Worfd Journs/
of Microbiology
and Biofecbnolo9y,
9, 24&242
Regulatory mutations affecting the synthesis of pectate lyase in Xanthomonas campestris B.E. Boboye* and 0.0. Shonukan Four classes of Xanthomonas campestris mutants were identified with respect to pectate lyase. Pectate lyase production in the wild-type and classes I and IIb mutants was partially dependent on the growth-phase whereas in classes IIa and III it was totally dependent. Enzyme activity in some of the mutants was constitutive and resistant to catabolite repression. fiy
words: Pectate lyase, polygalacturonic
acid, Xunfhomonus.
Xanthomonas cumpesfris is a phytopathogen particularly associated with the soft-rot of fruit and vegetables (Salle 1973; Liao & Wells 1986). The phytopathogenicity is due mainly to pectinolysis of the plant cell walls as a result of pectic enzyme activity (Horsfall & Dimond 1959). Pectate lyases (EC 4.2.2.2.) are extracellular non-hydrolytic pectic enzymes which cleave the 1 + 4 glycosidic linkage between adjacent uranic acid units by a frans-elimination mechanism, with the formation of a galacturonic acid residue having an unsaturated bond between carbons 4 and 5 (Manner 1982). Pectate lyase is probably inducible by 2-keto-3-deoxy gluconate and 2,5-diketo-3-deoxy gluconate, the intermediate products of polygalacturonic acid breakdown (Condemine et al. 1986). Pectate lyase production in some bacteria is subject to catabolite repression (Hubbard et al. 1978) and the formation of polymer-degrading enzymes, such as pectate lyase or cellulases, is growth-phasedependent (Hugouvieux-Cotte-Pattat et al. 1986). This report deals with regulatory mutations affecting the synthesis of pectate lyase in X. cumpesfris. We have isolated and characterized four classes of mutants affected in the pectate lyase synthesis.
Materials
and Methods
Medium and Growth Conditions Xanthomonas campeskis pathovar cavnpestris strain 2350 National Institute of Applied Sciences, Lyon, France,
was from and was
BE. Boboye is with the Microbiology Laboratory, Department of Biology, Federal University of Technology, Akure. Ondo State, Nigeria. 0.0. Shonukan is with the Department of Microbiology, Obafemi Awolowo University, Ile-lfe, Own State, Nigeria. *Corresponding author. @ 1993 Rapid Communications
240
of Oxford
Ltd
Workd ]oumal of Micmbiology and Biotechnology. Vol 9, 1993
grown in synthetic minimal medium M63 (Miller 1972) supplemented with glucose or glycerol (0.2% w/v) as carbon source and polygalacturonic acid or pectin at (0.4% w/v) as inducer. of Mufanfs Chemical mutagenesis of the microorganism was carried out with ethylmethane sulphonate by a modification of the method of Parkinson (1976). Overall, 150 colonies of the mutagenized cells were isolated and tested for pectate lyase activity. The affected colonies were designated ‘mutants’ and used for further studies. The mutants were grouped into classes based on their induction ratios with respect to induction by polygalacturonic acid. The survival percentages of the mutants were also determined.
Isolation
Regulation of Pectate Lyase Production during Growth The wild-type and mutants were grown separately in M63 supplemented with 0.2% (w/v) glycerol and 0.4% (w/v) polygalacturonic acid. Optical density (670 nm) and enzyme activity were determined at intervals. Enzyme Extraction Wild-type cells, cultured in supplemented M63 medium, were treated with toluene by the method of Moran & Starr (1969) and pelleted by centrifugation at 3500 x g for 15 min at 4°C. The supematant solution was then used as the intracellular source of enzymes.
Pectate Lyase Assay The total (intraand extra-cellular) pectate lyase activity was assayed, using cells grown to an optical density of 3.0 at 670 nm, by the modified thiobarbituric acid reagent method of Weissbach & Hurwitz (1959). The reaction was conducted at 35°C for I h and pectate lyase activity measured at 540 nm. The reaction mixture contained I% (w/v) polygalacturonic acid (grade III; Sigma) (3.5 ml), enzyme (1.0 ml), and CaCl, (I mM). One unit of pectate lyase activity was defined as the amount of pectate lyase required to produce an increase in absorbance of 0.01 per min under the specified assay conditions.
