Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) fromPseudomonas aeruginosa
10.1128/jb.152.1.431-440.1982
Crossref journal-article
American Society for Microbiology
Journal of Bacteriology (235)
Abstract

Phospholipase C (heat-labile hemolysin) ofPseudomonas aeruginosais a phosphate (Pi)-regulated extracellular protein which may be a significant virulence factor of this organism. The gene for this hemolytic enzyme was cloned on a 4.1-megadalton (Mdal) fragment from aBamHI digest ofP. aeruginosaPAO1 genomic DNA and was inserted into theBamHI sites of the multicopyEscherichia coli(pBR322) andP. aeruginosa(pMW79) vectors. TheE. coliandP. aeruginosarecombinant plasmids were designated pGV26 and pVB81, respectively. A restriction map of the 4.1-Mdal fragment from pGV26 was constructed, using double and single digestions withBamHI andEcoRI and several different restriction enzymes. Based on information from this map, a 2.4-MdalBamHI/BglII fragment containing the gene for phospholipase C was subcloned to pBR322. The hybrid plasmids pGV26 and pVB81 direct the synthesis of enzymatically active phospholipase C, which is also hemolytic. The plasmid-directed synthesis of phospholipase C inE. coliorP. aeruginosais not repressible by Pias is the chromosomally directed synthesis inP. aeruginosa. Data are presented which suggest that the synthesis of phospholipase C from pGV26 and pVB81 is directed from the tetracycline resistance gene promoter. The level of enzyme activity produced byE. coli(pGV26) is slightly higher than the levels produced byP. aeruginosa(pMW79) under repressed conditions. In contrast, the levels produced byP. aeruginosa(pVB81) are at least 600-fold higher than the levels produced byP. aeruginosa(pMW79) under repressed conditions and approximately 20-fold higher than those produced byP. aeruginosa(pMW79) under derepressed conditions. The majority (85%) of the enzyme produced byE. coli(pGV26) remained cell associated, whereas >95% of the enzyme produced byP. aeruginosa(pVB81) was extracellular. Analysis of extracellular proteins from cultures ofP. aeruginosa(pMW79) andP. aeruginosa(pVB81) by high-performance liquid chromotography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the phospholipase C gene was cloned intact, and it is likely that several additional genes were cloned on the 4.1-Mdal fragment of DNA. It was also found that some of these genes encode proteins which are the same molecular weight as some previously described Pi-repressible proteins ofP. aeruginosa. The existence of a Piregulon ofP. aeruginosais proposed. It is likely that one of these genes also regulates the level of pyocyanin production byP. aeruginosaand that one or more play a role in transport or binding of Pi. The availability of the hybrid plasmids described herein will be useful in further studies on the role of this hemolysin in the virulence ofP. aeruginosaand in the study of the genetics and physiology of Pi-regulated proteins.

Bibliography

Vasil, M. L., Berka, R. M., Gray, G. L., & Nakai, H. (1982). Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) fromPseudomonas aeruginosa. Journal of Bacteriology, 152(1), 431–440.

Authors 4
  1. Michael L. Vasil (first)
  2. Randy M. Berka (additional)
  3. Gregory L. Gray (additional)
  4. Hiroshi Nakai (additional)
References 38 Referenced 40
  1. Bachmann , B. J. , and K. B. Low . 1980 . Linkage map of Escherichia coli K-12, edition 6 . Microbiol. Rev. 44 : 1 - 56 . (10.1128/mr.44.1.1-56.1980) / Microbiol. Rev. / Linkage map of Escherichia coli K-12, edition 6 by Bachmann B. J. (1980)
  2. Berka , R. M. , G. L. Gray , and M. L. Vasil . 1981 . Studies of phospholipase C (heat-labile hemolysin) in Pseudomonas aeruginosa . Infect. Immun. 34 : 1071 - 1074 . (10.1128/IAI.34.3.1071-1074.1981) / Infect. Immun. / Studies of phospholipase C (heat-labile hemolysin) in Pseudomonas aeruginosa by Berka R. M. (1981)
  3. . Berka , R. M. , and M. L. Vasil . 1982 . Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization . J. Bacteriol. 152 : 239 - 245 . (10.1128/jb.152.1.239-245.1982) / J. Bacteriol. / Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization by Berka R. M. (1982)
  4. Bollvar , F. , and K. Beckman . 1979 . Plasmids ofEscherichia coli as cloning vectors . Methods Enzymol. 68 : 245 - 267 . (10.1016/0076-6879(79)68018-7) / Methods Enzymol. / Plasmids ofEscherichia coli as cloning vectors by Bollvar F. (1979)
  5. Bramccwl , M. G. , E. M. Twiddy , W. B. Balne , and R. K. Holmes . 1981 . Isolation and characterization of hypertoxinogenic (htx) mutants of Escherichia coli KL320 (pCG 86) . Infect. Immun. 32 : 1034 - 1044 . (10.1128/IAI.32.3.1034-1044.1981) / Infect. Immun. / Isolation and characterization of hypertoxinogenic (htx) mutants of Escherichia coli KL320 (pCG 86) by Bramccwl M. G. (1981)
