Biofilm Research Uncovers a Novel Nonenzymatic Signal Peptidase Function in Bacillus

The Gram-positive soil bacterium Bacillus subtilis is highly adaptable to environmental changes and insults. This adaptability is dearly needed since the soil is an ever-changing environment in terms of humidity, temperature, and nutrient availability. To respond appropriately to changing conditions, B. subtilis employs fast stress responses plus a series of relatively slower developmental programs that lead to cell differentiation as encountered in multicellular organisms. For example, subpopulations within a larger population of B. subtilis cells can become motile to move toward more attractive environments, or they can differentiate into spores that are highly resistant to harsh conditions. Another very effective survival strategy is the formation of thick multilayered biofilms in which cells are protected by a matrix of extracellular proteins and other macromolecules. B. subtilis can form such biofilms at air-liquid interfaces (i.e., pellicle biofilms) and on solid surfaces. Importantly, all these protective and developmental programs involve the transport of particular proteins from the cytoplasm across the cytoplasmic membrane to the membrane-cell wall interface, the cell wall, or the extracellular environment. Apparently, there has been a high selective pressure for protein export in B. subtilis and related bacilli, as all of them have a very high capacity for protein export (17). Most exported Bacillus proteins are synthesized with a signal peptide that is needed for their targeting to a translocation machinery in the membrane (17, 18, 19). Once the membrane translocation process is initiated or shortly thereafter, the signal peptide is proteolytically removed by so-called signal peptidases (8, 22). This is a prerequisite for the release of the translocated protein from the trans side of the membrane. During the evolution of Bacillus species, there must have been a particularly high selective pressure on efficient processing of exported proteins, as the signal peptidase-encoding (sip) genes have been multiplied several times. Thus, the B. subtilis genome contains five different sip genes, and up to seven sip genes are present in genomes of species belonging to the Bacillus cereus group (18, 22). Additional sip genes are found on otherwise cryptic endogenous plasmids of B. subtilis (6, 21). Quite unexpectedly, sequencing of the B. subtilis genome revealed that one of the five chromosomal sip genes, denoted as sipW, encodes a signal peptidase that is atypical for bacteria. Instead, SipW closely resembles the signal peptidases encountered in the plasma membrane of archaea and the endoplasmic reticular (ER) membrane of eukaryotes (18). The ER-type signal peptidase SipW differs from other prokaryotic (P)type signal peptidases in several respects. First, it employs a SerHis catalytic dyad or Ser-His-Asp catalytic triad, whereas the Ptype signal peptidases employ a Ser-Lys catalytic dyad (8, 20). Second, SipW has both N-terminal and C-terminal membrane anchors, whereas the majority of P-type signal peptidases have N-terminal membrane anchors only. Subsequent studies and, especially, large-scale genome sequencing efforts have shown that homologues of SipW can be found in various species belonging to the Actinobacteria (e.g., Arthrobacter, Rhodococcus, and Xylanimonas), Firmicutes (e.g., Bacillus, Clostridium, Desulfitobacterium, Eubacterium, and Ruminococcus), and Mollicutes (e.g., Sphaerobacter). The signal peptidases of B. subtilis have different but overlapping specificities. Accordingly, they are functionally redundant in the general secretion of proteins (18). However, SipW has a rather specific nonessential role in protein export (1, 3, 4, 18). The two major substrates processed by SipW are the precursors of the TapA (YqxM/YqhD) and TasA (CotN/YqhF) proteins, which are encoded by the tapA-sipW-tasA operon (13, 14). TasA was initially identified as a spore-associated protein with antibacterial activity and roles in the assembly and function of the endospore coat (11, 13, 14). Consistently, processing of the TasA precursor by SipW is required for the incorporation of mature TasA into spores (20). Subsequent studies revealed that SipW and its substrates TapA and TasA are also required for the formation of biofilms (2, 3, 4, 7). TasA is in fact a major protein component of B. subtilis biofilms that provides structural integrity through the formation of amyloid fibers (1, 10). In the present issue of the Journal of Bacteriology, Terra et al. (16) describe the intriguing observation that the SipW protein but not its enzymatic activity is needed for the formation of surfaceadhered biofilms. Specifically, they show that the 20 C-terminal residues of SipW, which localize to the cytoplasmic side of the membrane, are critically needed for solid-surface biofilm formation, whereas the catalytic Ser, His, and Asp residues are dispensable for this process. On the other hand, the 20 C-terminal residues turn out to be dispensable for SipW-catalyzed processing of TasA, showing unambiguously that these residues are needed for the second function of SipW in solid-surface biofilm formation. The first evidence for a second role of SipW in biofilm formation came from the earlier observation that strains lacking SipW were more strongly inhibited in solid-surface biofilm formation than strains lacking TasA and TapA (1, 4). While this might suggest a role for other, as-yet-unidentified substrates of SipW in solidsurface biofilm formation, Terra et al. entertained the more interesting hypothesis that SipW might have a second function specifically required in this process. This is now shown to be correct. A subsequent screen for sipW suppressors revealed that mutations in