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. 1996 Feb;178(4):1039–1046. doi: 10.1128/jb.178.4.1039-1046.1996

Molecular analysis of the amy gene locus of Thermoanaerobacterium thermosulfurigenes EM1 encoding starch-degrading enzymes and a binding protein-dependent maltose transport system.

K Sahm 1, M Matuschek 1, H Müller 1, W J Mitchell 1, H Bahl 1
PMCID: PMC177763  PMID: 8576036

Abstract

A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the maltose-binding protein of Escherichia coli was cloned and sequenced. It was located in the amy gene region of the chromosome downstream of the pullulanase-encoding amyB gene and upstream of amyDC, encoding membrane components of an ABC transport system, and the alpha-amylase gene amyA. The gene was designated amyE. Analysis of mRNA by Northern (RNA) blotting revealed that expression of the amy gene region is repressed during growth on glucose. Maximum levels of mRNA were detected with maltose as a substrate. An operon which was transcribed in the order amyBEDC was identified. However, an additional transcription start point was found in front of amyE. The amyA gene represented a monocistronic operon. Putative -35 and -10 promoter sites were deduced from the three transcription start sites of the amy gene region, and possible regulatory regions mediating induction by maltose and catabolite repression by glucose were identified by sequence analysis and comparison. The biochemical characterization of maltose uptake in T. thermosulfurigenes EM1 revealed two transport systems with Km values of 7 microM (high affinity) and 400 microM (low affinity). We conclude that the high-affinity system, which is specific for maltose and maltotriose, is a binding-protein-dependent transporter encoded by amyEDC. The gene for the putative ATP-binding protein has not yet been identified, and in contrast to similar systems in other bacteria, it is not located in the immediate vicinity of the chromosome.

