Abstract
Extracellular lipase activity detected on tributyrin agar has been identified in a cosmid clone, JMP3084, constructed from the chromosome of Aeromonas hydrophila and vector pHC79. This lipase, named apl-1, also exhibits nonhemolytic phospholipase C activity on lecithin and p-nitrophenylphosphorylcholine. Subcloning of the cosmid JMP3084 with partial Sau3a1 digestion localized the lipase gene to a 3.4-kb DNA fragment. Southern blot analysis shows the gene apl-1 to exist in single copy on the A. hydrophila chromosome. Expression of apl-1 in the pT7 system identified a single protein of molecular weight 70 kDa. Nucleotide sequencing of apl-1 has identified an open reading frame of 2055 bases predicting a protein of 73 kDa. The presence of an amino terminal signal sequence of 18 amino acids accounts for this molecular weight disparity. Further analysis of the lipase amino acid sequence revealed the presence of a classical serine active lipase site (Gly-X-Ser-X-Gly) located between residues 561 and 570. The A. hydrophila chromosomal copy of apl-1 has been inactivated by use of the mutagenesis vector pJP5603, resulting in the complete removal of phospholipase C activity and lowered levels of lipase activity detected on tributyrin agar.
Similar content being viewed by others
Literature Cited
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Anguita J, Rodriguez Aparicio LB, Naharro G (1993) Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from an Aeromonas hydrophila human isolate. Appl Environ Microbiol 59:2411–2417
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Buttke TM, Cuchens MA (1984) Inhibition of lymphocyte proliferation by free fatty acids. Immunology 53:507–514
Chrisope GL, Fox CW, Marshall RT (1976) Lecithin agar for detection of microbial phospholipases. Appl Environ Microbiol 31:784–786
Chung Chang M, Chang SY, Chen SL, Chuang SM (1992) Cloning and expression in Escherichia coli of the gene encoding an extracellular deoxyribonuclease from Aeromonas hydrophila. Gene 122:175–180
Eftimiadi C, Buzzi E, Tonetti M, Buffa P, Buffa D, van Steenbergen MTJ, de Graaff J, Botta GA (1987) Short-chain cal responses of human neutrophils to chemotactic peptide. J Infect 14:43–53
Gish W, States DJ (1993) Identification of protein coding regions by database similarity search. Nature Genet. 3:266–272
Gobius KS, Pemberton JM (1986) Use of plasmid pULB113 (RP4::Mini-Mu) to construct a genomic map of Aeromonas hydrophila. Curr Microbiol 13:111–115
Gobius KS, Pemberton JM (1988) Molecular cloning, characterization and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila. J Bacteriol 170:1325–1332
Herrero M, de Lorenzo V, Timmis KN (1990) Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosome insertion of foreign genes in Gram negative bacteria. J Bacteriol 170:6557–6567
Janda JM (1985) Biochemical and exoenzymatic properties of Aeromonas species. Diagn Microbiol Infect Dis 3:223–232
Kirov SM (1993) The public health significance of Aeromonas spp. in foods. Int J Food Microbiol 20:179–198
Kurioka S, Matsuda M (1976) Phospholipase C assay using p-nitrophenylphosphorylcholine together with sorbitol and its application to studying the metal and detergent requirement of the enzyme. Anal Biochem 75:281–289
Nath K (1991) A rapid DNA isolation procedure from Petri dish grown clinical bacterial isolates. Nucleic Acids Res 18:6462
Penfold RJ, Pemberton JM (1992) An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 118:145–146
Quigley NB, Reeves PR (1987) Chloramphenicol resistance vector based on pUC9. Plasmid 17:54–57
Rivero O, Anguita J, Paniagua C, Naharro G (1990) Molecular cloning and characterisation of an extracellular protease gene from Aeromonas hydrophila. J Bacteriol 172:3905–3908
Roffey PE, Pemberton JM (1990) Cloning and expression of an Aeromonas hydrophila chitinase gene in E. coli. Curr Microbiol 21:329–337
Russel M, Model P (1984) Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol 159:1034–1039
Tabor S, Richardson C (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078
Talmadge K, Gilbert W (1980) Construction of plasmid vectors with unique Pst1 cloning sites in a signal sequence coding region. Gene 12:235–241
Titball RW (1993) Bacterial phospholipases C. Microbiol Rev 57:347–366
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ingham, A.B., Pemberton, J.M. A lipase of Aeromonas hydrophila showing nonhemolytic phospholipase C activity. Current Microbiology 31, 28–33 (1995). https://doi.org/10.1007/BF00294630
Issue Date:
DOI: https://doi.org/10.1007/BF00294630