Abstract
A gene (brnQ), encoding a carrier for branched-chain amino acids in Lactobacillus delbrückii subsp. lactis DSM7290 was cloned in the low-copy-number vector pLG339 by complementation of a transport-deficient Escherichia coli strain. The plasmid carrying the cloned gene restored growth of an E. coli strain mutated in 4 different branched-chain amino acid transport genes at low concentrations of isoleucine, and increased its sensitivity to valine. Transport assays showed that leucine, isoleucine and valine are transported by this carrier and that transport is driven by the proton motive force. Nucleotide sequence analysis revealed an open reading frame of 1338 bp encoding a hydrophobic protein of 446 amino acids with a calculated molecular mass of 47864 Daltons. The start site of brnQ transcription was determined by primer extension analysis using mRNA from Lactobacillus delbrückii subsp. lactis DSM7290. The hydropathy profile suggests the existence of at least 12 hydrophobic domains that probably form membrane-associated α-helices. Comparisons of the nucleotide sequence of brnQ from Lactobacillus delbrückii subsp. lactis DSM7290, the amino acid sequence of its product and the topology of the hydrophobic domains with those of the respective carrier genes and proteins of Salmonella typhimurium and Pseudomonas aeruginosa revealed extensive homology.
Similar content being viewed by others
References
Adams MD, Wagner LM, Graddis TJ, Landick R, Antorucci TK, Gibson AL, Oxender DL (1990) Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of E. coli. J Biol Chem 265:11436–11443
Atlan D, Gilbert C, Blanc B, Portalier R (1994) Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrückii subsp. bulgaricus CNRZ 397. Microbiology 140:527–525
Bertani G (1951) Studies of lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Büchel DE, Gronenborn B, Müller-Hill B (1980) Sequence of the lactose permease gene. Nature 283:541–545
Chapot-Chartier MP, Nardi M, Chopin A, Gripon JC (1993) Cloning of pepC, a cysteine aminopeptidase from Lactococcus lactis subsp. cremoris AM2. Appl Env Microbiol 59:330–333
Davis BD, Mingioli ES (1950) Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol 60:17–28
De Crombrugghe B, Busby S, Buc H (1984) Cyclic AMP receptor protein: role in gene activation. Science 224:831–838
De Man JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of Lactobacilli. J Appl Bacteriol 23:130–135
Dower JW, Miller JW, Ragsdale W (1988) High efficiency transformation of Escherichia coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145
Eisenberg D, Weiss R, Terwilliger TC (1984) The hydrophobic moment detects periodicity in protein hydrophobicity. Proc Nat Acad Sci USA 81:140–144
Fernández L, Bhowmik T, Steele JL (1994) Characterization of the Lactobacillus helveticus CNRZ32 pepC gene. Appl Env Microbiol 60:333–336
Filip C, Fletcher G, Wulff JL, Earhart CK (1973) Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium lauryl sarcosinate. J Bacteriol 115:712–722
Freier SM, Kierzek R, Jaeger JA, Sugimoto N, Caruthers MH, Neilson T, Turner DH (1986) Improved free energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci USA 83:9373–9377
Hagting A, Kunji ERS, Leenhouts JJ, Poolman B, Konings WN (1994) The di- and tripeptide transport protein of Lactococcus lactis. J Biol Chem 269:11391–11399
Hawley DK, McClure WR (1983) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res 8:2237–2255
Henrich B, Monnerjahn U, Plapp R (1990) Peptidase D (pepD) of Escherichia coli K-12: nucleotide sequence, transcript mapping, and comparison with other peptidase genes. J Bacteriol 172:4641–4651
Holck A, Naes H (1992) Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracaseii subsp. paracaseii NCDO151. J Gen Microbiol 138:1352–1364
Hoshino T, Kose K, Uratani Y (1990) Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO. Mol Gen Genet 220:461–467
Hoshino T, Kose-Terai K, Uratani Y (1991) Isolation of the braZ gene encoding for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO. J Bacteriol 173:1855–1861
Klein P, Kanehisa M, DeLisi C (1985) The detection and classification of membrane-spanning proteins. Biochim Biophys Acta 869:197–214
Klein JR, Henrich B, Plapp R (1986) Cloning and expression of the pepD gene of Escherichia coli. J Gen Microbiol 132:2337–2343
Klein JR, Klein U, Schad M, Plapp R (1993) Cloning, DNA sequence analysis and partial characterization of pepN, a lysyl aminopeptidase from Lactobacillus delbrückii subsp. lactis DSM7290. Eur J Biochem 217:105–114
Klein JR, Schmidt U, Plapp R (1994a) Cloning, heterologous expression, and sequencing of a novel proline iminopeptidase gene, pepI, from Lactobacillus delbrückii subsp. lactis DSM7290. Mirobiology 140:1133–1139
Klein JR, Henrich B, Plapp R (1994b) Cloning and nucleotide sequence of Lactobacillus delbrückii subsp. lactis DSM7290 cysteine aminopeptidase gene pepC. FEMS Microbiol Lett 124:291–300
Klein JR, Dick A, Henrich B, Plapp R (1995) Molecular cloning and DNA sequence analysis of pepI, a leucyl aminopeptidase gene from Lactobacillus delbrückii subsp. lactis DSM7290. Eur J Biochem 228:570–578
Konings WN, Poolman B, Driessen AJM (1989) Bioenergetics and solute transport in lactococci. CRC Crit Rev Microbiol 16:419–476
Kyte J, Doolittle RF (1982) A simple method for displaying the hydrophobic character of protein. J Mol Biol 157:105–132
Laemmli UK (1970) Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227: 680–685