  6. Cash , H. A. , D. E. Woods , B. McCulough , W. G. Johanson , Jr. , and J. A. Ba . 1979 . A rat model of chronic respiratory infection with Pseudomonas aeruginosa . Am. Rev. Respir. Dis. 119 : 453 - 359 . / Am. Rev. Respir. Dis. / A rat model of chronic respiratory infection with Pseudomonas aeruginosa by Cash H. A. (1979)
  7. Clarke , L. , and J. Carbon . 1976 . A colony bank containing synthetic Col E 1 hybrid plasmids representative of the entire E. coli genome . Cell 9 : 91 - 99 . (10.1016/0092-8674(76)90055-6) / Cell / A colony bank containing synthetic Col E 1 hybrid plasmids representative of the entire E. coli genome by Clarke L. (1976)
  8. Evans , D. J. , D. G. Evans , C. Hohne , M. A. Noble , E. V. Holdane , H. Liar , and L. S. Young . 1981 . Hemolysin and K antigens in relation to serotype and hemagglutination type of Escherichia coli isolated from extraintestinal infection . J. Clin. Microbiol. 13 : 171 - 178 . (10.1128/jcm.13.1.171-178.1981) / J. Clin. Microbiol. / Hemolysin and K antigens in relation to serotype and hemagglutination type of Escherichia coli isolated from extraintestinal infection by Evans D. J. (1981)
  9. Gray , G. L. , R. M. Berka , and M. L. Vadl . 1981 . A Pseudomonas aeruginosa mutant nonderepressible for orthophosphate-regulated proteins . J. Bacteriol. 147 : 675 - 678 . (10.1128/jb.147.2.675-678.1981) / J. Bacteriol. / A Pseudomonas aeruginosa mutant nonderepressible for orthophosphate-regulated proteins by Gray G. L. (1981)
  10. Gray , G. L. , R. M. Berka , and M. L. Vadl . 1982 . Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins . J. Bacteriol. 150 : 1221 - 1226 . (10.1128/jb.150.3.1221-1226.1982) / J. Bacteriol. / Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins by Gray G. L. (1982)
  11. Gray , G. L. , and M. L. Vadl . 1981 . Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa . J. Bacteriol. 147 : 275 - 281 . (10.1128/jb.147.2.275-281.1981) / J. Bacteriol. / Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa by Gray G. L. (1981)
  12. Hedgs , R. W. , and A. E. Jacob . 1977 . In vivo translation of genes of Pseudomonas aeruginosa onto a promiscously transmissible plasmid . FEMS Microbiol. Lett. 2 : 15 - 19 . (10.1111/j.1574-6968.1977.tb00898.x) / FEMS Microbiol. Lett. / In vivo translation of genes of Pseudomonas aeruginosa onto a promiscously transmissible plasmid by Hedgs R. W. (1977)
  13. Hodges , R. W. , A. E. Jacob , and I. P. Crawford . 1977 . Wide ranging plasmid bearing the Pseudomonas aeruginosa tryptophan synthase genes . Nature (London) 267 : 283 - 284 . (10.1038/267283a0) / Nature (London) / Wide ranging plasmid bearing the Pseudomonas aeruginosa tryptophan synthase genes by Hodges R. W. (1977)
  14. Hirt , B. 1967 . Selective extraction of polyoma DNA from infected mouse cell cultures . J. Mol. Biol. 26 : 365 - 369 . (10.1016/0022-2836(67)90307-5) / J. Mol. Biol. / Selective extraction of polyoma DNA from infected mouse cell cultures by Hirt B. (1967)
  15. Ingledw , M. W. , and J. J. R. Campbell . 1969 . A new resuspension medium for pyocyanin production . Can. J. Microbiol. 15 : 595 - 598 . (10.1139/m69-101) / Can. J. Microbiol. / A new resuspension medium for pyocyanin production by Ingledw M. W. (1969)
  16. Johnson , M. K. , and D. Boese-Marrazzo . 1980 . Production and properties of heat-stable extracellular hemolysin from Pseudomonas aeruginosa . Infect. Immun. 28 : 1028 - 1033 . (10.1128/iai.29.3.1028-1033.1980) / Infect. Immun. / Production and properties of heat-stable extracellular hemolysin from Pseudomonas aeruginosa by Johnson M. K. (1980)
  17. rsapa, V. 1979 . DNA insertion mutagenesis in a Pseudomonas aeruginosa R plasmid . Plasmid 2 : 237 - 246 . (10.1016/0147-619X(79)90042-8) / Plasmid / DNA insertion mutagenesis in a Pseudomonas aeruginosa R plasmid (1979)
  18. Kurloka , S. , and P. V. Liu . 1967 . Effect of the hemolysin of Pseudomonas aeruginosa on phosphatides and on phospholipase C activity . J. Bacteriol. 93 : 670 - 674 . (10.1128/jb.93.2.670-674.1967) / J. Bacteriol. / Effect of the hemolysin of Pseudomonas aeruginosa on phosphatides and on phospholipase C activity by Kurloka S. (1967)
  19. La 1aronl, J. C. , and R. C. PortaHer . 1981 . Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12 . J. Bacteriol. 145 : 1351 - 1358 . (10.1128/jb.145.3.1351-1358.1981) / J. Bacteriol. / Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12 by La J. C. (1981)
  20. Un P. V. 1979. Toxin of Pseudomonas aeruginosa p. 63-68. In R. G. Doggett (ed.) Pseudomonas aeruginosa. Academic Press Inc. New York.
