 7.4. This KDG migrated 22.2 cm, as to a final concentration of 4 mM. At intervals, 150-.1. samples did an authentic standard kindly supplied by J. Preiss (Univer- [(GalUA)2] or 75-,ul samples [u(GalUA)2] were removed to tri- chloroacetic acid (5%, in a final volume of300 ,u1). After removal Table 1. Paper chromatography of pectic intermediates of the bacteria by centrifugation, 200 1,u of each sample was assayed by the TBA method. Products* Partial Purification ofPectic Enzymes. Detailed description Substrate Enzyme (GalUA)2 u(GalUA)2 GaIUA DK I will appear elsewhere. Briefly, the extracellular enzymes PL (GalUA)nt PL + and PG were resolved by gel filtration of concentrated loga- (GalUA)n PG + rithmic-phase culture supernatants. The intracellular enzymes u(GalUA)3 PG + + OGL and exopolygalacturonate lyase [exoPL; poly(1,4-a-D-gal- u(GalUA)4 exoPL + acturonide) exo-lyase, EC 4.2.2.9] were also resolved by gel u(GalUA)3 exoPL + + filtration, after streptomycin sulfate and ammonium sulfate frac- u(GalUA)2 OGL + tionation of sonicated cell extracts. Partially purified PL and (GalUA)2 OGL + + exoPL were 99% free ofthe otherpectic enzymes. PG contained * Reaction and chromatography conditions are described in Materials 4.5% PL activity. OGL had substantial PG and PL contamination. and Methods; PG reaction mixtures contained 50 mM potassium phosphate (pH 6.0) and 2 mM EDTA. t Cornell University Collection of Phytopathogenic Bacteria, R. S. tDGalacturonan [(GalUA)^] was digested by PL to u(GalUA)2 and Dickey, Department of Plant Pathology, Ithaca, NY 14853. larger unsaturated oligogalacturonides (not shown). 3922 Microbiology: Collmer and Bateman Proc. Natl. Acad. Sci. USA 78 (1981) sity ofCalifornia, Davis). GalUA migrated 10 cm in this system. exoPL Materials. The D-galacturonan used was product 3491 of u(GalUA)4 P 2 u(GalUA)2 Induction of Sunkist Growers (Corona, CA). Ethylmethane sulfonate, GalUA, u(GalUA)3 exoPL u(GalUA)2 PL TBA, cyclic AMP, Tris, and all antibiotics were obtained from DKI .. Sigma. OGL D -ISO u(GalUA)2 2 DKI \ RESULTS (r-alTTA)-kual QOGL mrw + Py+vate Induction ofPL by Pectic Intermediates. The rate ofPL syn- GalUA , KDG-'--- triose thesis increased at least 20-fold whenever CU 1 cultures were phosphate shifted from 0.5% glycerol to media containing 0.5% D-galact- GalUA------_-*------_I uronan. Paper chromatographic analyses of supernatants from Self-catabolite repression cyclic AMP t cultures in logarithmic-phase growth on D-galacturonan re- of PL vealed that the major products of extracellular pectic digestion were u(GalUA)2 and (GalUA)2. The potential of these dimers FIG. 2. Diagram of the postulated activity of intracellular pectic to mediate induction on D-galacturonan was tested by deter- enzymes and intermediates in extracellular PL regulation. Incoming mining the level of specific PL activity attained during incu- products [mostly u(GalUA)2 and (GalUA)2] of the extracellular diges- bation in different concentrations of the dimers, D-galactu- tion of D-galacturonan by PL and PG are degraded primarily to DK I ronan, or GalUA (Fig. 1). Because, for any given concentration, by OGL, then converted by 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase (ISO) to DK II, which is reduced by 2-keto-3-deoxy-D- the total amount of uronic acid was constant from treatment to gluconate dehydrogenase (DH) to KDG, an intermediate shared with treatment, the experiment compares the effect on PL induction GalUA catabolism (21). (Dashed arrows denote enzymes reported in ofthe degree ofpolymerization ofthe substrate and shows that other bacteria; solid arrows denote those confirmed in E. chrysan- the dimers were effective inducers at the low concentrations and themi.) Deoxyketuronic acid formation is necessary for induction