Vol. 57, No. 6
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1870-1872
0099-2240/91/061870-03$02.00/0 Copyright © 1991, American Society for Microbiology
Cloning of Alginate Lyase Gene (alxM) and Expression in Escherichia colit B. J.
Department
BROWN,'* J.
F. PRESTON,2 AND L.
0.
INGRAM2
Michigan Technological University, Houghton, Michigan 49931,1 and Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 326112
of Chemistry,
Received 8 March 1991/Accepted 16 April 1991
The alxM gene encoding a D-mannuronan-specific alginate lyase has been cloned from a marine bacterium isolated as an epiphyte on the brown alga, Sargassum fluitans. Expression of this gene in Escherichia coli provides a source of this enzyme for probing alginate structure and modifying the mannuronan-rich alginate polymers produced by bacterial pathogens.
Alginate is an acidic polysaccharide comprised of 1--4 linked P-D-mannuronic acid and cx-L-guluronic acid residues. It is the predominant structural component of brown algae (1) and is secreted as an extracellular polysaccharide by species of Azotobacter and Pseudomonas (11, 14, 16). The secretion of mannuronan-rich alginate by mucoid strains of Pseudomonas aeruginosa has been correlated with their relative virulence as opportunistic pathogens associated with cystic fibrosis (17). Similarly, secretion of alginate with mannuronic acid residues as the predominant if not exclusive component has been implicated in the phytopathogenic properties of several pathovars of Pseudomonas syringae (12). Alginate lyases (EC 4.2.2.3) cleave the 1->4 glycosidic linkage of the uronic acid polymer by a 1-elimination reaction resulting in an unsaturated nonreducing terminus. These enzymes have been detected in a variety of microorganisms (2, 9, 10, 15, 24), and known enzymes have a preference for either L-guluronic or D-mannuronic acid residues. The guluronan-specific alginate lyase of Klebsiella pneumoniae was cloned and expressed in Escherichia coli (5). The extracellular L-guluronan-specific alginate lyase from Klebsiella aerogenes type 25 has been used to investigate alginate structure (3, 6, 8). However, a mannuronan-specific enzyme is not readily available for complementary investigations. Bacteria associated with pelagic species of the brown algal genus Sargassum secrete alginate lyases which have a strong preference for either the D-mannuronan or L-guluronan components of alginate (18, 19, 21, 22). One of the facultative isolates, SFFB080483Alg-A (ATCC 433367), secreted an endolytic, mannuronan-specific lyase which was homogeneous on the basis of biochemical studies (21). This gramnegative bacterium was selected for construction of a gene library. Here, we report the cloning of the alxM gene encoding the mannuronan-specific lyase and its expression in E. coli. The gram-negative marine isolate, SFFBO80483AIg-A (19), was grown as previously described in Provasoli's enriched seawater medium containing 0.1% alginate (18). High-molecular-weight DNA was prepared by a combination of enzymatic digestion and cesium chloride centrifugation (4). Chromosomal DNA was partially digested with Sau3A and 4- to 6-kb fragments were purified by agarose gel * Corresponding author. t Florida Agricultural Experiment Station, Journal Series R00954.