  21. Maloy , S. B. , and W. D. Nunn . 1981 . Selection for loss of tetracycline resistance by Escherichia coli . J. Bacteriol. 145 : 1351 - 1358 . (10.1128/jb.145.2.1110-1111.1981) / J. Bacteriol. / Selection for loss of tetracycline resistance by Escherichia coli by Maloy S. B. (1981)
  22. Maendel , M. , and A. Hip . 1970 . Calcium-dependent bacteriophage DNA infection . J. Mol. Biol. 53 : 159 - 162 . (10.1016/0022-2836(70)90051-3) / J. Mol. Biol. / Calcium-dependent bacteriophage DNA infection by Maendel M. (1970)
  23. Mlusew , B. H. , J. jorgemson_, G. W. Counts , and S. Falkow . 1978 . Association of hemolysin production, hemagglutination of human erythrocytes, and virulence for chicken embryos of extraintestinal Escherichia coli isolates . Infect. Immun. 20 : 50 - 54 . (10.1128/iai.20.1.50-54.1978) / Infect. Immun. / Association of hemolysin production, hemagglutination of human erythrocytes, and virulence for chicken embryos of extraintestinal Escherichia coli isolates by Mlusew B. H. (1978)
  24. Mlyamato , Y. , Y. Obara , T. N. Nlkkawa , S. Yamal , T. Kato , Y. Yamada , and M. Ohash . 1980 . Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated hemolysin of Vibrio parahaemolyticus . Infect. Immun. 28 : 567 - 576 . (10.1128/iai.28.2.567-576.1980) / Infect. Immun. / Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated hemolysin of Vibrio parahaemolyticus by Mlyamato Y. (1980)
  25. Oakley , B. R. , 0. R. Kirsch , and N. R. Norris . 1980 . A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels . Anal. Biochem. 105 : 361 - 363 . (10.1016/0003-2697(80)90470-4) / Anal. Biochem. / A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels by Oakley B. R. (1980)
  26. Petit , J. C. , D. Sicord , S. Bago , and G. L. Daguet . 1981 . Effect of active and passive immunization on the development of experimental Pseudomonas aeruginosa pyelonephritis in mice . Can. J. Microbiol. 27 : 93 - 97 . (10.1139/m81-015) / Can. J. Microbiol. / Effect of active and passive immunization on the development of experimental Pseudomonas aeruginosa pyelonephritis in mice by Petit J. C. (1981)
  27. R Iddle , L. M. , T. E. Graham , and R. L. Amborski . 1981 . Medium for the accumulation of extracellular hemolysin and protease by Aeromonas hydrophila . Infect. Immun. 33 : 728 - 738 . (10.1128/IAI.33.3.728-733.1981) / Infect. Immun. / Medium for the accumulation of extracellular hemolysin and protease by Aeromonas hydrophila by Iddle L. M. (1981)
  28. Rlgby , P. W. , J. Dleckman , C. Rhodes , and P. Berg . 1977 . Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I . J. Mol. Biol. 113 : 237 - 251 . (10.1016/0022-2836(77)90052-3) / J. Mol. Biol. / Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I by Rlgby P. W. (1977)
  29. Ruvkun , G. B. , and F. M. Ausubel . 1981 . A general method for site-directed mutagenesis in prokaryotes . Nature (London) 289 : 85 - 88 . (10.1038/289085a0) / Nature (London) / A general method for site-directed mutagenesis in prokaryotes by Ruvkun G. B. (1981)
  30. Soutern , E. M. 1975 . Detection of specific sequences among DNA fragments separated by gel electrophoresis . J. Mol. Biol. 98 : 503 - 517 . (10.1016/S0022-2836(75)80083-0) / J. Mol. Biol. / Detection of specific sequences among DNA fragments separated by gel electrophoresis by Soutern E. M. (1975)