on relatively short incubation period used, but the polymer was oligomers, but high rates of formation lead to reduction in the cyclic not. Fig. 1 also shows that dimers were about 10 times more AMP concentration and subsequent self-catabolite repression of PL. Although GalUA is a less effective PL inducer than the oligomers (and PL than GalUA. These observations sug- effective inducers is not a major product of extracellular digestion of D-galacturonan), gested to us that the actual inducer ofPL was either the dimers our results do not exclude the possibility that KDG or a further metab- or a metabolite derived from them but not from GalUA. On the olite is the actual inducer. basis of previous reports of separate pathways for the initial catabolism of GalUA and u(GalUA)2 (15, 16, 21), metabolites Induction of PL in Mutants Deficient in OGL. In order to unique to dimer utilization (Fig. 2) were expected, and they clarify the role ofdimers and subsequent metabolites in PL in- were to cell-free extracts found be formed from u(GalUA)2 by duction we derived mutants deficient in OGL from CU 1. Cu and and Table of E. chrysanthemi (see Materials Methods 1). 2 (ogl-l) was one ofthese mutants. It produced wild-type levels The deoxyketuronic acids were tested for their PL-inducing of PL during incubation on DK II but was poorly induced by DK II was as effective as on an potential. (GalUA)2 equal weight (GalUA)2, u(GalUA)2, or D-galacturonan (Table 2). CU 2 was I had no TBA basis (Table 2). Exogenous DK inducing activity. unable to grow on dimers, but, as expected, could grow on assays revealed that 0.1 mM DK II was ofculture supernatants GalUA (Table 3). That CU 2 was indeed deficient in OGL was entirely removed from the medium during a 3.5-hr incubation, demonstrated by its failure to produce active OGL during in- DK I was not. DK II was used but under identical conditions cubation on DK II, which induced OGL in CU 1 (Table 4). in all subsequent studies. Because CU 2 reverted spontaneously (to CU 4) at a frequency of 1 x 10-8, we concluded that the CU 2 phenotype (Tables 3 and 4) resulted from a single mutation. The possibility remained that the failure ofthe dimers to induce PL in CU 2 resulted not from inability to generate the 0 deoxyketuronic acids but rather from a polar mutation affecting 0 0>-o both OGL and the transport ofoligogalacturonides. To address this possibility we obtained further evidence that deoxyket- ci uronic acid formation was necessary for PL induction by study- ing the response of CU 2 to larger unsaturated oligomers. Be- an to cause E. chrysanthemi produced intracellular exoPL that released DK I from u(GalUA)3 but not from u(GalUA)2 or u(GalUA)4 (Table 1), we postulated that PL production in CU

Table 2. Production of extracellular PL by CU 1 and CU 2 grown 1 2 on pectic compounds Uronic acid concentration, PL induced, umol/min ml* log(Q*g/ml) (GalUA)n DK II (GaIUA)2 (GalUA)nt FIG. 1. PL production during incubation ofE. chrysanthemi with Strain (0.1 mM) (0.2 mM) (0.1 mM) (0.5%) uronic acids differing in degree of polymerization and concentration. CU 1 0.219 0.203 0.122 0.176 A glycerol-grown culture (0.6 OD600) was centrifuged and resuspended CU 2 (ogl-1) 0.006 0.195 0.026 0.000 at 0.2 OD6N in mineral salts plus uronic acids. After 3-hr incubation, the ODow ofthe cultures were measured again and the PL activity in * Glycerol-grown cultures (0.3 OD600) were centrifuged and resus- culture supernatants was assayed. Samples lacking activity were ar- pended at the same OD65 in minimal salts plus the indicated pectic bitrarily plotted at the lowest ordinate value, which represents the compounds. After 4-hr incubation, PL activity in culture superna- limit of detection. *, (GalUA)2; o, u(GalUA)2; *, GalUA; o, D- tants was determined. galacturonan. t D-Galacturonan. Microbiology: Collmer and Bateman Proc. Natl. Acad. Sci. USA 78 (1981) 3923