1870
electrophoresis. This DNA was ligated into the dephosphorylated BamHI site of pUC18 (7). Transformants were selected in E. coli TC4 on Luria agar containing 50 jig of ampicillin per ml. A random sample of these indicated that more than 90% contained 4- to 6-kb DNA inserts. Approximately 2,400 clones were screened for alginate lyase activity. Colony lifts were made by using Whatman no. 1 filter paper circles cut to fit inside a petri plate. After exposure of the colonies to chloroform vapors for 5 min, the cell side of the filter was placed on the surface of petri plates containing 1% agarose and 0.1% alginate in buffer (30 mM potassium phosphate, 50 mM KCI, pH 7.5). Preparations were incubated overnight at 37°C. Filters were removed and the plates were stained by flooding with 0.05% ruthenium red (20). Clear zones of depolymerization on a red background were observed, indicating lyase activity. Twelve lyasepositive clones were isolated during the screening process. No zones of depolymerization were observed around control strains containing pUC18. The most active clone, pAL-A3, produced a 25-mm zone of clearing and was investigated further. A restriction map was constructed for pAL-A3 as shown in Fig. 1. The plasmid contains a 4.1-kb genomic DNA insert. The origin of the alxM-containing fragment of DNA was confirmed by Southern hybridization to chromosomal DNA from SFFBO80483Alg-A (Fig. 2) by using a 1.6-kb KpnI fragment of pAL-A3 as a probe (23). The pattern of hybridization to chromosomal digests was consistent with the presence of a single chromosomal copy of this gene in SFFBO80483Alg-A. The alginate lyase from strain SFFBO80483Alg-A has been reported to be specific for D-mannuronan (22), and this specificity was examined in the recombinant E. coli TC4(pAL-A3). Cells were grown to late exponential phase and treated to release periplasmic enzymes (25). The yield of enzyme from the transformed E. coli periplasmic preparation is 1.70 units per ml of culture broth, compared with 0.40 units per ml of culture broth from the parent organism (21). A232 was monitored to detect the unsaturated nonreducing terminus in the lyase-generated product. Homopolymeric substrates were prepared by mild acid hydrolysis of alginate (13). Reactions were run in 0.025 M sodium phosphate buffer (pH 7.5) containing 0.5 M NaCl and 0.02% uronic acid polymer (Fig. 3). The rate of product formation was higher with D-mannuronan than with mixed polymers of alginate. L-Guluronan was not degraded by the enzyme released from E. coli TC4(pAL-A3). Alginate lyase with L-guluronan spec-
VOL. 57, 1991
BamHl
Accl Sall Aval Kpnl
11
I
-
a
w
NOTES A3
Aval
BamHI
I
1.
1 3 4 kb 2 FIG. 1. Restriction endonuclease map of plasmid pAL-A3 carrying the D-mannuronan-specific alginate lyase gene.
ificity was prepared from another marine bacterium, SFFBO80483Alg-G (19), and assayed with the same protocol to verify the digestibility of the L-guluronan substrate. The substrate specificity of the recombinant enzyme encoded by pAL-A3 is identical to that reported previously for the dominant lyase present in SFFBO80483Alg-A (20), providing further evidence that the plasmid contains the alxM gene. The cloning and expression in E. coli of a gene coding for the endolytic D-mannuronan-specific alginate lyase will provide an abundant source of this enzyme to examine the relationships between enzyme structure, substrate specificity, mechanism, and regulation. The D-mannuronani
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8 16 0 8 0 Time, min FIG. 3. Evaluation of specificity of the alginate lyase expressed by E. coli TC4(pAL-A3). Lyase activity was measured as an increase in the A232. Reactions were initiated by addition of substrate as indicated by the arrows. From the left, tracing 1 resulted from the action of the recombinant enzyme (A3) on alginate from Macrocystis pyrifera (a). Tracing 2 shows the action of the recombinant enzyme on D-mannuronan (m). Tracing 3 demonstrates the lack of digestion of L-guluronan (g), although the enzyme preparation was active, as demonstrated by its subsequent action on D-mannuronan. Tracing 4 confirms the digestibility of L-guluronan by a guluronan-specific enzyme from another isolate, SFF B080483AIg-G (Alg G). 0
8
0
8
23-1specific enzyme also provides a complement to the L-guluronan-specific alginate lyase from K. aerogenes type 25, which has been used to probe the structural organization of alginate from brown algae (3), Azotobacter vinelandii (8), and pathogenic P. aeruginosa (6). The availability of the alxM gene product should be of particular value because of its specificity for D-mannuronan, which is the major component of alginate secreted by Pseudomonas sp. plant (12, 16) and animal pathogens (11, 17). We thank Donna Duarte and Martina Simmons for preparing the figures and text. This work was supported by the Gas Research Institute and the Institute of Food and Agricultural Sciences, University of Florida, as CRIS project MCS-02789.
REFERENCES 1. Baardstehl, E. 1966. Localization and structure of alginate gels, p. 19-28. In E. G. Young and J. L. McLachlan (ed.), Proceedings of the Fifth International Seaweed Symposium. Pergamon Press, Inc., New York. 2. Boyd, J., and J. R. Turvey. 1977. Isolation of a poly-oa-Lguluronate lyase from Klebsiella aerogenes. Carbohydr. Res.
FIG. 2. Southern hybrdization analysis of the genomic DNA from SFFBO80483Alg-A and E. coli. A 12P-labeled probe was prepared from the 1.6-kb Kpnl fragment of pAL-A3 by the random primer method. Lanes: 1, EcoRI digest of E. coli genomic DNA; 2, EcoRI digest; 3, KpnI digest; 4, HincIl digest of SFFBO80483AIg-A DNA; 5, Kpnl digest of pAL-A3 exposed for a shorter time.