Law J, Vos P, Hayes F, Daly C, De Vos WM, Fitzgerald G (1992) Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317. J General Microbiol 138:709–718
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
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
Mayo B, Kok J, Venema K, Bockelmann W, Teuber M, Reinke H, Venema G (1991) Molecular cloning and sequence analysis of the X-prolyl-dipeptidyl-aminopeptidase gene from Lactococcus lactis subsp. cremoris. Appl Env Microbiol 57:38–44
Meyer-Barton EC, Klein JR, Imam M, Plapp R (1993) Cloning and sequence analysis of the X-prolyl-dipeptidyl-aminopeptidase gene (pepX) from Lactobacillus delbrückii 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
Nardi M, Chopin MC, Chopin A, Cals MM, Gripon JC (1991) Cloning and DNA sequence analysis of an X-prolyl-dipeptidylaminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763. Appl Environ Microbiol 57:45–50
Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106
Ohnishi K, Hasegawa A, Matsubara K, Date T, Okada T, Kiritani K (1988) Cloning and nucleotide sequence of the brnQ gene, the structural gene for a membrane-associated component of the LIVII transport system for branched-chain amino acids in Salmonella typhimurium. Jpn J Genet 63:343–357
Olson ER, Dunyak DS, Jurss LM, Poorman RA (1991) Identification and characterization of dppA, an E coli gene encoding a periplasmic dipeptide transport protein. J Bacteriol 173:234–244
Poolman B, Modderman R, Reizer J (1992) Lactose transport system of Streptococcus thermophilus. J Biol Chem 267:9150–9157
Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Gen 13:319–353
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sankar P, Hutton ME, VanBogelen RA, Clark RL, Neidhardt FC (1993) Expression analysis of cloned chromosomal segments of Escherichia coli. J Bacteriol 75:5145–5152
Sankar P, Hutton ME, VanBogelen RA, Clark RL, Neidthardt FC (1994) Authors' correction. J Bacteriol 176:939
Sarid S, Berger A, Katchalski E (1959) Proline iminopeptidase. J Biol Chem 234:1740–1746
Sarid S, Berger A, Katchalski E (1962) Proline iminopeptidase. J Biol Chem 237:2207–2212
Schröder U (1990) Untersuchungen an der Peptidase D und der Dipeptidyl Carboxypeptidase von Escherichia coli K-12. Ph. D. Thesis, University of Kaiserslautern, Germany
Schüller A (1990) Klonierung and Sequenzierung des Genes der Peptidase Q von Escherichia coli K-12. Ph. D. Thesis, University of Kaiserslautern, Germany
Seiffer D, Klein JR, Plapp R (1993) EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli. FEMS Microbiol Lett 107:175–178
Smid EJ, Poolman B, Konings WN (1991) Casein utilization by Lactococci. Appl Env Microbiol 57:2447–2452
Stoker NG, Fairweather NF, Spratt BG (1982) Versatile low copy number vectors for cloning in Escherichia coli. Gene 18:335–341
Strøman P (1992) Sequence of a gene (lap) encoding a 953 kDa aminopeptidase from Lactococcus lactis subsp. cremoris Wg2. Gene 113:107–112
Stucky K, Klein JR, Schüller A, Matern H, Henrich B, Plapp R (1995) Cloning and DNA sequence analysis of pepQ, a prolidase gene from Lactobacillus delbrückii subsp. lactis DSM7290 and partial characterization of its product. Mol Gen Genet 247:494–500
Studier FW, Moffatt BA (1985) Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes. J Mol Biol 189:113–130
Tan PST, Poolman B, Konings WN (1993) Proteolytic enzymes of Lactococcus lactis. J Dairy Res 60:269–286
Tatum EL (1946) Biochemical mutations in bacteria. Cold Spring Harbour Symp Quant Biol 11:278–283
Timoco 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 264:40–41
Tynkkynen S, Buist G, Kunji E, Kok J, Poolman B, Venema G, Haandrikman A (1993) Genetic and biochemical characterization of the oligopeptide transport system of Lactococcus lactis. J Bacteriol 175:7523–7532
Vongerichten KF, Klein JR, Matern H, Plapp R (1994) Cloning and DNA sequence analysis of pepV, a carnosinase gene from Lactobacillus delbrückii subsp. lactis DSM7290. Microbiology 140:2591–2600
Weber K, Osborn M (1969) The reliability of molecular weight determinations by sodium dodecylsulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412
Weiss N, Schillinger U, Kandler O (1983) Lactobacillus lactis, Lactobacillus leichmannii and Lactobacillus bulgaricus, subjective synonyms of Lactobacillus delbrückii subsp. lactis comb. nov. and Lactobacillus delbrückii subsp. bulgaricus comb. nov. Syst Appl Microbiol 4:552–557
Yamato I, Ohki M, Anraku Y (1979) Genetical and biochemical studies of transport sytems for branched-chain amino acids in Escherichia coli. J Bacteriol 138:24–32
Yamato I, Anraku Y (1980) Genetical and biochemical studies of transport sytems for branched-chain amino acids in Escherichia coli K-12: isolation and properties of mutants defective in leucine repressible transport activities. J Bacteriol 144:36–44
Yanisch-Perron CJ, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103–119
Zuker M, Stiegler P (1980) Optimal computer foldings of large RNA sequences using thermodynamics and auxiliary information. Nuclic Acids Res 9:133–148
Author information
Authors and Affiliations
Additional information
Communicated by W. Goebel
Rights and permissions
About this article
Cite this article
Stucky, K., Hagting, A., Klein, J.R. et al. Cloning and characterization of brnQ, a gene encoding a low-affinity, branched-chain amino acid carrier in Lactobacillus delbrdückii subsp. lactic DSM7290. Molec. Gen. Genet. 249, 682–690 (1995). https://doi.org/10.1007/BF00418038
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00418038