Table 3. Specific growth rates on pectic intermediates Table 5. Production of extracellular PL by CU 1 and CU 2 grown Growth rate, hr-1 on 4,5-unsaturated oligogalacturonides Strain (GalUA)2 u(GalUA)2 GalUA PL induced, ,umol/min ml CU 1 0.25 0.28 0.31 Strain u(GalUA)2 u(GalUA)3 u(GalUA)4 CU 2 (ogl-1) 0.00 0.03 0.33 CU 1 0.306 0.455 0.676 CU 3 (ogl-2) 0.06 0.18 0.30 CU 2 (ogl-1) 0.081 0.310 0.111 CU 4 0.24 0.26 0.32 Cultures were prepared and assayed as in Table 2 but at 0.45 OD600 Cultures in logarithmic growth on glycerol were centrifuged and in 0.2 mM unsaturated oligomer. resuspended in minimal salts plus 4 mM dimers or 8 mM GalUA. The specific growth rate was determined during logarithmic growth on the induction to occur, the cell must be able to generate a signal pectic compounds. from a large and potentially unassimilable substrate. Second, for the enzyme to be secreted at appropriate rates into envi- 2 would be stimulated by u(GalUA)3 but not by u(GalUA)2 or ronments that may vary widely in their effects on the activity u(GalUA)4. The data in Table 5 confirm this. Because it is un- and longevity ofthe enzyme, the rate ofsynthesis must be sen- likely that u(GalUA)3 was transported in preference to u(GalUA)2 sitive to the specific activity of the enzyme. Accordingly, it is or u(GalUA)4, we concluded that the failure of the dimers to generally assumed that extracellular depolymerases are "prod- induce PL in CU 2 was entirely a consequence of the OGL uct induced" by the action ofthe same enzyme synthesized and deficiency (and not ofinducer exclusion) and that exoPL activity secreted at basal levels (22-25), and that, after induction, the could be an alternate route for generating inducers. accumulation of high concentrations of products can lead to Self-Catabolite Repression of PL. Fig. 1 shows that at con- ,'self-catabolite repression" (11). These concepts are supported centrations above 11 ug/ml (0.03 mM), (GalUA)2 stimulated by physiological observations but to our knowledge have not much higher levels of PL production than did u(GalUA)2. That been tested before by genetic means with any extracellular this effect was due to catabolite repression exerted by u(GalUA)2 depolymerase. and not by (GalUA)2 was suggested by three observations. Un- The plant pathogen E. chrysanthemi increases its rate ofex- der the conditions described in Fig. 1, CU 1 produced the same tracellular PL production when shifted into media containing level ofspecific PL activity on 0.2 mM u(GalUA)2 plus 0.2 mM pectic polymers, and PL synthesis is inhibited by high concen- (GalUA)2 as it did on 0.2 mM u(GalUA)2 alone (data not shown), trations ofits product, u(GalUA)2 (Fig. 1). At least four enzymes indicating that u(GalUA)2 catabolism could abolish the differ- may participate in this regulatory process. As indicated by the ential stimulation by (GalUA)2. Addition of 5 mM cyclic AMP phenotype oftwo mutants, the key enzyme in determining the to 0.5 mM u(GalUA)2 resulted in an increase in PL synthesis level of PL synthesis is the intracellular enzyme OGL: it is the to a level equal to that induced by 0.5 mM (GalUA)2 without primary generator ofthe apparent PL inducer and its high rate cyclic AMP (Table 6). Finally, the difference between (GalUA)2 of activity on u(GalUA)2 promotes self-catabolite repression of and u(GalUA)2 becomes apparent only at concentrations sup- PL. porting bacterial growth (Fig. 1). Our observations can be integrated into a regulatory model The phenotype ofCU 3 (ogl-2) provides genetic confirmation (Fig. 2) based on the separate pathways for monomeric and that u(GalUA)2 could act as a catabolite repressor