57:163-171. 3. Boyd, J., and J. R. Turvey. 1978. Structural studies of alginic acid using a bacterial poly-aX-L-guluronate lyase. Carbohydr. Res. 66:187-194. 4. Byun, M. O.-K., J. B. Kaper, and L. 0. Ingram. 1986. Construction of a new vector for the expression of foreign genes in Zymomonas mobilis. J. Ind. Microbiol. 1:9-15. 5. Caswell, R. C., P. Gacesa, K. E. Luttrell, and A. J. Weightman. 1989. Molecular cloning and heterologous expression of Klebsiella pneumoniae gene coding for alginate lyase. Gene 75:127134. 6. Chitnis, C. E., and D. E. Ohman. 1990. Cloning of Pseudomonas aeruginosa algG, which controls alginate structure. J. Bacte-
1872
.NOTES
riol. 172:2894-2900. 7. Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffmann, and L. 0. Ingram. 1987. Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase. J. Bacteriol. 169: 949-954. 8. Currie, A. J., and J. R. Turvey. 1982. An enzymic method for the assay of D-mannuronan C-5 epimerase activity. Carbohydr. Res. 107:156-161. 9. Doubet, R. S., and R. S. Quatrano. 1984. Properties of alginate lyases from marine bacteria. Appl. Environ. Microbiol. 47:600703. 10. Dunne, W. M., Jr., and F. L. A. Buckmire. 1985. Partial purification of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis. Appl. Environ. Microbiol. 50:562567. 11. Evans, L. R., and A. Linker. 1973. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J. Bacteriol. 116:915-924. 12. Fett, W. F., and M. F. Dunn. 1989. Exopolysaccharides produced by phytopathogenic Pseudomonas syringe pathovars in infected leaves of susceptible hosts. Plant Physiol. 89:5-9. 13. Haug, A., B. Larsen, and 0. Smidsrod. 1967. Studies on the sequence of uronic acid residues in alginic acid. Acta Chem. Scand. 21:691-704. 14. Haug, A., B. Larsen, and 0. Smidsrod. 1974. Uronic acid sequence in alginate from different sources. Carbohydr. Res. 32:217-225. 15. Linker, A., and L. R. Evans. 1984. Isolation and characterization of an alginase from mucoid strains of Pseudomonas aeruginosa. J. Bacteriol. 159:958-964. 16. Osman, S. F., W. F. Fett, and M. L. Fishman. 1986. Exopolysaccharides of the phytopathogen Pseudomonas syringe pv. glycinea. J. Bacteriol. 166:66-71.
APPL. ENVIRON. MICROBIOL. 17. Pederson, S. S., F. Espersen, N. Hoiby, and G. H. Shand. 1989. Purification, characterization, and immunological cross-reactivity of alginates produced by mucoid Pseudomonas aeruginosa from patients with cystic fibrosis. J. Clin. Microbiol. 27:691699. 18. Preston, J. F., III, T. Romeo, and J. C. Bromley. 1986. Selective alginate degradation by marine bacteria associated with the algal genus Sargassum. J. Ind. Microbiol. 1:235-244. 19. Preston, J. F., III, T. Romeo, J. C. Bromley, R. W. Robinson, and H. C. Aldrich. 1985. Alginate lyase-secreting bacteria associated with the algal genus Sargassum. Dev. Ind. Microbiol. 26:727-740. 20. Ried, J. L., and A. Collmer. 1985. Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. Appl. Environ. Microbiol. 51:615-622. 21. Romeo, T., and J. F. Preston III. 1986. Purification and structural properties of an extracellular (1-4)-p-D-mannuronanspecific alginate lyase from a marine bacterium. Biochemistry
25:8385-8391. 22. Romeo, T., and J. F. Preston III. 1986. Depolymerization of alginate by an extracellular alginate lyase from a marine bacterium: substrate specificity and accumulation of reaction products. Biochemistry 25:8391-8396. 23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Sutherland, I. W., and G. A. Keen. 1981. Alginases from Benekia pelagia and Pseudomonas spp. J. Appl. Biochem. 3:48-57. 25. Witholt, B., M. Boekhout, M. Brock, J. Kingma, H. van Heerkhuizen, and L. de Leij. 1976. An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal. Biochem. 74:160-170.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1870-1872
0099-2240/91/061870-03$02.00/0 Copyright © 1991, American Society for Microbiology
Cloning of Alginate Lyase Gene (alxM) and Expression in Escherichia colit B. J.