Pe&te
Results
and Discussion
The survival rate of the mutants produced by treatment with ethylmethane sulphonate (EMS) was 2.6% of the wild-type. Pectate lyase activity of the wild-type was approximately 200-fold higher than that of 50 of the mutants under various growth conditions (Table 1). This is attributed to the marked effect of EMS on the gene controlling pectate lyase synthesis in the mutants. This mutagen causes mutations by producing ethylation in the purine ring of the DNA (De Robertis & De Robertis 1980). All the mutations allowed constitutive production of the enzyme, either because the mutant no longer produced a repressor protein or because the mutant’s operator regions had been altererd so that they no longer responded adequately to the action of the repressor protein (Brock et nl. 1984). The mutants were derepressed at different levels. Polygalacturonic acid was a better inducer than pectin in all mutants except those of Class IIb; in Table
1. Pectate
Class
lyase Strains
activity
in the mutants
Wild-type
No. 2350
GIY GIY GIY Glu Glu Glu
Mu 29 and 25 others
Ilb
Ill
Mu 5 and 12 others
Mu 47 only
Mu 48 and nine others
Pectate lyase activity (Units/ml)
Inducer None PGA
20 37
Pet None PGA
31.8 22 34
Pet
30
GIY
None
0.7
GIY GIY Glu
PGA Pet
8.3 6.0
None PGA
0.75 3.0
Glu Glu Ila
campesfrfs.*
Growth* Substrate
Pet
2.0
None
0.1 5.0
GIY GIY
PGA
GIY Glu Glu
Pet None PGA
4.7
Glu
Pet
1.0
GIY
None
‘JY GIY Glu Glu
PGA PeC None
0.1 5.1
Glu
Pet
GIY
None PGA
0.1 10.4
Pet None PGA Pet
5.0 0.1 6.2 3.5
GIY GIY Glu Glu Glu
X. campestris
agreement with the phenomenon of enzyme-substrate specificity (Sato & Kaji 1977; Weber & Canale-Parola 1984). Four classes of mutants were recognized. About half (52%) 26% and 20% of the mutants constituted classes I, IIa and III, with induction ratios of
of Microbiology
and Biofecbnolo9y,
9, 24&242
Regulatory mutations affecting the synthesis of pectate lyase in Xanthomonas campestris B.E. Boboye* and 0.0. Shonukan Four classes of Xanthomonas campestris mutants were identified with respect to pectate lyase. Pectate lyase production in the wild-type and classes I and IIb mutants was partially dependent on the growth-phase whereas in classes IIa and III it was totally dependent. Enzyme activity in some of the mutants was constitutive and resistant to catabolite repression. fiy
words: Pectate lyase, polygalacturonic
acid, Xunfhomonus.
Xanthomonas cumpesfris is a phytopathogen particularly associated with the soft-rot of fruit and vegetables (Salle 1973; Liao & Wells 1986). The phytopathogenicity is due mainly to pectinolysis of the plant cell walls as a result of pectic enzyme activity (Horsfall & Dimond 1959). Pectate lyases (EC 4.2.2.2.) are extracellular non-hydrolytic pectic enzymes which cleave the 1 + 4 glycosidic linkage between adjacent uranic acid units by a frans-elimination mechanism, with the formation of a galacturonic acid residue having an unsaturated bond between carbons 4 and 5 (Manner 1982). Pectate lyase is probably inducible by 2-keto-3-deoxy gluconate and 2,5-diketo-3-deoxy gluconate, the intermediate products of polygalacturonic acid breakdown (Condemine et al. 1986). Pectate lyase production in some bacteria is subject to catabolite repression (Hubbard et al. 1978) and the formation of polymer-degrading enzymes, such as pectate lyase or cellulases, is growth-phasedependent (Hugouvieux-Cotte-Pattat et al. 1986). This report deals with regulatory mutations affecting the synthesis of pectate lyase in X. cumpesfris. We have isolated and characterized four classes of mutants affected in the pectate lyase synthesis.
