Advertisement

Molecular and General Genetics MGG

, Volume 245, Issue 1, pp 117–125 | Cite as

Promotor analysis and transcriptional regulation of Lactobacillus pentosus genes involved in xylose catabolism

  • B. Christien Lokman
  • Rob J. Leer
  • Renée van Sorge
  • Peter H. Pouwels
Original Paper

Abstract

The xyl genes in Lactobacillus pentosus are induced by xylose and repressed by glucose, ribose, and arabinose. Northern blot analysis showed that regulation is mediated at the transcriptional level. Under inducing conditions, two xylA transcripts were detected, a major transcript of 1·5 kb and a minor transcript of 3 kb. The 3 kb transcript also comprises sequences from xylB, suggesting that xylA and xylB are transcribed together. A 1·2 kb xylR transcript was found under inducing and non-inducing conditions. In the presence of xylose, a second xylR transcript (>7 kb) was detected, which includes sequences from two upstream genes, xylQ and xylP. The transcription start sites for xylA and xylR were mapped by primer extension and S1 nuclease experiments at 42 and 83 nucleotides, respectively upstream of the translation start sites. Induction by xylose of the chloramphenicol acetyltransferase (CAT) gene under control of the xylA promoter, on a multicopy plasmid, was 60 to 80-fold, but only 3 to 10-fold in the presence of glucose and xylose. Expression of CAT under control of the xylR promoter was constitutive at a level tenfold less than that observed under control of the xylA promoter. Sequence analysis suggests the presence of two operator-like elements, one overlapping with the promoter — 35 region of xylA and controlling the expression of xylA by binding factors involved in catabolite repression, and a second operator downstream of the promoter — 10 region of xylA, which may bind the product of xylR, the repressor. Titration experiments with multiple copies of these elements showed that, under inducing conditions, expression of xylA in wild-type L. pentosus is suboptimal.