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Selected References

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  1. Angerer A., Gaisser S., Braun V. Nucleotide sequences of the sfuA, sfuB, and sfuC genes of Serratia marcescens suggest a periplasmic-binding-protein-dependent iron transport mechanism. J Bacteriol. 1990 Feb;172(2):572–578. doi: 10.1128/jb.172.2.572-578.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bahl H., Burchhardt G., Spreinat A., Haeckel K., Wienecke A., Schmidt B., Antranikian G. alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases. Appl Environ Microbiol. 1991 May;57(5):1554–1559. doi: 10.1128/aem.57.5.1554-1559.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bahl H., Burchhardt G., Wienecke A. Nucleotide sequence of two Clostridium thermosulfurogenes EM1 genes homologous to Escherichia coli genes encoding integral membrane components of binding protein-dependent transport systems. FEMS Microbiol Lett. 1991 Jun 1;65(1):83–87. doi: 10.1016/0378-1097(91)90476-q. [DOI] [PubMed] [Google Scholar]
  4. Berish S. A., Kapczynski D. R., Morse S. A. Nucleotide sequence of the Fbp gene from Neisseria meningitidis. Nucleic Acids Res. 1990 Aug 11;18(15):4596–4596. doi: 10.1093/nar/18.15.4596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coleman R. D., Yang S. S., McAlister M. P. Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis. J Bacteriol. 1987 Sep;169(9):4302–4307. doi: 10.1128/jb.169.9.4302-4307.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cook G. M., Janssen P. H., Morgan H. W. Uncoupler-Resistant Glucose Uptake by the Thermophilic Glycolytic Anaerobe Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum). Appl Environ Microbiol. 1993 Sep;59(9):2984–2990. doi: 10.1128/aem.59.9.2984-2990.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dahl M. K., Francoz E., Saurin W., Boos W., Manson M. D., Hofnung M. Comparison of sequences from the malB regions of Salmonella typhimurium and Enterobacter aerogenes with Escherichia coli K12: a potential new regulatory site in the interoperonic region. Mol Gen Genet. 1989 Aug;218(2):199–207. doi: 10.1007/BF00331269. [DOI] [PubMed] [Google Scholar]
  9. Duplay P., Bedouelle H., Fowler A., Zabin I., Saurin W., Hofnung M. Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12. J Biol Chem. 1984 Aug 25;259(16):10606–10613. [PubMed] [Google Scholar]
  10. Gilson E., Alloing G., Schmidt T., Claverys J. P., Dudler R., Hofnung M. Evidence for high affinity binding-protein dependent transport systems in gram-positive bacteria and in Mycoplasma. EMBO J. 1988 Dec 1;7(12):3971–3974. doi: 10.1002/j.1460-2075.1988.tb03284.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hayashi S., Wu H. C. Lipoproteins in bacteria. J Bioenerg Biomembr. 1990 Jun;22(3):451–471. doi: 10.1007/BF00763177. [DOI] [PubMed] [Google Scholar]
  12. Higgins C. F. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  13. Hueck C. J., Hillen W., Saier M. H., Jr Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria. Res Microbiol. 1994 Sep;145(7):503–518. doi: 10.1016/0923-2508(94)90028-0. [DOI] [PubMed] [Google Scholar]
  14. Hyun H. H., Zeikus J. G. Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum. J Bacteriol. 1985 Dec;164(3):1146–1152. doi: 10.1128/jb.164.3.1146-1152.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kerppola R. E., Ames G. F. Topology of the hydrophobic membrane-bound components of the histidine periplasmic permease. Comparison with other members of the family. J Biol Chem. 1992 Feb 5;267(4):2329–2336. [PubMed] [Google Scholar]
  16. Kimmich G. A., Randles J., Brand J. S. Assay of picomole amounts of ATP, ADP, and AMP using the luciferase enzyme system. Anal Biochem. 1975 Nov;69(1):187–206. doi: 10.1016/0003-2697(75)90580-1. [DOI] [PubMed] [Google Scholar]
  17. Lacks S. A., Dunn J. J., Greenberg B. Identification of base mismatches recognized by the heteroduplex-DNA-repair system of Streptococcus pneumoniae. Cell. 1982 Dec;31(2 Pt 1):327–336. doi: 10.1016/0092-8674(82)90126-x. [DOI] [PubMed] [Google Scholar]
  18. Liebl W., Feil R., Gabelsberger J., Kellermann J., Schleifer K. H. Purification and characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli. Eur J Biochem. 1992 Jul 1;207(1):81–88. doi: 10.1111/j.1432-1033.1992.tb17023.x. [DOI] [PubMed] [Google Scholar]
  19. Matuschek M., Burchhardt G., Sahm K., Bahl H. Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface. J Bacteriol. 1994 Jun;176(11):3295–3302. doi: 10.1128/jb.176.11.3295-3302.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Melasniemi H., Paloheimo M., Hemiö L. Nucleotide sequence of the alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum. J Gen Microbiol. 1990 Mar;136(3):447–454. doi: 10.1099/00221287-136-3-447. [DOI] [PubMed] [Google Scholar]
  21. Melasniemi H. Purification and some properties of the extracellular alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum. Biochem J. 1988 Mar 15;250(3):813–818. doi: 10.1042/bj2500813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ng T. K., Zeikus J. G. Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum. J Bacteriol. 1982 Jun;150(3):1391–1399. doi: 10.1128/jb.150.3.1391-1399.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Overduin P., Boos W., Tommassen J. Nucleotide sequence of the ugp genes of Escherichia coli K-12: homology to the maltose system. Mol Microbiol. 1988 Nov;2(6):767–775. doi: 10.1111/j.1365-2958.1988.tb00088.x. [DOI] [PubMed] [Google Scholar]
  24. Ramesh M. V., Podkovyrov S. M., Lowe S. E., Zeikus J. G. Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli. Appl Environ Microbiol. 1994 Jan;60(1):94–101. doi: 10.1128/aem.60.1.94-101.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Russell R. R., Aduse-Opoku J., Sutcliffe I. C., Tao L., Ferretti J. J. A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism. J Biol Chem. 1992 Mar 5;267(7):4631–4637. [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sutcliffe I. C., Tao L., Ferretti J. J., Russell R. R. MsmE, a lipoprotein involved in sugar transport in Streptococcus mutans. J Bacteriol. 1993 Mar;175(6):1853–1855. doi: 10.1128/jb.175.6.1853-1855.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tam R., Saier M. H., Jr Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria. Microbiol Rev. 1993 Jun;57(2):320–346. doi: 10.1128/mr.57.2.320-346.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  30. von Heijne G. The structure of signal peptides from bacterial lipoproteins. Protein Eng. 1989 May;2(7):531–534. doi: 10.1093/protein/2.7.531. [DOI] [PubMed] [Google Scholar]

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