To further characterize the function of the Borrelia burgdorferi C-terminal protease CtpA, we used sitedirected mutagenesis to alter the putative CtpA cleavage site of one of its known substrates, the outer membrane (OM) porin P13. These mutations resulted in only partial blockage of P13 processing. Ectopic expression of a C-terminally truncated P13 in B. burgdorferi indicated that the C-terminal peptide functions as a safeguard against misfolding or mislocalization prior to its proteolytic removal by CtpA. In a parallel study of Borrelia burgdorferi lipoprotein sorting mechanisms, we observed a lower-molecular-weight variant of surface lipoprotein OspC that was particularly prominent with OspC mutants that mislocalized to the periplasm or contained C-terminal epitope tags. Further investigation revealed that the variant resulted from C-terminal proteolysis by CtpA. Together, these findings indicate that CtpA rather promiscuously targets polypeptides that lack structurally constrained C termini, as proteolysis appears to occur independently of a specific peptide recognition sequence. Low-level processing of surface lipoproteins such as OspC suggests the presence of a CtpA-dependent quality control mechanism that may sense proper translocation of integral outer membrane proteins and surface lipoproteins by detecting the release of C-terminal peptides.Borrelia spirochetes, the etiological agents of vector-borne Lyme borreliosis and relapsing fever, are diderm bacteria (bacteria with both an inner and outer membrane) with an unusual envelope structure (reviewed in reference 6). As in Gramnegative bacteria such as Escherichia coli, a periplasmic space separates an inner cytoplasmic membrane (IM) from an outer membrane (OM) (26), but flagella remain sequestered in the periplasm, where they provide both motility and determine bacterial shape (34). Similarly, the Borrelia OM also contains integral membrane proteins (23,43,56), including the porins P66 (15, 41, 51) and P13 (39, 45), as well as BesC, a component of an envelope-spanning type I secretion system involved in virulence and antibiotic resistance (11). However, the cell envelope lacks the lipopolysaccharide coat that typifies Gramnegative bacteria (54). Instead, the borrelial surface is covered by abundant glycolipids (4, 5, 22) and surface lipoproteins (9) such as OspC, a virulence factor that is required for the establishment of mammalian infection upon arthropod-borne transmission (20,44,53,55).Our current understanding of posttranslational protein processing and modification in Borrelia burgdorferi is limited.Based on studies in other Gram-negative or diderm model organisms, lipoproteins are likely modified in a three-step process occurring on the periplasmic face of the IM, involving cleavage by the signal II peptidase Lsp (encoded by open reading frame [ORF] BB0469). Surprisingly, the small B. burgdorferi genome contains genes encoding three signal peptidase I paralogues, LepB1 to LepB3 (BB0030, BB0031, and BB0263) with currently undescribed functions; at...