Materials
and Methods
Medium and Growth Conditions Xanthomonas campeskis pathovar cavnpestris strain 2350 National Institute of Applied Sciences, Lyon, France,
was from and was
BE. Boboye is with the Microbiology Laboratory, Department of Biology, Federal University of Technology, Akure. Ondo State, Nigeria. 0.0. Shonukan is with the Department of Microbiology, Obafemi Awolowo University, Ile-lfe, Own State, Nigeria. *Corresponding author. @ 1993 Rapid Communications
240
of Oxford
Ltd
Workd ]oumal of Micmbiology and Biotechnology. Vol 9, 1993
grown in synthetic minimal medium M63 (Miller 1972) supplemented with glucose or glycerol (0.2% w/v) as carbon source and polygalacturonic acid or pectin at (0.4% w/v) as inducer. of Mufanfs Chemical mutagenesis of the microorganism was carried out with ethylmethane sulphonate by a modification of the method of Parkinson (1976). Overall, 150 colonies of the mutagenized cells were isolated and tested for pectate lyase activity. The affected colonies were designated ‘mutants’ and used for further studies. The mutants were grouped into classes based on their induction ratios with respect to induction by polygalacturonic acid. The survival percentages of the mutants were also determined.
Isolation
Regulation of Pectate Lyase Production during Growth The wild-type and mutants were grown separately in M63 supplemented with 0.2% (w/v) glycerol and 0.4% (w/v) polygalacturonic acid. Optical density (670 nm) and enzyme activity were determined at intervals. Enzyme Extraction Wild-type cells, cultured in supplemented M63 medium, were treated with toluene by the method of Moran & Starr (1969) and pelleted by centrifugation at 3500 x g for 15 min at 4°C. The supematant solution was then used as the intracellular source of enzymes.
Pectate Lyase Assay The total (intraand extra-cellular) pectate lyase activity was assayed, using cells grown to an optical density of 3.0 at 670 nm, by the modified thiobarbituric acid reagent method of Weissbach & Hurwitz (1959). The reaction was conducted at 35°C for I h and pectate lyase activity measured at 540 nm. The reaction mixture contained I% (w/v) polygalacturonic acid (grade III; Sigma) (3.5 ml), enzyme (1.0 ml), and CaCl, (I mM). One unit of pectate lyase activity was defined as the amount of pectate lyase required to produce an increase in absorbance of 0.01 per min under the specified assay conditions.
Pe&te
Results
and Discussion
The survival rate of the mutants produced by treatment with ethylmethane sulphonate (EMS) was 2.6% of the wild-type. Pectate lyase activity of the wild-type was approximately 200-fold higher than that of 50 of the mutants under various growth conditions (Table 1). This is attributed to the marked effect of EMS on the gene controlling pectate lyase synthesis in the mutants. This mutagen causes mutations by producing ethylation in the purine ring of the DNA (De Robertis & De Robertis 1980). All the mutations allowed constitutive production of the enzyme, either because the mutant no longer produced a repressor protein or because the mutant’s operator regions had been altererd so that they no longer responded adequately to the action of the repressor protein (Brock et nl. 1984). The mutants were derepressed at different levels. Polygalacturonic acid was a better inducer than pectin in all mutants except those of Class IIb; in Table
1. Pectate
Class
lyase Strains
activity
in the mutants
Wild-type
No. 2350
GIY GIY GIY Glu Glu Glu
Mu 29 and 25 others
Ilb
Ill
Mu 5 and 12 others
Mu 47 only
Mu 48 and nine others
Pectate lyase activity (Units/ml)
Inducer None PGA
20 37
Pet None PGA
31.8 22 34
Pet
30
GIY
None
0.7
GIY GIY Glu
PGA Pet
8.3 6.0
None PGA
0.75 3.0
Glu Glu Ila
campesfrfs.*
Growth* Substrate
Pet
2.0
None
0.1 5.0
GIY GIY
PGA
GIY Glu Glu
Pet None PGA
4.7
Glu
Pet
1.0
GIY
None
‘JY GIY Glu Glu
PGA PeC None
0.1 5.1
Glu
Pet
GIY
None PGA
0.1 10.4
Pet None PGA Pet
5.0 0.1 6.2 3.5
GIY GIY Glu Glu Glu
X. campestris
agreement with the phenomenon of enzyme-substrate specificity (Sato & Kaji 1977; Weber & Canale-Parola 1984). Four classes of mutants were recognized. About half (52%) 26% and 20% of the mutants constituted classes I, IIa and III, with induction ratios of
