Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens

Tang, Leslie B.; Lenstra, Reijer; Borchert, Torben V.; Nagarajan, Vasantha

doi:10.1016/0378-1119(90)90345-r

Cited by 52 publications

(30 citation statements)

References 13 publications

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“…B. subtilis BG4103 carrying pBE504 was grown in synthetic medium, and cells were induced with 2% sucrose for 30 min when the OD600 reached 0. IZZI (A [13-19J) i T29 V29 Q29 S29 P29 E29 W29 T30 V30 G30 A30 L30 E30 R30 W30 C30 H30 F30 D30 GTA (27), TTT (28) GGC (27), TTT (28) CCT (27), TTT (28) GAG (27), TTT (28) CGC (27), TTT (28) TTG (27), TTT (28) ACG (29) GTT (29) CAG (29) TCG (29) CCG (29) GAG (29) TGG (29) ACA (30) GTG (30) GGC (30) GCG (30) CTC (30) GAA (30) CGT (30) GCT (29), TGG (30) TGC (30) CAT (30) TTT (30) GAC (30) a All mutants were derived from pBE504 ( Fig. 1) except for 1415 and 1415N8, which were derived from the D415 template.…”

Section: Resultsmentioning

confidence: 99%

“…subtilis shuttle vector constructed by ligating HindIII digested pTZ18R (Pharmacia) and pC194. The EcoRV-XbaI fragment containing sacB-[BamP] from pBE301 (27) was subcloned into SmaI-XbaIdigested pBE20, resulting in pBE501.…”

Section: Methodsmentioning

confidence: 99%

“…B. subtilis was grown in medium B, and when the culture had reached an OD6. of 0.5, a 10-ml culture was induced with 2% sucrose for 2 h. The levansucrase in the extracellular medium was partially purified and assayed (27). In order to account for the variability in the medium and the reagents, the wild-type strain was grown along with the mutants.…”

Section: Methodsmentioning

confidence: 99%

“…(i) The levansucrase gene is inducible in B. subtilis, and thus studies of signal sequence mutations that may be lethal to B. subtilis should be feasible (27). (ii) The conversion of levansucrase precursor to mature protein involves a single processing event (signal peptide cleavage); this is in contrast to the multiple processing events involved in the conversion of precursor to mature protein for several Bacillus secreted proteins (protease and RNase) due to the presence of a propeptide between the signal peptide and mature protein (19,29).…”

mentioning

confidence: 99%

“…We earlier reported the isolation of the levansucrase gene from B. amyloliquefaciens (sacB [BamP]) and its sucroseinducible expression in B. subtilis (27). The levansucrase signal peptide is 29 amino acids long and has three positively charged residues at the N-terminus, followed by the hydrophobic core, comprising a stretch of 13 amino acids.…”

mentioning

confidence: 99%

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Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis

Borchert

Nagarajan

1991

J Bacteriol

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A series of alterations in the Bacillus amyloliquefaciens levansucrase signal peptide were made by in vitro mutagenesis, and their effect on the secretion of levansucrase in Bacillus subtilis was studied. Some of the alterations resulted in a completely defective signal peptide. These included the removal of positively charged residues from the N-terminus and disruption of the hydrophobic core of the signal peptide either by introducing a charged residue or by deleting five or more amino acids. Analysis of the signal peptide processing-site alterations revealed that small residues are preferred at the -1 and -3 positions. However, a wide variety of amino acids are tolerated at the + 1 position.Most of our current knowledge on protein secretion in bacteria is based on studies on Escherichia coli. Signal sequence mutants of E. coli have aided both in understanding the role of signal sequences in the secretion process and in identifying E. coli genes involved in protein secretion (1,3,5,25). The mechanism of protein secretion in Bacillus subtilis has not been extensively studied, and few signal sequence mutants of B. subtilis have been reported (18,21). We were interested in determining whether signal sequence mutants of B. subtilis will have the same phenotype as analogous mutants of E. coli. The Bacillus signal peptides have some of the common features observed in signal peptides from both procaryotes and eucaryotes (22,28,30). The signal peptides of Bacillus secreted proteins are 28 to 34 residues long; the number of positively charged residues at the N-terminus varies from two to five, and the hydrophobic core consists of 12 to 18 residues. The signal peptide processing site is between two alanine residues in the majority of cases.Bacillus amyloliquefaciens levansucrase signal peptide was chosen as a model Bacillus signal peptide for structurefunction studies for several reasons. (i) The levansucrase gene is inducible in B. subtilis, and thus studies of signal sequence mutations that may be lethal to B. subtilis should be feasible (27). (ii) The conversion of levansucrase precursor to mature protein involves a single processing event (signal peptide cleavage); this is in contrast to the multiple processing events involved in the conversion of precursor to mature protein for several Bacillus secreted proteins (protease and RNase) due to the presence of a propeptide between the signal peptide and mature protein (19,29). Thus, construction of levansucrase signal peptide processing site mutants is feasible. (iii) A genetic system to isolate suppressors of signal sequence mutants based on the secretion of levansucrase can be developed.We earlier reported the isolation of the levansucrase gene from B. amyloliquefaciens (sacB [BamP]) and its sucroseinducible expression in B. subtilis (27). The levansucrase signal peptide is 29 amino acids long and has three positively * Corresponding author. t Present address: Department of Microbiology, The Technical University of Denmark, Bygning 221, DK2800 Lyngby, Denmark. charged...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis

Borchert

Nagarajan

1991

J Bacteriol

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Structural characterization ofAcetobacter diazotropicus levansucrase by matrix-assisted laser desorption/ionization mass spectrometry: identification of an N-terminal blocking group and a free-thiol cysteine residue

Betancourt¹,

Takao²,

Hernández³

et al. 1999

J. Mass Spectrom.

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Construction of Bacteriophage P1 Libraries with Large Inserts

Sternberg

Shepherd

1994

CP Human Genetics

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The bacteriophage P1 cloning system was originally developed as an alternative to YAC and cosmid systems for cloning high-molecular-weight genomic DNA. This unit details the preparation of the bacteriophage P1 library. Three support protocols provide the raw materials for the basic procedure, including the vector (pAd10sacBII), the mammalian DNA inserts, and the two packaging extracts that contain the viral proteins necessary to construct a P1 bacteriophage incorporating the vector and insert. A fourth support protocol describes how to induce replication of the plasmids cloned in the basic protocol, isolate the cloned DNA, and analyze the final products.

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Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens

Cited by 52 publications

References 13 publications

Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis

Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis

Structural characterization ofAcetobacter diazotropicus levansucrase by matrix-assisted laser desorption/ionization mass spectrometry: identification of an N-terminal blocking group and a free-thiol cysteine residue

Construction of Bacteriophage P1 Libraries with Large Inserts

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