Key words

Lactobacillus pentosus Regulation of transcription Promoter analysis Glucose repression cis elements 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Amster-Choder O, Houman F, Wright A (1989) Protein phosphorylation regulates transcription of the β-glucoside utilization operon in E. coli. Cell 58:847–855Google Scholar
  2. Aslanidis C, Schmitt R (1990) Regulatory elements of the raffinose operon: nucleotide sequences of operator and repressor genes. J Bacteriol 172:2178–2180Google Scholar
  3. Belasco JG, Nilsson G, von Gabain A, Cohen SN (1986) The stability of E. coli gene transcripts is dependent on determinants localized to specific segments. Cell 46:245–251Google Scholar
  4. Brückner R (1992) A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene 122:187–192Google Scholar
  5. Davis EO, Henderson PJF (1987) The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli K12. J Biol Chem 262:13928–13932Google Scholar
  6. deMan JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23:130–135Google Scholar
  7. Dodd IB, Egan JB (1990) Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. Nucleic Acids Res 18:5019–5026Google Scholar
  8. Friedman PJ, Imperiale MJ, Adhya SL (1987) RNA 3′-end formation in the control of gene expression. Annu Rev Genet 21:453–488Google Scholar
  9. Fujita Y, Fujita T (1987) The gluconate operon gnt of Bacillus subtilis encodes its own transcriptional negative regulator. Proc Natl Acad Sci USA 84:4524–4528Google Scholar
  10. Gärtner D, Geissendörfer M, Hillen W (1988) Expression of the Bacillus subtilis xyl operon is repressed at the level of transcription and is induced by xylose. J Bacteriol 170:3102–3109Google Scholar
  11. Gärtner D, Degenkolb J, Ripperger JAE, Allmansberger R, Hillen W (1992) Regulation of the Bacillus subtilis W23 xylose utilization operon: interaction of the Xyl repressor with the xyl operator and the inducer xylose. Mol Gen Genet 232:415–422Google Scholar
  12. Graves MC, Rabinowitz JC (1986) In vivo and in vitro transcription of the Clostridium pasteuranicum ferredoxin gene. J Biol Chem 261:11409–11415Google Scholar
  13. Harley CB, Reynolds RP (1987) Analysis of E. coli promoter sequences. Nucleic Acids Res 15:2343–2361Google Scholar
  14. Hastrup S (1988) Analysis of the Bacillus subtilis xylose regulon. In: Ganesan AT, Hoch JA (eds) Genetics and biotechnology of bacilli, vol 2, Academic Press, New York, pp 79–83Google Scholar
  15. Jacob S, Allmansberger R, Gärtner D, Hillen W (1991) Catabolite repression of the operon for xylose utilization from Bacillus subtilis W23 is mediated at the level of transcription and depends on a cis site in the xylA reading frame. Mol Gen Genet 229:189–196Google Scholar
  16. Jahreis K, Postma PW, Lengeler JW (1991) Nucleotide sequence of the ilvH-fruR gene region of Escherichia coli and Salmonella typhimurium LT2. Mol Gen Genet 226:332–336Google Scholar
  17. Kreuzer P, Gärtner D, Allmansberger R, Hillen W (1989) Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator. J Bacteriol 171:3840–3845Google Scholar
  18. Leer RJ, van Luijk N, Posno M, Pouwels PH (1992) Structural and functional analysis of two cryptic plasmids from Lactobacillus plantarum ATCC 8014. Mol Gen Genet 234:265–274Google Scholar
  19. Leong-Morgenthaler P, Zwahlen MC, Hottinger H (1991) Lactose metabolism in Lactobacillus bulgaricus: Analysis of the primary structure and expression of the genes involved. J Bacteriol 173:1951–1957Google Scholar
  20. Lokman BC, van Santen P, Verdoes JC, Krüse J, Leer RJ, Posno M, Pouwels PH (1991) Organization and characterization of three genes involved in d-xylose catabolism in Lactobacillus pentosus. Mol Gen Genet 230:161–169Google Scholar
  21. Malezka R, Wang PY, Schneider H (1982) A ColEl hybrid plasmid containing Escherichia coli genes complementing d-xylose negative mutants of Escherichia coli and Salmonella typhimurium. Can J Biochem 60:144–151Google Scholar
  22. Melin L, Fridén H, Dehlin E, Rutberg L, von Gabain A (1990) The importance of the 5′-region in regulating the stability of sdh mRNA in Bacillus subtilis. Mol Microbiol 4:1881–1889Google Scholar
  23. Newbury SF, Smith NH, Robinson EC, Hiles ID, Higgins CF (1987) Stabilization of translationally active mRNA by prokaryotic REP sequences. Cell 48:297–310Google Scholar
  24. Platt T (1986) Transcription termination and regulation of gene expression. Annu Rev Biochem 55:339–372Google Scholar
  25. Plumbridge JA (1989) Sequence of the nagBACD opereon in Escherichia coli K12 and pattern of transcription within the nag operon. Mol Microbiol 3:505–515Google Scholar
  26. Poolman B, Royer TJ, Mainzer SE, Schmidt BF (1989) Lactose transport system of Streptococcus thermophilus: a hybrid protein with homology to the melibiose carrier and enzyme III of phophoenolpyruvate-dependent phosphotransferase systems. J Bacteriol 171:244–253Google Scholar
  27. Posno M, Leer RJ, van Luijk N, van Giezen MJF, Heuvelmans PTHM, Lokman BC, Pouwels PH (1991a) Incompatibility of Lactobacillus vectors with replicons derived from small cryptic Lactobacillus plasmids and segregational instability of the introduced vectors. Appl Environ Microbiol 57:1822–1828Google Scholar
  28. Posno M, Heuvelmans PTHM, van Giezen MJF, Lokman BC, Leer RJ, Pouwels PH (1991b) Complementation of the inability of Lactobacillus strains to utilize d-xylose with d-xylose catabolism-encoding genes of Lactobacillus pentosus. Appl Environ Micro biol 57:2764–2766Google Scholar
  29. Pouwels PH, van Luijk N, Leer RJ, Posno M (1994) Control of replication of the Lactobacillus pentosus plasmid p353-2: evidence for a mechanism involving transcriptional attenuation of the gene coding for the replication protein. Mol Gen Genet 242:614–622Google Scholar
  30. Rygus T, Hillen W (1992) Catabolite repression of the xyl operon in Bacillus megaterium. J Bacteriol 174:3049–3055Google Scholar
  31. Rygus T, Scheler A, Allmansberger R, Hillen W (1991) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. Arch Microbiol 155:535–542Google Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  33. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  34. Sauer RT, Pabo CO (1984) Protein-DNA recognition. Annu Rev Biochem 53:293–321Google Scholar
  35. Scheler A, Rygus T, Allmansberhger R, Hillen W (1991) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus licheniformis encoded regulon for xylose utilization. Arch Microbiol 155:526–534Google Scholar
  36. Schleif R (1987) The l-arabinose operon. Cell Mol Biol 2:1473–1481Google Scholar
  37. Shaw WV (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol 43:737–755Google Scholar
  38. Sizemore C, Buchner E, Rygus T, Witke C, Götz F, Hillen W (1991) Organization, promoter analysis and transcriptional regulation of the Staphylococcus xylosus xylose utilization operon. Mol Gen Genet 227:377–384Google Scholar
  39. Sizemore C, Wieland B, Götz F, Hillen W (1992) Regulation of Staphylococcus xylosus xylose utilization genes at the molecular level. J Bacteriol 174:3042–3048Google Scholar
  40. Stern MJ, Ames GFL, Smith NH, Robinson EC, Higgins CF (1984) Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell 37:1015–1026Google Scholar
  41. Stevis PE, HO NWY (1987) Positive selection vectors based on xylose utilization suppression. Gene 55:67–74Google Scholar
  42. Sumiya M, Henderson PJF (1989) The d-xylose binding protein of Escherichia coli. Biochem Soc Trans 17:553–554Google Scholar
  43. Weickert MJ, Chambliss GH (1990) Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis. Proc Natl Acad Sci USA 87:6238–6242Google Scholar
  44. Yazyu H, Shiota-Niiya S, Shimamoto T, Kanazawa H, Futai M, Tsuchiya T (1984) Nucleotide sequence of the melB gene and characterization of deduced amino acid sequence of the melibiose carrier in Escherichia coli. J Biol Chem 259:4320–4326Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • B. Christien Lokman
    • 1
  • Rob J. Leer
    • 1
  • Renée van Sorge
    • 1
  • Peter H. Pouwels
    • 1
  1. 1.Department of Molecular Genetics and GenetechnologyTNO Nutrition and Food ResearchHV RijswijkThe Netherlands

Personalised recommendations