Abstract
Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.
Similar content being viewed by others
References
Aslandis C, Schmid K, Schmitt R (1989) Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli. J. Bacteriol 171:6753–6763
Bockmann J, Heuel H, Lengeler JW (1992) Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132. Mol Gen Genet 235:22–32
Brass JM, Manson MD, Larson TJ (1984) Transposon Tn 10-dependent expression of the lamB gene in Escherichia coli. J Bacteriol 159:93–99
Curtliss R III, Jagueztyn-Krynicka EK, Hansen JB, Smorawinska M, Abiko Y, Cardineau G (1982) In: Mitsuhashis (ed) Microbial drug resistance, vol 3. Scientific Societies, Tokyo, 1115–27
Death A, Notley L, Ferenci T (1993) Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress. J Bacteriol 175:1475–1483
Diderichsen B. Poulsen GB, Jorgensen PL (1991) Cloning and expression of an amylase gene from Bacillus stearothermophilus. Res Microbiol 142:793–796
Feng DF, Doolittle RF (1987) Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol 25:351–360
Ferenci T, Lee KS (1986) Temperature-sensitive binding of α-glucans by Bacillus stearothermophilus. J Bacteriol 166:95–99
Ferenci T, Stretton S (1989) Cysteine-22 and cysteine-38 are not essential for the functions of maltoporin (LamB protein). FEMS Microbiol Lett 61:335–340
French BA, Valdez BC, Younathan ES, Chang SH (1987) High-level expression of Bacillus stearothermophilus 6-phosphofructo-1-kinase in Escherichia coli. Gene 59:279–83
Gilson E, Alloing G, Schmidt T, Claverys J-P, Dudler R, Hofnung M (1988) Evidence for high affinity binding-protein dependent transport systems in gram-positive bacteria and in Mycoplasma. EMBO J 7:3971–3974
Gough JA, Murray NE (1983) Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol 166:1–19
Gray GL, Mainzer SE, Rey MW, Lamsa MH, Kindle KL, Carmona C, Requadt C (1986) Structural genes encoding the thermophilic α-amylases of Bacillus stearothermophilus and Bacillus lichenoformis. J Bacteriol 166:635–643
Hatfield D, Hofnung M, Schwartz M (1969) Genetic analysis of the maltose A region in Escherichia coli K12. J Bacteriol 89:28–40
Higgins DG, Sharp PM (1988) Clustal: a package for performing multiple alignment on a microcomputer. Gene 73:237–244
Hofnung M, Schwartz M, Hatfield D (1971) Complementation studies in the maltose A region of Escherichia coli K12 genetic map. J Mol Biol 61:681–694
Ihara H, Sasaki T, Tsuboi A, Yanagata H, Tsukagoshi N, Udaka S (1985) Complete nucleotide sequence of thermophilic α-iamylase gene: homology between prokaryotic and eukaryotic α-amylases at the active sites. J Biochem 98:95–103
Inagaki K, Tanizawa K, Badet B, Walsh CT, Tanaka H, Soda K (1986) Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification and characterization. Biochemistry 25:3268–3274
Kaback HR, Bibi E, Roepe PD (1990) β-galactosidase transport in Escherichia coli: a functional dissection of lac permease. Trends Biochem Sci 15:309–314
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Lengeler JW, Bockmann J, Heuel H, Titgemeyer F (1992) The enzymes II of the PTS as carbohydrate transport systems: what the evolutionary studies tell us on their structure and function. In: Quagliariello E, Palmieri F (eds) Molecular mechanisms of transport. Elsevier, pp 77–85
Liu ML, Kondo JK, Barnes MB, Bartholomew DT (1988) Plasmid-linked maltose utilization in Lactobacillus ssp. Biochimie 70:351–356
Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Marger MD, Saier MH Jr (1993) A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci 18:13–20
Marmur J (1961) A procedure for the isolation of deoxribonucleic acid from microorganisms. J Mol Biol 3:208–218
Mead DA, Szczesna-Skorupa E, Kemper B (1986) Single-stranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng 1:67–74
Mielenz JR (1983) Bacillus stearothermophilus containing a plasmid borne gene for α-amylase. Proc Natl Acad Sci USA 80:5975–5979
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Murray NE, Brammar WJ, Murray K (1977) Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet 150:53–61
Papavinasasundaram KG (1989) Maltose-inducd functions in B. stearothermophilus ATCC7953: properties, regulation and genetic instability. Ph D thesis. Sydney University, Sydney, Australia
Pearson WR (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol 183:63–98
Pfueller SL, Elliott WH (1969) The extracellular α-amylase of Bacillus stearothermophilus. J Biol Chem 244:48–54
Prentki P, Karch F, Iida S, Meyer J (1981) The plasmid cloning vector pBR325 containing a 482 base pair long inverted duplication. Gene 14:289–299
Puyet A, Espinosa M (1993) Structure of the maltodextrin-uptake locus of Streptococcus pneumoniae. Correlation to the Escherichia coli maltose regulon. J Mol Biol 230:800–811
Reidl J, Boos W (1991) The malXmalY operon of Escherichia coli encodes a novel enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system. J Bacteriol 173:4862–4876
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schwartz M (1987) The maltose regulon. In: Neidhart FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (1987) Escherichia coli and Salmonella typhimurium Cellular and molecular biology. American Society for Microbiology, pp 1482–1502
Sen S, Oriel P (1989) Multiple amylase genes in two strains of B. stearothermophilus. Gene 76:137–144
Shuman HA (1982) Active transport of maltose in Escherichia coli K12. J Biol Chem 257:5455–5461
Staden R (1990) Searching for protein patterns in protein and nucleic acid sequences. Methods Enzymol 183:193–211
Stassi DL, Dunn JJ, Lacks SA (1982) Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae. Gene 20:359–366
Sutcliffe JG (1979) Complete nucleotide sequence of the E. coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–90
Szmeleman S, Hofnung M (1975) Maltose transport in Escherichia coli K-12. Involvement of the bacteriophage lambda receptor. J Bacteriol 124:112–118
Tangney M, Buchanan CJ, Priest FG, Mitchell WJ (1992a) Maltose uptake and its regulation in Bacillus subtilis. FEMS Microbiol Lett 97:191–196
Tangney M, Smith P, Priest FG, Mitchell WJ (1992b) Maltose transport in Bacillus licheniformis NCIB 6346. J Gen Microbiol 138:1821–1827
Taylor DC, Costilow RN (1977) Uptake of glucose and maltose by Bacillus popillae. Appl Environ Microbiol 34:102–104
Wada K, Wada Y, Doi H, Ishibashi F, Gotobori T, Ikemura T (1991) Codon usage tabulated from Genbank genetic sequence data. Nucleic Acids Res 19:1981–1992
Wursch P, Koellreutter B (1985) Maltotriitol inhibition of maltose metabolism in Streptococcus mutans via maltose transport, amylomaltase and phospho-α-glucosidase activities. Caries Res 19:439–449
Zagursky RJ (1985) pBR322 plasmids that yield single-stranded DNA. BRL Focus 7:4–6
Author information
Authors and Affiliations
Additional information
Communicated by J. Lengeler
Rights and permissions
About this article
Cite this article
Liong, E.C., Ferenci, T. Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12. Molec. Gen. Genet. 243, 343–352 (1994). https://doi.org/10.1007/BF00301070
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00301070