Department
BROWN,'* J.
F. PRESTON,2 AND L.
0.
INGRAM2
Michigan Technological University, Houghton, Michigan 49931,1 and Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 326112
of Chemistry,
Received 8 March 1991/Accepted 16 April 1991
The alxM gene encoding a D-mannuronan-specific alginate lyase has been cloned from a marine bacterium isolated as an epiphyte on the brown alga, Sargassum fluitans. Expression of this gene in Escherichia coli provides a source of this enzyme for probing alginate structure and modifying the mannuronan-rich alginate polymers produced by bacterial pathogens.
Alginate is an acidic polysaccharide comprised of 1--4 linked P-D-mannuronic acid and cx-L-guluronic acid residues. It is the predominant structural component of brown algae (1) and is secreted as an extracellular polysaccharide by species of Azotobacter and Pseudomonas (11, 14, 16). The secretion of mannuronan-rich alginate by mucoid strains of Pseudomonas aeruginosa has been correlated with their relative virulence as opportunistic pathogens associated with cystic fibrosis (17). Similarly, secretion of alginate with mannuronic acid residues as the predominant if not exclusive component has been implicated in the phytopathogenic properties of several pathovars of Pseudomonas syringae (12). Alginate lyases (EC 4.2.2.3) cleave the 1->4 glycosidic linkage of the uronic acid polymer by a 1-elimination reaction resulting in an unsaturated nonreducing terminus. These enzymes have been detected in a variety of microorganisms (2, 9, 10, 15, 24), and known enzymes have a preference for either L-guluronic or D-mannuronic acid residues. The guluronan-specific alginate lyase of Klebsiella pneumoniae was cloned and expressed in Escherichia coli (5). The extracellular L-guluronan-specific alginate lyase from Klebsiella aerogenes type 25 has been used to investigate alginate structure (3, 6, 8). However, a mannuronan-specific enzyme is not readily available for complementary investigations. Bacteria associated with pelagic species of the brown algal genus Sargassum secrete alginate lyases which have a strong preference for either the D-mannuronan or L-guluronan components of alginate (18, 19, 21, 22). One of the facultative isolates, SFFB080483Alg-A (ATCC 433367), secreted an endolytic, mannuronan-specific lyase which was homogeneous on the basis of biochemical studies (21). This gramnegative bacterium was selected for construction of a gene library. Here, we report the cloning of the alxM gene encoding the mannuronan-specific lyase and its expression in E. coli. The gram-negative marine isolate, SFFBO80483AIg-A (19), was grown as previously described in Provasoli's enriched seawater medium containing 0.1% alginate (18). High-molecular-weight DNA was prepared by a combination of enzymatic digestion and cesium chloride centrifugation (4). Chromosomal DNA was partially digested with Sau3A and 4- to 6-kb fragments were purified by agarose gel * Corresponding author. t Florida Agricultural Experiment Station, Journal Series R00954.
1870
electrophoresis. This DNA was ligated into the dephosphorylated BamHI site of pUC18 (7). Transformants were selected in E. coli TC4 on Luria agar containing 50 jig of ampicillin per ml. A random sample of these indicated that more than 90% contained 4- to 6-kb DNA inserts. Approximately 2,400 clones were screened for alginate lyase activity. Colony lifts were made by using Whatman no. 1 filter paper circles cut to fit inside a petri plate. After exposure of the colonies to chloroform vapors for 5 min, the cell side of the filter was placed on the surface of petri plates containing 1% agarose and 0.1% alginate in buffer (30 mM potassium phosphate, 50 mM KCI, pH 7.5). Preparations were incubated overnight at 37°C. Filters were removed and the plates were stained by flooding with 0.05% ruthenium red (20). Clear zones of depolymerization on a red background were observed, indicating lyase activity. Twelve lyasepositive clones were isolated during the screening process. No zones of depolymerization were observed around control strains containing pUC18. The most active clone, pAL-A3, produced a 25-mm zone of clearing and was investigated further. A restriction map was constructed for pAL-A3 as shown in Fig. 1. The plasmid contains a 4.1-kb genomic DNA insert. The origin of the alxM-containing fragment of DNA was confirmed by Southern hybridization to chromosomal DNA from SFFBO80483Alg-A (Fig. 2) by using a 1.6-kb KpnI fragment of pAL-A3 as a probe (23). The pattern of hybridization to chromosomal digests was consistent with the presence of a single chromosomal copy of this gene in SFFBO80483Alg-A. The alginate lyase from strain SFFBO80483Alg-A has been reported to be specific for D-mannuronan (22), and this specificity was examined in the recombinant E. coli TC4(pAL-A3). Cells were grown to late exponential phase and treated to release periplasmic enzymes (25). The yield of enzyme from the transformed E. coli periplasmic preparation is 1.70 units per ml of culture broth, compared with 0.40 units per ml of culture broth from the parent organism (21). A232 was monitored to detect the unsaturated nonreducing terminus in the lyase-generated product. Homopolymeric substrates were prepared by mild acid hydrolysis of alginate (13). Reactions were run in 0.025 M sodium phosphate buffer (pH 7.5) containing 0.5 M NaCl and 0.02% uronic acid polymer (Fig. 3). The rate of product formation was higher with D-mannuronan than with mixed polymers of alginate. L-Guluronan was not degraded by the enzyme released from E. coli TC4(pAL-A3). Alginate lyase with L-guluronan spec-
VOL. 57, 1991
BamHl
Accl Sall Aval Kpnl
11
I
-
a
w
NOTES A3
Aval
BamHI
I
1.
