A Proteomic View on Genome-Based Signal Peptide Predictions

Abstract

The availability of complete genome sequences has allowed the prediction of all exported proteins of the corresponding organisms with dedicated algorithms. Even though numerous studies report on genome-based predictions of signal peptides and cell retention signals, they lack a proteomic verification. For example, 180 secretory and 114 lipoprotein signal peptides were predicted recently for the Gram-positive eubacterium Bacillus subtilis. In the present studies, proteomic approaches were used to define the extracellular complement of the B. subtilis secretome. Using different growth conditions and a hyper-secreting mutant, ∼200 extracellular proteins were visualized by two-dimensional (2D) gel electrophoresis, of which 82 were identified by mass spectrometry. These include 41 proteins that have a potential signal peptide with a type I signal peptidase (SPase) cleavage site, and lack a retention signal. Strikingly, the remaining 41 proteins were predicted previously to be cell associated because of the apparent absence of a signal peptide (22), or the presence of specific cell retention signals in addition to an export signal (19). To test the importance of the five type I SPases and the unique lipoprotein-specific SPase of B. subtilis, the extracellular proteome of (multiple) SPase mutants was analyzed. Surprisingly, only the processing of the polytopic membrane protein YfnI was strongly inhibited in Spase I mutants, showing for the first time that a native eubacterial membrane protein is a genuine Spase I substrate. Furthermore, a mutation affecting lipoprotein modification and processing resulted in the shedding of at least 23 (lipo-)proteins into the medium. In conclusion, our observations show that genome-based predictions reflect the actual composition of the extracellular proteome for ∼50%. Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.As soil microorganisms, Bacillus species secrete numerous enzymes, enabling them to degrade a variety of substrates and survive in a complex and continuously changing environment. The best-studied representative of this genus is Bacillus subtilis. The genome sequence of this organism was published in 1997, representing the first complete genome sequence of a Gram-positive eubacterium (Kunst et al. 1997). Since then, much Bacillus-related research has been focused on the functional analysis of the identified genes and their expression (see the Micado and JAFAN databases,

Global secretome analysis identifies novel mediators of bone metastasis

“…While the earliest secretome analyses were performed in bacteria [46], investigations into the mammalian secretome have also become prevalent. The first cancer secretome analysis identified 145 proteins that were differentially expressed between tumorigenic and nontumorigenic pancreatic epithelial cells, and the first www.cell-research.com | Cell Research…”

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

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“…The identification of the 'mature-like' lipoprotein forms indicates that the build-up of lipoprotein precursors in the membranes of lgt and lsp mutant strains could result in alternative processing by other peptidases such as the recently recognized Eep peptidase [36], Lsp (in Lgt mutant backgrounds) [28,29] or type 1 signal peptidases. The release of 'mature-like' forms from some lipoprotein precursors in lgt or lsp deletion mutants has been termed 'shaving', whereas the release of either lipoprotein precursors or mature, lipidated lipoproteins can be considered as 'shedding' [37]. Shaving most likely reflects protein-specific proteolytic cleavage events because N-terminal sequencing of released lipoprotein products, from both mutant and wild-type backgrounds, shows differing cleavage positions with respect to the N-terminal cysteine [2,29,37,38].…”

“…The release of 'mature-like' forms from some lipoprotein precursors in lgt or lsp deletion mutants has been termed 'shaving', whereas the release of either lipoprotein precursors or mature, lipidated lipoproteins can be considered as 'shedding' [37]. Shaving most likely reflects protein-specific proteolytic cleavage events because N-terminal sequencing of released lipoprotein products, from both mutant and wild-type backgrounds, shows differing cleavage positions with respect to the N-terminal cysteine [2,29,37,38].…”

Two-dimensional gel electrophoresis in bacterial proteomics

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