Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili
2000; Springer Nature; Volume: 19; Issue: 23 Linguagem: Inglês
10.1093/emboj/19.23.6408
ISSN1460-2075
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle1 December 2000free access Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili Matthew Wolfgang Matthew Wolfgang Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, 48109 USA Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Jos P.M. van Putten Jos P.M. van Putten Laboratory of Microbial Structure and Function, Hamilton, MT, 59840 USA Present address: Department of Bacteriology, Institute of Infectious Diseases and Immunology, University of Utrecht, NL-3584 CL Utrecht, The Netherlands Search for more papers by this author Stanley F. Hayes Stanley F. Hayes Microscopy Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, 59840 USA Search for more papers by this author David Dorward David Dorward Microscopy Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, 59840 USA Search for more papers by this author Michael Koomey Corresponding Author Michael Koomey Biotechnology Centre of Oslo and Institute of Pharmacy, University of Oslo, 0316 Oslo, Norway Search for more papers by this author Matthew Wolfgang Matthew Wolfgang Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, 48109 USA Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Jos P.M. van Putten Jos P.M. van Putten Laboratory of Microbial Structure and Function, Hamilton, MT, 59840 USA Present address: Department of Bacteriology, Institute of Infectious Diseases and Immunology, University of Utrecht, NL-3584 CL Utrecht, The Netherlands Search for more papers by this author Stanley F. Hayes Stanley F. Hayes Microscopy Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, 59840 USA Search for more papers by this author David Dorward David Dorward Microscopy Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, 59840 USA Search for more papers by this author Michael Koomey Corresponding Author Michael Koomey Biotechnology Centre of Oslo and Institute of Pharmacy, University of Oslo, 0316 Oslo, Norway Search for more papers by this author Author Information Matthew Wolfgang1,2, Jos P.M. van Putten3,4, Stanley F. Hayes5, David Dorward5 and Michael Koomey 6 1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, 48109 USA 2Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, 02115 USA 3Laboratory of Microbial Structure and Function, Hamilton, MT, 59840 USA 4Present address: Department of Bacteriology, Institute of Infectious Diseases and Immunology, University of Utrecht, NL-3584 CL Utrecht, The Netherlands 5Microscopy Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, 59840 USA 6Biotechnology Centre of Oslo and Institute of Pharmacy, University of Oslo, 0316 Oslo, Norway *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:6408-6418https://doi.org/10.1093/emboj/19.23.6408 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Type IV pili (Tfp) are a unique class of multifunctional surface organelles in Gram-negative bacteria, which play important roles in prokaryotic cell biology. Although components of the Tfp biogenesis machinery have been characterized, it is not clear how they function or interact. Using Neisseria gonorrhoeae as a model system, we report here that organelle biogenesis can be resolved into two discrete steps: fiber formation and translocation of the fiber to the cell surface. This conclusion is based on the capturing of an intermediate state in which the organelle is retained within the cell owing to the simultaneous absence of the secretin family member and biogenesis component PilQ and the twitching motility/pilus retraction protein PilT. This finding is the first demonstration of a specific translocation defect associated with loss of secretin function, and additionally confirms the role of PilT as a conditional antagonist of stable pilus fiber formation. These findings have important implications for Tfp structure and function and are pertinent to other membrane translocation systems that utilize a highly related set of components. Introduction Type IV pili (Tfp) are a unique class of filamentous appendages defined by their shared structural, biochemical, antigenic and morphological features (Strom and Lory, 1993), which are expressed by Gram-negative bacteria of medical, environmental and industrial importance. The pilin subunits of Tfp display a high degree of identity to each other within their N-termini, the domain that functions in inner membrane insertion, proteolytic processing, subunit–subunit interaction and is predicted to form the central helical core of the pilus filament (Parge et al., 1995). Further evidence for the relatedness of these organelles can be found in the conservation of genes and gene products required for their expression (Tønjum and Koomey, 1997). These include prepilin peptidases (which proteolytically process the N-termini of pilins), soluble proteins with essential nucleotide-binding