1 3 4 kb 2 FIG. 1. Restriction endonuclease map of plasmid pAL-A3 carrying the D-mannuronan-specific alginate lyase gene.
ificity was prepared from another marine bacterium, SFFBO80483Alg-G (19), and assayed with the same protocol to verify the digestibility of the L-guluronan substrate. The substrate specificity of the recombinant enzyme encoded by pAL-A3 is identical to that reported previously for the dominant lyase present in SFFBO80483Alg-A (20), providing further evidence that the plasmid contains the alxM gene. The cloning and expression in E. coli of a gene coding for the endolytic D-mannuronan-specific alginate lyase will provide an abundant source of this enzyme to examine the relationships between enzyme structure, substrate specificity, mechanism, and regulation. The D-mannuronani
'00Q:'.t:.'0
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-4,:f
i
A3 m
a
i
i
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i
1871
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m
i
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N
0.4
0.2
-zz
F v//
I
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I
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8 16 0 8 0 Time, min FIG. 3. Evaluation of specificity of the alginate lyase expressed by E. coli TC4(pAL-A3). Lyase activity was measured as an increase in the A232. Reactions were initiated by addition of substrate as indicated by the arrows. From the left, tracing 1 resulted from the action of the recombinant enzyme (A3) on alginate from Macrocystis pyrifera (a). Tracing 2 shows the action of the recombinant enzyme on D-mannuronan (m). Tracing 3 demonstrates the lack of digestion of L-guluronan (g), although the enzyme preparation was active, as demonstrated by its subsequent action on D-mannuronan. Tracing 4 confirms the digestibility of L-guluronan by a guluronan-specific enzyme from another isolate, SFF B080483AIg-G (Alg G). 0
8
0
8
23-1specific enzyme also provides a complement to the L-guluronan-specific alginate lyase from K. aerogenes type 25, which has been used to probe the structural organization of alginate from brown algae (3), Azotobacter vinelandii (8), and pathogenic P. aeruginosa (6). The availability of the alxM gene product should be of particular value because of its specificity for D-mannuronan, which is the major component of alginate secreted by Pseudomonas sp. plant (12, 16) and animal pathogens (11, 17). We thank Donna Duarte and Martina Simmons for preparing the figures and text. This work was supported by the Gas Research Institute and the Institute of Food and Agricultural Sciences, University of Florida, as CRIS project MCS-02789.
REFERENCES 1. Baardstehl, E. 1966. Localization and structure of alginate gels, p. 19-28. In E. G. Young and J. L. McLachlan (ed.), Proceedings of the Fifth International Seaweed Symposium. Pergamon Press, Inc., New York. 2. Boyd, J., and J. R. Turvey. 1977. Isolation of a poly-oa-Lguluronate lyase from Klebsiella aerogenes. Carbohydr. Res.
FIG. 2. Southern hybrdization analysis of the genomic DNA from SFFBO80483Alg-A and E. coli. A 12P-labeled probe was prepared from the 1.6-kb Kpnl fragment of pAL-A3 by the random primer method. Lanes: 1, EcoRI digest of E. coli genomic DNA; 2, EcoRI digest; 3, KpnI digest; 4, HincIl digest of SFFBO80483AIg-A DNA; 5, Kpnl digest of pAL-A3 exposed for a shorter time.
