Membrane‐fusion protein homologues in Gram‐positive bacteria (2000)

Sir, We previously characterized a family of Gram-negative bacterial auxiliary transport proteins that we designated the ‘membrane-fusion protein’ (MFP) family (TC #8.A.1; see our website: http://www-biology.ucsd.edu/~msaier/transport) (Dinh et al. 1994, J Bacteriol176: 3825–3831; Saier et al. 1994, Mol Microbiol11: 841–847, and references cited therein). MFPs are generally encoded within operons that also encode a primary cytoplasmic transporter of the major facilitator superfamily (MFS, TC #2.A.1; Saier et al. 1999, J Mol Microbiol Biotechnol1: 257–279), the ATP-binding cassette (ABC) superfamily (TC #3.A.1; Saurin et al. 1999, J Mol Evol.48: 22–41) or the resistance/nodulation/division (RND) superfamily (TC #2.A.6; Tseng et al. 1999, J Mol Microbiol Biotechnol1: 107–125). MFPs were thought to allow export of macromolecules and toxic substances across both the inner and the outer membranes of the Gram-negative bacterial cell envelope in a single energy-coupled transport step, possibly by providing a bridge between the inner membrane transporter and an outer membrane porin factor (OMF, TC #1.B.17; Binet et al. 1997, Gene192: 7–11; Paulsen et al. 1997, FEMS Microbiol Lett156: 1–8). Structural analyses of MFPs suggested that their N-termini are in the cytoplasm, but that a single N-terminal TMS spans the cytoplasmic membrane. Adjacent and C-terminal to this TMS were three domains: (i) a fairly well-conserved region that was proposed to interact with the permease; (ii) a poorly conserved α-helical region, thought possibly to span the periplasm; and (iii) a well-conserved C-terminal region, rich in β structure, thought to interact with outer membrane constituents (Dinh et al. 1994, J Bacteriol176: 3825–3831). Extensive evidence now shows that MFPs are: (i) asymmetric proteins capable of spanning the periplasm (Zgurskaya and Nikaido, 1999a, J Mol Biol285: 409–420); (ii) mediate substrate-induced assembly of the proposed heterotrimeric protein complex (Létofféet al. 1996, EMBO J15: 5804–5811; Thanabalu et al. 1998, EMBO J17: 6487–6496); and (iii) can bring two proteoliposomal membranes together (Zgurskaya and Nikaido, 1999b, Proc Natl Acad Sci USA96: 7190–7195). These reports clearly support the original proposal that MFPs can function as bridges between the inner and the outer membranes of Gram-negative bacteria. No other function for the MFPs has been proposed. Recently, structural homologues of the MFPs have been identified in low G+C Gram-positive bacteria that lack an outer membrane. In this MicroCorrespondence, we use computer approaches to: (i) identify the members of this Gram-positive bacterial subfamily; (ii) establish that these proteins are homologous to the Gram-negative bacterial MFPs; (iii) summarize functional information currently available; (iv) provide a probable structural description of these proteins; and (v) identify internally shortened forms of these proteins that appear to lack the central 270 residue α-helical region. Gram-positive bacterial MFPs must serve a function distinct from that currently recognized for Gram-negative bacterial MFPs, and this fact implies that the latter proteins may serve a corresponding function in addition to those supported by current experimental evidence. Table 1 lists the Gram-positive bacterial MFP protein homologues that were retrieved using the psiblast program (Altschuhl et al. 1997, Nucleic Acids Res25: 3389–3402). These proteins exhibit comparison scores of greater than 20 standard deviations (SD) with each other using the gap program with 100 random shuffles (Deveraux et al. 1984, Nucleic Acids Res12: 387–395). They fall into two size ranges: 10 of the proteins are of nearly uniform size (449–474 amino acyl residues), but two of them, both from lactobacilli, are much shorter (188–197 residues) (Fremaux et al. 1993, Appl Environ Microbiol59: 3906–3915; Kawai and Saitoh, 1999, GenBank accession number A3029612). A partial multiple alignment of the two short proteins (LafY Lac and Orf2 Lga) with a full-length protein (PlnH Lpl; see Table 1) revealed that homology is observed for most of the N-terminal 90 residues and the C-terminal 110 residues of the latter protein (data not shown). An average hydropathy plot of the multiply aligned full-length proteins revealed: (i) a hydrophilic N-terminus (residue positions 1–19); (ii) a single putative TMS (positions 20–40); (iii) an adjacent short, moderately hydrophilic region (positions 41–80); (iv) a long strongly hydrophilic region (positions 81–330); and (v) a moderately hydrophilic region (positions 331–end). Average similarity plots, average amphipathicity and secondary structure predictions (data not shown) revealed the following for the five regions cited above: Region i forms a putative amphipathic α- helix with almost all phenylalanine and tyrosine residues on the hydrophobic side of the helix; region ii, the TMS (Franke et al. 1996, J Bacteriol178: 1766–1769), is less well conserved than the two flanking regions; region iii exhibits a high degree of conservation, moderate amphipathicity and mixed (α and β) predicted secondary structure; region iv is the least well conserved but is predominantly α-helical and exhibits striking regions of amphipathicity when portrayed as an α-helix, and region v is relatively well conserved with a large percentage of β-structure relative to the rest of these proteins. These structural features are similar to those predicted for Gram-negative MFPs (Dinh et al. 1994, J Bacteriol176: 3825–3831). One of the proteins listed in Table 1 has been reported to exhibit limited sequence similarity with established Gram-negative bacterial MFPs (Franke et al. 1999, J Biol Chem274: 8484–8490). In fact, homology of the Gram-positive bacterial MFPs with the Gram-negative bacterial MFPs was established using the GAP program (comparison scores of up to 13.5 SD.). Although Gram-positive bacterial MFPs function as essential transport accessory proteins with transporters of the ABC superfamily (Axelsson and Holck, 1995, J Bacteriol177: 2125–2137; Venema et al. 1996, Appl Environ Microbiol62: 1689–1692; Quadri et al. 1997, J Bacteriol179: 6163–6171), the functions they provide have yet to be determined. Further studies will be required to establish the functional characteristics of these interesting proteins.