motifs (the GspE/TrbB-like molecules), polytopic inner membrane proteins and a family of outer membrane proteins termed secretins which can be isolated in some instances as oligomeric ring-shaped structures. Homologs of all of these Tfp biogenesis proteins are found as components of the type II secretion machinery, also termed the secreton, which is responsible for toxin and hydrolase excretion in Gram-negative bacteria (Pugsley, 1993). In particular, the secreton utilizes five pseudopilins which are homologous to Tfp pilin subunits (Nunn, 1999), and two observations have demonstrated the functional inter-relatedness of the Tfp biogenesis and secreton machineries. First, Pseudomonas aeruginosa shares a Tfp and secreton pathway, and mutants failing to express PilA, the Tfp subunit, are defective in protein secretion (Lu et al., 1997). Secondly, overexpression of the pullulanase secreton in Escherichia coli was found to lead to the expression of the PulG pseudopilin as pilus-like bundles, while low-level secreton expression was sufficient to incorporate an endogenous E.coli Tfp pilin into fibers (Sauvonnet et al., 2000). Filamentous phage morphogenesis (Russel et al., 1997), natural competence for genetic transformation (Dubnau, 1997; Dougherty and Smith, 1999) as well as the type III toxin translocation/secretion systems also require homologs of some of the Tfp biogenesis components (Hueck, 1998). With the exception of the prepilin peptidases that proteolytically process the pilin subunit (Lory and Strom, 1997), the precise functions served by any of the conserved biogenesis components are not known. This situation stems from the fact that defects in the expression of any one lead to the same null phenotype defined by the lack of expression of pilus filaments. An analogous situation occurs in the type II secretion systems, in that defects in any one of the Tfp biogenesis homologs lead to the phenotype in which target molecule(s) accumulate in the periplasmic space rather than being secreted (Nunn, 1999). This lack of distinctiveness in any class of Tfp biogenesis mutant could be interpreted as an indication that fiber formation and cell surface localization are intrinsically coupled events and that most biogenesis factors act at this crucial, but as yet ill-defined, step. The most well understood system for the biogenesis of pili is the chaperone-assisted or chaperone/usher secretion pathway, which is responsible for the assembly of >30 distinct forms of adhesive organelles in various Gram-negative species (Thanassi et al., 1998a). In these systems, the release of nascent pilus subunits from the cytoplasmic membrane is mediated by binding to immunoglobulin-like periplasmic chaperones (Hung and Hultgren, 1998). These subunit–chaperone complexes are then targeted to the outer membrane where interaction of the complexes with the usher outer membrane protein appears to lead to chaperone dissociation and exposure of subunit-interactive surfaces, which in turn drives assembly into pilus fibers (Thanassi et al., 1998b; Sauer et al., 1999). Although details of the process remain unclear, it appears that fiber polymerization and membrane translocation are highly coupled events. Flagellar biogenesis is a more complicated process that involves >30 protein components (MacNab, 1996). Flagellin subunits are extruded through a channel in the filament and hook and are incorporated into the distal end of the nascent structure so that fiber assembly clearly occurs subsequent to membrane translocation. Attempts to rationalize Tfp biogenesis in the context of either the chaperone/usher or flagellar systems, however, do not appear to be tenable since none of the Tfp biogenesis components are structurally related to those found there. Neisseria gonorrhoeae, the etiological agent of gonorrhea, has proven to be a particularly attractive species in which to study Tfp biology because organelle expression is constitutive under conditions of in vitro growth and the organism is highly amenable to genetic manipulation. Moreover, Tfp expression is tightly coupled to the biology of the organism since it is associated with the ability of the microorganism to: (i) colonize humans and cause disease; (ii) take up DNA in a sequence-specific manner during transformation; (iii) express multicellular aggregative behavior; and (iv) exhibit a novel form of flagella-independent, cell locomotion termed twitching motility (Tønjum and Koomey, 1997). Tfp-associated twitching motility has been identified in many Gram-negative species including Pseudomonas aeruginosa and Myxo coccus xanthus (Henrichsen, 1983; Wu and Kaiser, 1995) and originally was hypothesized to involve pilus retraction (Bradley, 1980). Mutations in the highly conserved pilT genes encoding members of a GspE/TrbB-like family of proteins lead to a piliated but non-motile phenotype in all species (Whitchurch et al., 1991; Wu et al., 1997; Wolfgang et al., 1998a). A recent biophysical study