57:163-171. 3. Boyd, J., and J. R. Turvey. 1978. Structural studies of alginic acid using a bacterial poly-aX-L-guluronate lyase. Carbohydr. Res. 66:187-194. 4. Byun, M. O.-K., J. B. Kaper, and L. 0. Ingram. 1986. Construction of a new vector for the expression of foreign genes in Zymomonas mobilis. J. Ind. Microbiol. 1:9-15. 5. Caswell, R. C., P. Gacesa, K. E. Luttrell, and A. J. Weightman. 1989. Molecular cloning and heterologous expression of Klebsiella pneumoniae gene coding for alginate lyase. Gene 75:127134. 6. Chitnis, C. E., and D. E. Ohman. 1990. Cloning of Pseudomonas aeruginosa algG, which controls alginate structure. J. Bacte-
1872
.NOTES
riol. 172:2894-2900. 7. Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffmann, and L. 0. Ingram. 1987. Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase. J. Bacteriol. 169: 949-954. 8. Currie, A. J., and J. R. Turvey. 1982. An enzymic method for the assay of D-mannuronan C-5 epimerase activity. Carbohydr. Res. 107:156-161. 9. Doubet, R. S., and R. S. Quatrano. 1984. Properties of alginate lyases from marine bacteria. Appl. Environ. Microbiol. 47:600703. 10. Dunne, W. M., Jr., and F. L. A. Buckmire. 1985. Partial purification of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis. Appl. Environ. Microbiol. 50:562567. 11. Evans, L. R., and A. Linker. 1973. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J. Bacteriol. 116:915-924. 12. Fett, W. F., and M. F. Dunn. 1989. Exopolysaccharides produced by phytopathogenic Pseudomonas syringe pathovars in infected leaves of susceptible hosts. Plant Physiol. 89:5-9. 13. Haug, A., B. Larsen, and 0. Smidsrod. 1967. Studies on the sequence of uronic acid residues in alginic acid. Acta Chem. Scand. 21:691-704. 14. Haug, A., B. Larsen, and 0. Smidsrod. 1974. Uronic acid sequence in alginate from different sources. Carbohydr. Res. 32:217-225. 15. Linker, A., and L. R. Evans. 1984. Isolation and characterization of an alginase from mucoid strains of Pseudomonas aeruginosa. J. Bacteriol. 159:958-964. 16. Osman, S. F., W. F. Fett, and M. L. Fishman. 1986. Exopolysaccharides of the phytopathogen Pseudomonas syringe pv. glycinea. J. Bacteriol. 166:66-71.
APPL. ENVIRON. MICROBIOL. 17. Pederson, S. S., F. Espersen, N. Hoiby, and G. H. Shand. 1989. Purification, characterization, and immunological cross-reactivity of alginates produced by mucoid Pseudomonas aeruginosa from patients with cystic fibrosis. J. Clin. Microbiol. 27:691699. 18. Preston, J. F., III, T. Romeo, and J. C. Bromley. 1986. Selective alginate degradation by marine bacteria associated with the algal genus Sargassum. J. Ind. Microbiol. 1:235-244. 19. Preston, J. F., III, T. Romeo, J. C. Bromley, R. W. Robinson, and H. C. Aldrich. 1985. Alginate lyase-secreting bacteria associated with the algal genus Sargassum. Dev. Ind. Microbiol. 26:727-740. 20. Ried, J. L., and A. Collmer. 1985. Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. Appl. Environ. Microbiol. 51:615-622. 21. Romeo, T., and J. F. Preston III. 1986. Purification and structural properties of an extracellular (1-4)-p-D-mannuronanspecific alginate lyase from a marine bacterium. Biochemistry
25:8385-8391. 22. Romeo, T., and J. F. Preston III. 1986. Depolymerization of alginate by an extracellular alginate lyase from a marine bacterium: substrate specificity and accumulation of reaction products. Biochemistry 25:8391-8396. 23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Sutherland, I. W., and G. A. Keen. 1981. Alginases from Benekia pelagia and Pseudomonas spp. J. Appl. Biochem. 3:48-57. 25. Witholt, B., M. Boekhout, M. Brock, J. Kingma, H. van Heerkhuizen, and L. de Leij. 1976. An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal. Biochem. 74:160-170.