of PL. CU 3, oligomeric GalUA catabolism previously reported for a Pseu- though lacking detectable OGL (Table 4), was able to grow domonas species (14, 15) and forE. carotovora (16) and partially slowly on u(GalUA)2 (Table 3). In comparison with CU 1 it was confirmed in E. chrysanthemi (Table 1 and Materials and hyperinduced on 0.5 mM u(GalUA)2 but poorly induced when Methods). catabolite repression was relieved in both strains by addition We believe that PL induction is dependent on the generation of5 mM cyclic AMP or imposed on both strains by logarithmic ofdeoxyketuronic acids. A mutant deficient in OGL (CU 2) was growth on glycerol (Table 6). The latter two observations, along poorly induced unless supplied with DK II or with a substrate with the poor ability of (GalUA)2 to stimulate PL synthesis in [u(GalUA)3] that could be converted by another intracellular CU 3 (Table 6), provide further evidence for the necessity of enzyme (exoPL) to DK I. We cannot determine from our data the deoxyketuronic acids for PL induction. whether the actual inducer is DK I or DK II because the two deoxyketuronic acids are freely interconverted intracellularly by 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase (EC DISCUSSION 5.3.1.17) (15). Our conclusion that the inducer is not an inter- The efficient regulation ofextracellular enzymes digesting poly- mediate common with GalUA catabolism-e.g., KDG-is meric substrates presents bacteria with two problems not en- based on the inducing potential of GalUA of only 1/10th ob- countered in the regulation ofintracellular enzymes. First, for served even at low concentrations at which the confounding

Table 4. OGL activity in E. chrysanthemi strains Activity, AA548/min-ml Table 6. Induction of extracellular PL in CU 1 and CU 3 Strain u(GalUA)2 (GalUA)2 PL induced, ,umol/min ml CU 1 0.110 0.035 u(GalUA)2 u(GalUA)2 CU 2 (ogl-1) 0.000 0.000 (0.5 mM) (0.5 mM) CU 3 (ogl-2) 0.000 0.000 + + CU 4 0.088 0.028 (GalUA)2 u(GalUA)2 cyclic AMP glycerol Strain (0.5 mM) (0.5 mM) (5 mM) (50 mM) After 4-hr preincubation in 0.2 mM DK II, cultures (all 0.51 OD600) CU 1 0.429 0.072 0.439 0.092 were harvested and assayed for the rate ofincrease ofTBA-detectable CU 3 0.043 0.137 0.208 material per ml oftoluene-treated cells in the presence ofthe indicated (ogl-2) 0.019 substrates. Cultures were prepared and assayed as for Table 2. 3924 Microbiology: Collmer and Bateman Proc. Natl. Acad. Sci. USA 78 (1981) effects of self-catabolite repression are not likely to be a factor PL, however, appears to be neither the substrate nor the prod- (Fig. 1). The inducing potential that was possessed by Ga1UA uct but a further metabolite of oligogalacturonide catabolism. we tentatively attribute to either reversibility of the 2-keto-3- We thank C. H. Whalen for excellent assistance with parts of this deoxy-D-gluconate dehydrogenase (EC 1.1.1.126) reaction (15) work, D. E. Matthews for critical review of the manuscript, and S. V. or to structural similarities between the deoxyketuronic acids Beer for thoughtful guidance and encouragement. and GalUA and its unique catabolites, D-tagaturonic acid and D-altronic acid (21). Mutants deficient in other enzymes ofpec- 1. Bateman, D. F. & Basham, H. G. (1976) in Encyclopedia ofPlant Physiology, eds. Heitefuss, R. & Williams, P. H. (Springer, Ber- tic catabolism are needed to resolve these ambiguities. lin), Vol. 4, pp. 316-55. Our data indicate that induction on D-galacturonan requires 2. McClendon, J. H. (1964) Am. J. Bot. 51, 628-633. the release ofdimers from the polymer as an intermediate step 3. Keegstra, K., Talmadge, K. W., Bauer, W. D. & Albersheim, P. in deoxyketuronic acid formation. The dimeric products of PL (1973) Plant Physiol. 