Conserved serine and histidine residues are critical for activity of the ER-type signal peptidase SipW of Bacillus subtilis.

Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane. Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally equivalent, but structurally quite different, forming two distinct SPase families that share only few conserved residues. P-type SPases were, so far, exclusively identified in eubacteria and organelles, whereas ER-type SPases were found in the three kingdoms of life. Strikingly, the presence of ER-type SPases appears to be limited to sporulating Gram-positive eubacteria. The present studies were aimed at the identification of potential active site residues of the ER-type SPase SipW of Bacillus subtilis, which is required for processing of the spore-associated protein TasA. Conserved serine, histidine, and aspartic acid residues are critical for SipW activity, suggesting that the ER-type SPases employ a Ser-His-Asp catalytic triad or, alternatively, a Ser-His catalytic dyad. In contrast, the P-type SPases employ a Ser-Lys catalytic dyad (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). Notably, catalytic activity of SipW was not only essential for pre-TasA processing, but also for the incorporation of mature TasA into spores.

Functional regions of the Bacillus subtilis spore coat morphogenetic protein CotE.

The Bacillus subtilis spore is encased in a resilient, multilayered proteinaceous shell, called the coat, that protects it from the environment. A 181-amino-acid coat protein called CotE assembles into the coat early in spore formation and plays a morphogenetic role in the assembly of the coat's outer layer. We have used a series of mutant alleles of cotE to identify regions involved in outer coat protein assembly. We found that the insertion of a 10-amino-acid epitope, between amino acids 178 and 179 of CotE, reduced or prevented the assembly of several spore coat proteins, including, most likely, CotG and CotB. The removal of 9 or 23 of the C-terminal-most amino acids resulted in an unusually thin outer coat from which a larger set of spore proteins was missing. In contrast, the removal of 37 amino acids from the C terminus, as well as other alterations between amino acids 4 and 160, resulted in the absence of a detectable outer coat but did not prevent localization of CotE to the forespore. These results indicate that changes in the C-terminal 23 amino acids of CotE and in the remainder of the protein have different consequences for outer coat protein assembly.

Control of synthesis and secretion of the Bacillus subtilis protein YqxM.

yqxM is a Bacillus subtilis gene of unknown function residing in an operon with sipW, which encodes a signal peptidase, and tasA, which encodes an antibiotic protein secreted in a sipW-dependent manner. YqxM was undetectable during growth in a variety of rich media, including Luria-Bertani (LB) medium, or in minimal media or under heat shock or ethanol stress conditions but was synthesized and secreted during growth in LB medium supplemented with 1.2 M NaCl. Consistent with the possible involvement of sipW in YqxM secretion, inactivation of sipW prevented YqxM secretion. YqxM was produced and secreted in a sipW-dependent manner during growth in LB medium when the sequences upstream of yqxM were replaced with those of the inducible P(spac) promoter. Coexpression of yqxM and sipW in Escherichia coli resulted in a decrease in the apparent molecular mass of YqxM, consistent with the removal of a signal peptide. These experiments suggest that YqxM production is induced by a high concentration of salt and that YqxM is secreted under the control of SipW. We hypothesize that during most conditions of growth, YqxM is present at very low levels or is not synthesized at all and that this low level or absence is due, at least in part, to posttranscriptional repression.

Regulation of synthesis of the Bacillus subtilis transition-phase, spore-associated antibacterial protein TasA.

Previously, we identified a novel component of Bacillus subtilis spores, called TasA, which possesses antibacterial activity. TasA is made early in spore formation, as cells enter stationary phase, and is secreted into the medium as well as deposited into the spore. Here, we show that tasA expression can occur as cells enter stationary phase even under sporulation-repressing conditions, indicating that TasA is a transition-phase protein. tasA and two upstream genes, yqxM and sipW, likely form an operon, transcription of which is under positive control by the transition-phase regulatory genes spo0A and spo0H and negative control by the transition phase regulatory gene abrB. These results are consistent with the suggestion that yqxM, sipW, and tasA constitute a transition phase operon that could play a protective role in a variety of cellular responses to stress during late-exponential-phase and early-stationary-phase growth in B. subtilis.

Secretion, localization, and antibacterial activity of TasA, a Bacillus subtilis spore-associated protein.

The synthesis and subcellular localization of the proteins that comprise the Bacillus subtilis spore are under a variety of complex controls. To better understand these controls, we have identified and characterized a 31-kDa sporulation protein, called TasA, which is secreted into the culture medium early in sporulation and is also incorporated into the spore. TasA synthesis begins approximately 30 min after the onset of sporulation and requires the sporulation transcription factor genes spo0H and spo0A. The first 81 nucleotides of tasA encode a 27-amino-acid sequence that resembles a signal peptide and which is missing from TasA isolated from a sporulating cell lysate. In B. subtilis cells unable to synthesize the signal peptidase SipW, TasA is not secreted, nor is it incorporated into spores. Cells unable to produce SipW produce a 34-kDa form of TasA, consistent with a failure to remove the N-terminal 27 amino acids. In cells engineered to express sipW and tasA during exponential growth, TasA migrates as a 31-kDa species and is secreted into the culture medium. These results indicate that SipW plays a crucial role in the export of TasA out of the cell and its incorporation into spores. Although TasA is dispensable for sporulation under laboratory conditions, we find that TasA has a broad-spectrum antibacterial activity. We discuss the possibility that during the beginning of sporulation as well as later, during germination, TasA inhibits other organisms in the environment, thus conferring a competitive advantage to the spore.

Growth inhibition of Candida albicans hyphae by CD8+ lymphocytes.

We have shown previously that IL-2-activated splenocytes can inhibit the growth of Candida albicans hyphae in vitro. Herein we demonstrate that plastic nonadherent lymphocytes that are CD8+ mediate the antifungal activity. Enrichment for CD8+ cells markedly enhanced the antifungal activity of the IL-2-activated lymphocyte population for C. albicans and the cytotoxic activity of the lymphocytes for an NK-resistant cell line. Depletion of CD8+ cells reduced the lymphocyte population's antifungal activity and cytotoxic activity for the NK-resistant cell line. Enrichment for NK1.1+ cells markedly reduced the antifungal activity of the lymphocyte population for C. albicans and increased the cytotoxic activity of the lymphocytes for an NK-sensitive cell line. Depletion of NK1.1+ cells increased the lymphocyte population's antifungal activity and cytotoxic activity for the NK-resistant cell line. Generation of the antifungal lymphocytes in culture required IL-2 and was not replaced with IFN-gamma. These data show that IL-2-activated CD8+ T lymphocytes exert the greatest amount of antifungal effect against the hyphal form of C. albicans, whereas IL-2- or IFN-gamma-activated NK cells have little or no effect against the hyphae.