Abstract
From a genomic library of Lactobacillus delbrueckii subsp. lactis (DSM7290) DNA, in the low-copy-number vector pLG339, a recombinant clone was selected, which complemented a mutation in the prolidase gene (pepQ) of Escherichia coli UK173. Nucleotide sequence analysis revealed an open reading frame of 1104 nucleotides corresponding to a protein of 368 amino acids with a calculated pI of 4.64 and a molecular mass of 41087 Da. The start site of pepQ transcription was determined by primer extension analysis with mRNA prepared from L. delbrueckii. Based on homology of the gene product to various peptidases and on the substrate specificity determined, the peptidase was designated PepQ. The influence of various protease inhibitors and cations on peptidase activity indicated that PepQ is a metalloprotease. The absence of a membrane-spanning domain and a signal peptide sequence argues for a cytoplasmic localization of the enzyme.
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References
Ben-Bassat A, Bauer K, Chang SY, Myambo K, Boosman A, Chang S (1987) Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure. J Bacteriol 169:751–757
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Bouchez D, Tourneur J (1991) Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes. Plasmid 25:27–39
Butler MJ, Bergeron A, Soostmeyer G, Zimny T, Malek LT (1993) Cloning and characterization of an aminopeptidase P-encoding gene from Streptomyces lividans. Gene 123:115–119
Casey MG, Meyer J (1985) Presence of X-prolyl-dipeptidel-peptidase in lactic acid bacteria. J Dairy Sci 58:3212–3215
Daniels DL, Plunkett G III, Burland V, Blattner FR (1992) Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science 257:771–778
DeFelice MJ, Guardiola A, Lamberti A, Iaccarino M (1973) Escherichia coli K-12 mutants altered in the transport system for oligo- and dipeptides. J Bacteriol 116:751–756
Dunn BM (1989) Proteolytic enzymes. A practical approach. IRL Press, Oxford
El Soda M (1993) The role of lactic acid bacteria in accelerated cheese ripening. FEMS Microbiol Rev 12:239–252
Endo F, Tanoue A, Nakai H, Hata A, Indo Y, TitaniK, Matsuda I (1989) Primary structure and gene localization of the human prolidase. J Biol Chem 264:4476–4481
Feng DF, Doolittle RF (1987) Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol 25:351–360
Jongeneel CV, Bouvier J, Bairoch A (1989) A unique signature identifies a family of zinc-dependent metallopeptidases. FEBS Lett 242:211–214
Kleckner N, Barker DF, Ross DG, Botstein D (1978) Properties of the translocatable tetracycline-resistance element Tn10 in Escherichia coli and bacteriophage lambda. Genetics 90:427–461
Klein JR, Klein U, Schad M, Plapp R (1993) Cloning, DNA sequence analysis and partial characterization of pepN, a lysyl aminopeptidase from Lactobacillus delbrueckii subsp. lactis DSM7290. Eur J Biochem 217:105–114
Klein JR, Schmidt U, Plapp R (1994) Cloning, heterologous expression, and sequencing of a novel proline iminopeptidase gene, pepI, from Lactobacillus delbrueckii subsp. lactis DSM7290. Microbiology 140:1133–1139
Larsen KS, Auld DS (1988) The mechanism of zinc inhibition of bovine carboxypeptidase A. FASEB J 2:A1114
Lengeler J (1979) Streptozotocin, an antibiotic superior to penicillin in the selection of rare bacterial mutations. FEMS Microbiol Lett 5:417–419
Matern HT, Klein JR, Henrich B, Plapp R (1994) Determination and comparison of Lactobacillus delbrückii subsp. lactis DSM7290 promoter sequences. FEMS Microbiol Lett 122:121–128
McKay L, Baldwin K (1990) Applications for biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbiol Rev 87:3–14
Meyer-Barton EC, Klein JR, Imam M, Plapp R (1993) Cloning and sequence analysis of the X-prolyl-dipeptidyl-aminopeptidase gene (pepX) from Lactobacillus delbrueckii subsp. lactis DSM7290. Appl Microbiol Biotechnol 40:82–89
Miller CG, Schwartz G (1978) Peptidase deficient mutants of Escherichia coli. J Bacteriol 135:603–611
Miller CG, Mackinnon K (1974) Peptidase mutants of Salmonella typhimurium. J Bacteriol 120:355–365
Movva NR, Semon D, Meyer C, Kawashima E, Wingfield P, Miller JL, Miller CG (1990) Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene. Mol Gen Genet 223:345–348
Nakamura K, Nakamura A, Takamatsu H, Yoshikawa H, Yamane K (1990) Cloning and characterization of a Bacillus subtilis gene homologous to Escherichia coli secY. J Biochem 107:603–607
Rao MJK, Argos P (1986) A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta 869:197–214
Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353
Smid EJ, Poolman B, Konings WN (1991) Casein utilization by lactococci. Appl Environ Microbiol 57:2447–2452
Stoker NG, Fairweather NF, Spratt BG (1982) Versatile low copy number plasmid vectors for cloning in Escherichia coli. Gene 18:335–341
Sugiura M, Ito Y, Hirano K, Sawaki S (1977) Detection of dipeptidase and tripeptidase activities on polyacrylamide gel and cellulose acetate gel by reduction of tetrazolium salts. Anal Biochem 81:481–484
Tatum EL (1946) Biochemical mutations in bacteria. Cold Spring Harbor Symp Quant Biol 11:278–283
Tinoco I, Borer PN, Dengler B, Levine MD, Uhlenbeck OC, Crothers DM, Gralla J (1973) Improved estimation of secondary structure in ribonucleic acids. Nature New Biol 246:40–41
Van der Vossen JMBM, Van der Lelie D, Venema G (1987) Isolation and characterization of Streptococcus cremoris WG2-specific promoters. Appl Environ Microbiol 53:2452–2457
Vongerichten KF, Klein JR, Matern H, Plapp R (1994) Cloning and DNA sequence analysis of pepV, a carnosinase gene from Lactobacillus delbrueckii subsp. lactis DSM7290 and partial characterization of the enzyme. Microbiology 140:2591–2600
Von Heijne G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690
Weiss N, Schillinger U, Kandler O (1983) Lactobacillus lactis, Lactobacillus leichmanii and Lactobacillus bulgaricus, subjective synonyms of Lactobacillus delbrueckii subsp. lactis comb. nov. and Lactobacillus delbrueckii subsp. bulgaricus comb. nov. Syst Appl Microbiol 4:552–557
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of M13mp18 and pUC19 vectors. Gene 33:103–119
Yoshimoto T, Tone H, Honda T, Osatomi K, Kobayashi R, Tsuru D (1989) Sequencing and high expression of aminopeptidase P from Escherichia coli HB101. J Biochem 105:412–416
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Communicated by W. Goebel
Prof. Plapp died in November 1994
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Stucky, K., Robert Klein, J., Schüller, A. et al. Cloning and DNA sequence analysis of pepQ, a prolidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290 and partial characterization of its product. Molec. Gen. Genet. 247, 494–500 (1995). https://doi.org/10.1007/BF00293152
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DOI: https://doi.org/10.1007/BF00293152