51, 188-196. and PG were effective inducers at concentrations 1/1000th of 4. Basham, H. G. & Bateman, D. F. (1975) Physiol. Plant Pathol. the concentration required by D-galacturonan. The inducing 5, 249-262. 5. Stephens, G. J. & Wood, R. K. S. (1975) Physiol. Plant Pathol. potential of different concentrations of D-galacturonan corre- 5, 165-181. lated with the Km of PL (100 pug/ml) and PG (500 Ag/ml), sug- 6. Chatterjee, A. K. & Starr, M. P. (1977) J. Bacteriol. 132, 862- gesting that extracellular digestion was a limiting step in the 869. induction process. Dimers specifically appear to be key inter- 7. Rexova-Benkova, L. & Markovic, 0. (1976) Adv. Carbohydr. mediates in the induction process as observed by the failure of Chem. Biochem. 33, 323-385. CU 2 (ogl-l) to be induced by D-galacturonan, even though it 8. Tsuyumu, S. (1977) Nature (London) 269, 237-238. 9. Hubbard, J. P., Williams, J. D., Niles, R. M. & Mount, M. S. could be induced by u(GalUA)3 (Tables 2 and 5). OGL activity (1978) Phytopathology 68, 95-99. was maximal on dimers and negligible on D-galacturonan (Table 10. Mount, M. S., Berman, P. M., Mortlock, R. P. & Hubbard, J. 4 and unpublished results); yet OGLwas required forinduction, P.. (1979) Phytopathology 69, 117-120. again suggesting that dimers mediated induction on the poly- 11. Tsuyumu, S. (1979)J. Bacteriol. 137, 1035-1036. mer. Further evidence that extracellular digestion precedes in- 12. Garibaldi, A. & Bateman, D. F. (1971) Physiol. Plant Pathol. 1, duction will be presented elsewhere. 2540. 13. Collmer, A. & Bateman, D. F. (1979) Phytopathology 69, 1025 We have also observed that PL is subject to self-catabolite (abstr.). repression exerted by high concentrations of its product, 14. Preiss, J. & Ashwell, G. (1963)J. Biol. Chem. 238, 1571-1576. u(GalUA)2 (Fig. 1 and Table 6). The cyclic AMP-reversible na- 15. Preiss, J. & Ashwell, G. (1963)J. Biol. Chem. 238, 1577-1583. ture of this repression suggests that it results from the rapid 16. Moran, F., Nasuno, S. & Starr, M. P. (1968) Arch. Biochem. Bio- catabolism of u(GalUA)2 in a manner analogous to the glucose phys. 125, 734-741. repression ofother bacterial catabolic enzymes (26). To account 17. Zucker, M. & Hankin, L. (1970)J. Bacteriol. 104, 13-18. 18. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold for the ability of u(GalUA)2 to both induce and catabolite re- Spring Harbor Laboratory, Cold Spring Harbor, NY). press, we postulate that induction is saturated at deoxyketuronic 19. Warren, L. (1960) Nature (London) 186, 237. acid concentrations lower than those required for maximal rates 20. Anet, E. F. L. G. & Reynolds, T. M. (1954) Nature (London) 174, of catabolism (Fig. 2). Accordingly, partially reduced rates of 903. 21. Kilgore, W. W. & Starr, M. P. (1959)J. Biol. Chem. 234, 2227- dimer degradation resulting from substrate limitation (Fig. 1), 2235. OGL mutation (Table 6), or the inherently lower rate of OGL 22. Glenn, A. R. (1976) Annu. Rev. Microbiol. 30, 41-62. activity on (GalUA)2 relative to u(GalUA)2 (Table 4) result in 23. Stanier, R. Y., Adelberg, E. A. & Ingraham, J. L. (1976) The greater PL production. Microbial World (Prentice-Hall, Englewood Cliffs, NJ). The regulation ofextracellular PL appears to share features 24. Priest, F. G. (1977) Bacteriol. Rev. 41, 711-753. 25. Ingle, M. B. & Erickson, R. J. (1978) Adv. Appl. Microbiol. 24, with the regulation of well-studied intracellular catabolic en- 257-278. zymes such as (-galactosidase (27). The rate of synthesis is re- 26. Rickenberg, H. V. (1974) Annu. Rev. Microbiol. 28, 353-369. duced by cyclic AMP-mediated catabolite repression and stim- 27. Beckwith, J. R. & Zipser, D. (1970) The Lactose Operon (Cold ulated by the presence of the substrate. The actual inducer of Spring Harbor Laboratory, Cold Spring Harbor, NY).