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

Abstract

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...

Mutational analysis of the early forespore/mother-cell signalling pathway in Bacillus subtilis

“…Because of the lack of specific probes against SpoIIR, the effects of the GV21-AM23 mutation on protein release were tested by fusing the first 30 aa of either the wildtype or the mutated SpoIIR to the soluble domain of levansucrase (lacking the N-terminal signal peptide) (Borchert & Nagarajan, 1991 ;Petit-Glatron et al, 1990). An antiserum specific for levansucrase (kindly supplied by M. F. Petit-Glatron) was used for the detection of the SpoIIR-levansucrase hybrid proteins in culture supernatants or membrane protein extracts.…”

High Incidence of Disorder-Promoting Amino Acids in the Amino Terminus of Mature Proteins in <i>Bacillus subtilis</i>

“…Mutations in the positively charged NH 2 region of the levansucrase signal peptide have shown that the secretion efficiency became slow when the 3 positive charges were reduced to 2 and the signal peptide was no longer processed when the charges were reduced to zero (Borchert and Nagarajan, 1991) and we showed the NH 2 -terminal regions of the mature domains had contrary charged residues to NH 2 -terminus of the signal peptide. These observations may implicate that the negatively charged residues in the NH 2 -terminal regions of the mature domains interacted with the positively charged N-terminus of the signal peptide.…”

Escherichia coli signal peptides direct inefficient secretion of an outer membrane protein (OmpA) and periplasmic proteins (maltose-binding protein, ribose-binding protein, and alkaline phosphatase) in Bacillus subtilis

“…At various times after initiation of chase, 1-ml samples were placed in 0.5 ml of ice-cold 15% trichloroacetic acid. Processing of radiolabeled cells for immunoprecipitation has been described elsewhere (4,39). Rabbit polyclonal antisera specific for MBP, OmpA, PhoA, and RBP were a generous gift from P. J. Bassford, Jr. Immunoprecipitates were resolved by SDS-PAGE (10% acrylamide-0.27% bisacrylamide gel) and visualized by fluorography of gels which had been soaked in an autoradiography enhancer (Entensify; DuPont) and dried.…”