Phytoplasmas ("CandidatusPhytoplasmaThe phylogenetic tree of mollicutes is composed of two major clades that diverged early in evolution (51). One clade contains the orders Acholeplasmatales and Anaeroplasmatales (AAA clade mollicutes), and the other clade contains the orders Mycoplasmatales and Entomoplasmatales (SEM clade mollicutes) (9). Phytoplasmas, formerly known as mycoplasma-like organisms of plants, form a monophyletic group in the order Acholeplasmatales (51) and were recently assigned to a novel genus, "Candidatus Phytoplasma" (41). Approximately 20 phytoplasma phylogenetic groups have been proposed based on 16S rRNA gene sequences, and new branches are continuously being discovered (69,85). Members of the order Acholeplasmatales are distinct from other mollicutes in several ways. For instance, whereas most mollicutes use UGA as a tryptophan codon instead of a stop codon, a feature they share with mitochondria, the acholeplasmas and phytoplasmas retained UGA as a stop codon (80).

Protein export signals from the low-passage 297 strain of Borrelia burgdorferi were cloned as fusions with an Escherichia coli alkaline phosphatase (PhoA) reporter lacking a signal sequence. One PhoA+ clone (BbK2.10-PhoA) was derived from a borrelial lipoprotein. Although the polypeptide encoded by the full-length bbk2.10 gene had 76% similarity and 56% identity to outer surface protein F (OspF) from B. burgdorferi strain N40, antibodies directed against recombinant forms of the two proteins revealed that they were not cross-reactive. The nucleotide sequences of bbk2.10 and ospF from the N40 and 297 strains, respectively, were determined to confirm that the N40 and 297 strains each contained both genes. Southern blot analysis revealed that bbk2.10 is a single-copy gene and that the B. burgdorferi strain 297 and N40 genomes appeared to contain one other gene more closely related to ospF than bbk2.10. It was particularly noteworthy that ospF, but not bbk2.10, was expressed in vitro while B. burgdorferi-infected mice generated antibodies reactive with both lipoproteins. To help confirm that the BbK2.10-reactive antibodies produced by the B. burgdorferi-infected mice were specific for that protein, a second gene, bbk2.11, which hybridized with the ospF probe was cloned; the corresponding polypeptide reacted strongly with OspF antisera but failed to react with BbK2.10-specific antisera. Taken together, these data demonstrate that BbK2.10, BbK2.11, and OspF comprise a B. burgdorferi lipoprotein family and that at least one member (BbK2.10) appears to be expressed only during infection.

Venereal syphilis is a chronic, multisystem infectious disorder caused by the spirochetal bacterium Treponema pallidum subsp. pallidum (T. pallidum). Like all spirochetes, T. pallidum is an elongated, highly motile organism that consists of a fragile outer membrane surrounding a periplasmic space, a peptidoglycan-cytoplasmic membrane (PG-CM) complex, and a protoplasmic cylinder (36). There is now a substantial body of evidence that the outer membranes of T. pallidum and enteric gram-negative bacteria differ considerably with respect to composition and molecular architecture (52, 57). One of the key differences concerns the relative abundance of proteins with membrane-spanning domains. Whereas outer membranes of gram-negative bacteria contain high densities of such polypeptides (48), freeze-fracture electron microscopy and cell fractionation studies have shown that they are sparse in T. pallidum, hence the designation rare outer membrane proteins (52,57,59,77).Based on the assumption that T. pallidum rare outer membrane proteins are important in disease pathogenesis, molecular characterization of these polypeptides has become a major objective of contemporary syphilis research. One strategy recently developed to accomplish this goal is to isolate T. pallidum outer membranes for partial amino acid sequencing of candidate rare outer membrane proteins (9, 60). Using this approach, Blanco et al. (6) identified a 31-kDa protein (Tromp1) in isolated outer membranes which formed ion-conducting channels in planar lipid bilayers. More recently, the same investigators reported that recombinant Tromp1 expressed in Escherichia coli is surface exposed and that the recombinant protein possesses porin-like properties (7). However, to date, no evidence for either outer membrane location or surface exposure of native Tromp1 within motile or intact treponemes has been presented (6, 7). Moreover, Tromp1 has extensive sequence homology with the periplasmic substratebinding proteins of known ATP-binding cassette (ABC) transporters and the tromp1 gene is transcriptionally linked to open reading frames which encode homologs for ABC transporter components which, in gram-negative bacteria, are cytoplasmic membrane associated (30,33). The apparent discrepancy between the studies of Blanco and coworkers (6, 7) and these newer genetic data prompted efforts to clarify the physicochemical properties and cellular location of Tromp1 in T. pallidum. Here we present evidence that Tromp1, rather than being an outer membrane-spanning protein, actually is anchored by an uncleaved signal sequence to the T. pallidum cytoplasmic membrane.