  31. Southrn , P. M. , B. B. Mays , A. D. Perce , and J. P. Sanford . 1970 . Pulmonary clearance of Pseudomonas aeruginosa . J. Lab. Clin. Med. 76 : 548 - 559 . / J. Lab. Clin. Med. / Pulmonary clearance of Pseudomonas aeruginosa by Southrn P. M. (1970)
  32. Stlerltz , D. D. , and I. A. Holder . 1975 . Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: description of a burned mouse model . J. Infect. Dis. 131 : 688 - 691 . (10.1093/infdis/131.6.688) / J. Infect. Dis. / Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: description of a burned mouse model by Stlerltz D. D. (1975)
  33. Wanner , B. L. , and P. Lattrl . 1980 . Mutants affected in allcaine phosphatase expression: evidence for multiple positive regulators for the phosphate regulon in Escherichia coli . Genetics 96 : 353 - 366 . (10.1093/genetics/96.2.353) / Genetics / Mutants affected in allcaine phosphatase expression: evidence for multiple positive regulators for the phosphate regulon in Escherichia coli by Wanner B. L. (1980)
  34. Welch , R. A. , E. P. Deger , B. Mlnshew , and S. Falkow . 1981 . Haemolysin contributes to virulence of extra-intestinal E. coli infections . Nature (London) 294 : 665 - 667 . (10.1038/294665a0) / Nature (London) / Haemolysin contributes to virulence of extra-intestinal E. coli infections by Welch R. A. (1981)
  35. Wood , D. 0. , M. F. Hoflnger , and M. B. Tlndof . 1981 . Versatile cloning vector for Pseudomonas aeruginosa . J. Bacteriol. 145 : 1448 - 1451 . (10.1128/jb.145.3.1448-1451.1981) / J. Bacteriol. / Versatile cloning vector for Pseudomonas aeruginosa by Wood D. (1981)
  36. Wretlnd , B. , R. MolIby , and T. Wadstrom . 1971 . Separation of two hemolysins from Aeromonas hydrophila by isoelectric focusing . Infect. Immun. 4 : 503 - 505 . (10.1128/IAI.4.4.503-505.1971) / Infect. Immun. / Separation of two hemolysins from Aeromonas hydrophila by isoelectric focusing by Wretlnd B. (1971)
  37. Young , L. S. 1980 . The role of exotoxins in the pathogenesis of Pseudomonas aeruginosa . J. Infect. Dis. 142 : 626 - 630 . (10.1093/infdis/142.4.626) / J. Infect. Dis. / The role of exotoxins in the pathogenesis of Pseudomonas aeruginosa by Young L. S. (1980)
  38. Ziegler , E. J. , and B. H. Douglas . 1979 . Pseudomonas aeruginosa vasculitis and bacteremia following conjunctivitis: a simple model of fatal Pseudomonas infection in neutropenia . J. Infect. Dis. 139 : 288 - 296 . (10.1093/infdis/139.3.288) / J. Infect. Dis. / Pseudomonas aeruginosa vasculitis and bacteremia following conjunctivitis: a simple model of fatal Pseudomonas infection in neutropenia by Ziegler E. J. (1979)
Dates
Type When
Created 4 years, 2 months ago (June 2, 2021, 7:15 p.m.)
Deposited 2 years, 7 months ago (Dec. 29, 2022, 9:06 a.m.)
Indexed 1 year, 5 months ago (March 20, 2024, 5:56 a.m.)
Issued 42 years, 10 months ago (Oct. 1, 1982)
Published 42 years, 10 months ago (Oct. 1, 1982)
Published Print 42 years, 10 months ago (Oct. 1, 1982)
Funders 0

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@article{Vasil_1982, title={Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) fromPseudomonas aeruginosa}, volume={152}, ISSN={1098-5530}, url={http://dx.doi.org/10.1128/jb.152.1.431-440.1982}, DOI={10.1128/jb.152.1.431-440.1982}, number={1}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Vasil, Michael L. and Berka, Randy M. and Gray, Gregory L. and Nakai, Hiroshi}, year={1982}, month=oct, pages={431–440} }