Summary auto-generated
Researchers isolated and characterized the lsp gene encoding signal peptidase II (SPase II) from Bacillus subtilis. Signal peptidase II is a membrane-bound enzyme that removes signal peptides from lipid-modified proteins during their secretion across the cell membrane. The B. subtilis lsp gene was identified by screening a genomic library for the ability to confer globomycin resistance and complement a temperature-sensitive SPase II mutation in E. coli. The gene encodes a 154-amino acid protein, making it the smallest known SPase II. Sequence analysis revealed four predicted membrane-spanning regions and three conserved domains. The enzyme was confirmed to be functional through pulse-labeling experiments in E. coli. Genomic mapping showed the lsp gene is located near the isoleucyl-tRNA synthetase gene and the pyrimidine biosynthetic cluster at position 139° on the B. subtilis chromosome. Unlike Gram-negative bacteria where lsp is part of a larger operon, in B. subtilis and other Gram-positive bacteria, lsp appears to form an operon with an adjacent gene encoding a hypothetical protein. The identification and characterization of lsp(Bsu) enables future studies on lipoprotein export and protein secretion in B. subtilis.
Key findings
- The B. subtilis signal peptidase II gene was successfully cloned and sequenced, encoding a 154-amino acid protein that is the smallest known SPase II enzyme
- B. subtilis SPase II contains four transmembrane domains and shows functional activity in E. coli, confirming its enzymatic capability
- The lsp gene is organized in an operon with a gene encoding a hypothetical protein, differing from Gram-negative bacteria organization
- Three conserved aspartic acid residues in SPase II suggest these enzymes belong to a novel class of aspartic proteases with catalytic sites in extracytoplasmic loops
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Abstract
The gene encoding the type II signal peptidase (SPase II) of Bacillus subtilis was isolated by screening a genomic DNA library of this bacterium for the ability to increase the levels of globomycin resistance in Escherichia coli, and to complement the growth deficiency at the non-permissive temperature of E. coli strain Y815 carrying a temperature-sensitive mutation in its Isp gene for SPase II. The deduced amino acid sequence of the B. subtilis SPase II showed significant similarity with those of other known SPase II enzymes. Activity of the B. subtilis SPase II was demonstrated by a pulse- labelling experiment in E. coli. In B. subtilis, the Isp gene is flanked by the isoleucyl-tRNA synthetase (ileS) gene and the pyrimidine biosynthetic (pyr) gene cluster, which is known to map at 139 degrees of the chromosome. In the Gram-positive bacteria studied thus far, Isp appears to be the first gene in an operon. The promoter-distal gene ("orf4') of this operon specifies a hypothetical protein in bacteria and yeast.