using N.gonorrhoeae has shown that Tfp do retract and that PilT is essential for this property (Merz et al., 2000). We previously demonstrated that that loss-of-function mutations in pilT alleviated the biogenesis requirement for PilC, indicating that PilT can function as an antagonist of organelle expression (Wolfgang et al., 1998b). This result led us in turn to examine what influence PilT might have in Tfp mutants failing to express the secretin Tfp biogenesis protein PilQ (Drake and Koomey, 1995; Tønjum et al., 1998). We show that the absence of PilT in this background leads to the expression of Tfp fibers, which fail to reach the cell surface and remain localized in membrane protrusions. This result confirms that PilT acts as a conditional antagonist of fiber formation, and accompanying data show that Tfp biogenesis entails three genetically dissociable steps: (i) fiber formation; (ii) fiber stabilization; and (iii) surface localization of the intact organelle. The findings make it possible to assign empirically the step at which various biogenesis factors function and, based on tests of epistasis, to establish the order in which they function. Results Tfp fiber formation independent of cell surface localization defines a unique step in organelle biogenesis PilQ is a member of the pIV/PulD family of outer membrane proteins, which in multimeric form are proposed to act as gated channels through which macromolecules are translocated (Russel et al., 1997). In order to determine what influence the twitching motility protein PilT might have on the phenotypes of a PilQ− Tfp biogenesis mutant (Drake and Koomey, 1995), mutants simultaneously lacking both molecules were made by recombination of a pilQ transposon insertion mutation into a strain that carries the pilT gene under control of an inducible promoter. In contrast to the mutant carrying only the pilQ lesion, the double mutants displayed extreme defects in growth, which were reflected in their inability to form colonies of normal size and morphology (Figure 1A, top panel). When pilT expression was restored, these strains behaved identically to strains bearing only the pilQ mutation in that they were defective in Tfp expression and grew normally in a fashion typical of non-piliated mutants (Figure 1A, lower left panel) (Drake and Koomey, 1995). The pilT gene and the pilU gene immediately distal to it, which encodes a protein highly related to PilT, are expressed as a single transcriptional unit (H.S.Park and M.Koomey, manuscript in preparation) and it thus was formally possible that the growth defect seen was related to the absence of PilU and not of PilT alone. Strains carrying both the pilQ transposon mutation and an insertion mutation in pilU were phenotypically identical to those possessing only the pilQ mutation (data not shown), ruling out this possibility. To examine the growth defect displayed by the pilQ/T mutant in a more quantitative manner, we took advantage of the inducibility of the pilT allele to measure the efficiency of plating in the presence and absence of PilT. This assay showed that the absence of PilT in the pilQ background led to a >30-fold reduction in plating efficiency (Figure 1B), indicating that reduced viability contributed significantly to the growth defect observed. Figure 1.Simultaneous loss of PilQ and PilT expression leads to defects in growth reflected in altered colony size and plating efficiencies. (A) Neisseria gonorrhoeae colonies photographed after 24 h growth on solid agar at a magnification of 30× using a stereomicroscope. Top panel: strain MW11 (pilQ::mTncm21, pilTind). Lower left panel: relief of the growth defect in MW11 by derepression of pilT expression (+IPTG). Lower right panel: variants that suppress the growth defect can be isolated based on their ability to yield colonies of normal size, regardless of pilT expression. (B) Effects of pilT derepression on the plating efficiencies of wild-type and mutant N.gonorrhoeae strains. The ratio of colony forming seen after 24 h in the absence and presence of pilT de-repression was determined. Data represent the average of three experiments. Strain designation and genotype for the mutants presented are as follows: wt (N400), pilT (MW4, pilTind), pilQ (GQ21, pilQ::mTncm21) and pilQ/pilT (MW11, pilQ::mTncm21, pilTind). Download figure Download PowerPoint The morphology and surface structure of pilQ/T cells growing in colonies were assessed by electron microscopy. Using scanning electron microscopy (SEM), Tfp form lateral aggregates which radiate from the cell surface and form a fibrous network in which cells are interconnected (Figure 2A). These structures were found in greater abundance in the pilT mutant but were absent in the pilQ mutant. In contrast, the pilQ/T mutant cells were irregular in shape, often lacking the characteristic diplococcal morphology, and were covered with membranous protrusions that spanned individual cells in the colony. In many instances, the