Automated Edman degradation was used to obtain N-terminal and internal amino acid sequences from a 26-kDa protein in isolated Treponema pallidum outer membranes (OMs). The resulting sequences enabled us to PCR amplify from T. pallidum DNA a 275-bp fragment of the corresponding gene. The complete nucleotide sequence of the gene was determined from fragments amplified by long-distance PCR. Primer extension verified the assigned translational start of the open reading frame (ORF) and putative upstream promoter elements. The ORF encoded a highly basic (pI 9.6) 26-kDa protein which contained an N-terminal 25-aminoacid leader peptide terminated by a signal peptidase I cleavage site. The mature protein contained seven tandemly spaced copies (as well as an eighth incomplete copy) of a leucine-rich repeat (LRR), a motif previously identified in a number of prokaryotic and eukaryotic proteins. Accordingly, the polypeptide was designated T. pallidum leucine-rich repeat protein (TpLRR). Although Triton X-114 phase partitioning showed that TpLRR was hydrophilic, cell localization studies showed that most of the antigen was associated with the peptidoglycan-cytoplasmic membrane complex rather than being freely soluble in the periplasmic space. Immunoblot studies showed that syphilis patients develop a weak antibody response to the antigen. Lastly, the lrr T. pallidum gene was mapped to a 60-kb SfiI-SpeI fragment of the T. pallidum chromosome which also contains the rrnA and flaA genes. The function(s) of TpLRR is currently unknown; however, protein-protein and/or protein-lipid interactions mediated by its LRR motifs may facilitate interactions between components of the T. pallidum cell envelope.Syphilis is a sexually transmitted, multistage infection caused by the noncultivatable spirochetal pathogen Treponema pallidum (26). Like all spirochetes, T. pallidum is a highly motile bacterium in which an outer membrane (OM) surrounds the periplasmic space, peptidoglycan (PG)-cytoplasmic membrane (CM) complex, and protoplasmic cylinder (PC); within the periplasmic space are endoflagella, the organelles of motility (19). Compared with conventional gram-negative bacteria, we know comparatively little about the structure and composition of the T. pallidum cell envelope and how its diverse components promote survival of the syphilis spirochete in the demanding milieu of the mammalian host. Studies from a number of laboratories have provided compelling evidence that OMs of T. pallidum and gram-negative bacteria differ markedly with respect to physical properties, composition, and molecular architecture (6,29,32,34,42). While the paucity of surfaceexposed proteins appears to explain, at least in part, how this extracellular pathogen so successfully evades host immune responses during persistent infection, it also raises intriguing and fundamental questions regarding the mechanisms by which the bacterium acquires nutrients from its environment (32). In contrast to the protein-deficient OM, the vast majority of T. pallidum cell envelope con...

In this study, we characterized candidate rare outer membrane (OM) proteins with apparent molecular masses of 19, 27, 38, and 38.5 kDa, which had been identified previously in OM fractions from Treponema pallidum (J. D. Radolf et al., Infect. Immun. 63:4244-4252, 1995). Using N-terminal and internal amino acid sequences, a probe for the 19-kDa candidate was PCR amplified and used to screen a T. pallidum genomic library in Lambda Zap II. The corresponding gene (tlp) encoded a homolog for periplasmic thioredoxin-like proteins (Tlp), which reduce c-type cytochromes. A degenerate oligonucleotide derived from the N terminus of the 27-kDa protein was used to PCR amplify a duplex probe from a T. pallidum genomic library in pBluescript II SK؉. With this probe, the corresponding gene (ppiB) was identified and found to code for a presumptive periplasmic cyclophilin B-type peptidyl prolyl cis-trans isomerase (PpiB). We postulate that PpiB assists the folding of proteins within the T. pallidum periplasmic space. The N terminus of the 38-kDa candidate was blocked to Edman degradation. However, internal sequence data revealed that it was basic membrane protein (Bmp), a previously characterized, signal peptidase I-processed protein. Triton X-114 phase partitioning revealed that despite its name, Bmp is hydrophilic and therefore likely to be periplasmic. The final candidate was also blocked to Edman degradation; as before, a duplex probe was PCR amplified with degenerate primers derived from internal sequences. The corresponding gene (glpQ) coded for a presumptively lipid-modified homolog of glycerophosphodiester phosphodiesterase (GlpQ). Based upon findings with other treponemal lipoproteins, the hydrophilic GlpQ polypeptide is thought to be anchored by N-terminal lipids to the periplasmic leaflet(s) of the cytoplasmic membrane and/or OM. The discovery of T. pallidum periplasmic proteins with potentially defined functions provides fresh insights into a poorly understood aspect of treponemal physiology. At the same time, however, these findings also raise important issues regarding the use of OM preparations for identifying rare OM proteins of T. pallidum.