membranous appendages displayed variability in diameter along their length, with alternating patterns of constriction and expansion (Figure 2A, lower right panel). Figure 2.Electron microscopic analysis of pilQ/pilT mutants shows altered cell surface structures comprised of membrane-bound pilus filaments. (A) Scanning electron micrographs of wild-type and mutant N.gonorrhoeae strains. wt (N400); pilT (MW4, pilTind); pilQ (GQ21, pilQ::mTncm21); pilQ/pilT (MW11, pilQ::mTncm21, pilTind). In the lower center and lower right panels, wt and pilQ/pilT micrographs are at 50 000× magnification; all others are at 25 000×. Individual N.gonorrhoeae cells are ∼1 μm in diameter. (B) Transmission electron micrographs showing membrane-bound pilus fibers in a pilQ/T mutant (strain MW11). Note that bulges seen in the membranous protrusions contain coiled fibers detected by TEM (center and right panels) and that they correspond to analogous structures seen in SEM. Micrographs are taken at a magnification of 90 000× and inset panels show digitally enlarged images at a 3× higher magnification. (C) Immunolabeling of fibers associated with disrupted blebs with antiserum raised against purified pili (135 000×). Download figure Download PowerPoint Examination by negative staining and transmission electron microscopy (TEM) showed that the membranous protrusions in the pilQ/T mutant contained pilus fibers (Figure 2B). The pilus fibers were indistinguishable in diameter and morphology from those seen on the surface of wild-type cells and could be immunolabeled in areas where they are exposed by virtue of ruptures in the blebs using antisera raised against purified pili (Figure 2C). The arrangement of Tfp within these structures was consistent with the view that fiber extrusion or growth was responsible for distortion of the membrane (Figure 2B, left panel). In addition, Tfp were often seen in coiled configurations within membrane bulges, suggesting that fiber growth or extrusion was constricted (Figure 2B, center and right panels). It appeared then that an intermediate state in Tfp biogenesis, in which intact pilus fibers were retained inside the cell, was captured due to the simultaneous absence of PilQ and PilT. We conclude that: (i) the outer membrane protein PilQ is non-essential to pilus fiber formation and functions specifically in Tfp biogenesis by facilitating translocation of the fiber to the cell surface; and (ii) PilT is responsible for the absence of Tfp fibers in pilQ mutants. PilT is required for fiber subunit degradation in pilQ mutants In most N.gonorrhoeae Tfp biogenesis mutants characterized to date, the pilin subunit protein PilE is proteolytically degraded into a stable, truncated form lacking the first 39 residues of the mature polypeptide, termed S-pilin (Haas et al., 1987; Koomey et al., 1991). As seen by immunoblotting of whole-cell samples, degradation of pilin occurred in the pilQ mutant strain but was absent in the strain lacking both PilQ and PilT and was restored in the latter background following derepression of pilT expression (Figure 3, lanes 3–5). In light of the potential influence of PilU expression on these findings, pilin degradation was examined in a pilQ/U mutant and proteolysis was not perturbed (Figure 3, lanes 7 and 8). These results indicate that PilT is required for degradation of the pilin subunit in pilQ biogenesis mutants and that the presence of fibers in their membrane-bound form together with associated growth defects were inversely correlated with subunit degradation. Figure 3.PilT is required for fiber subunit degradation in pilQ mutants. Abolition of fiber expression and associated growth defects in the pilQ/pilT mutant by de-repression of pilT expression leads to subunit degradation. An immunoblot of whole-cell lysates probed with the pilin-specific monoclonal antibodies (mAb MC02) is shown. Lane 1, wild-type (N400, recA6); lane 2, pilT (MW4, pilTind); lane 3, pilQ (GQ21, pilQ::mTncm21); lanes 4 and 5, pilQ/pilT (MW11, pilQ::mTncm21, pilTind); lane 6, pilU (MW35, pilU::kan); lane 7, pilQ (GQ21, pilQ::mTncm21); lane 8, pilQ/pilU (MW36, pilQ::mTncm21, pilU::kan). (+) denotes lysates derived from strains propagated in the presence of pilT expression (plus IPTG). S-pilin denotes the migration of the truncated species of PilE lacking the first 39 residues present in the mature molecule. Download figure Download PowerPoint Suppression of intracellular Tfp fiber formation and associated growth defects by alterations in pilE, encoding the fiber subunit The severe toxicity of the combined defects in PilT and PilQ expression made it possible to isolate suppressor variants in which normal growth was restored, providing an opportunity to examine the relationship between intracellular Tfp fiber expression and the growth defects (Figure 1A, lower right). Twelve independently derived variants of this type were chosen for further analysis. By SEM and TEM, all suppressor variants lacked the membranous protrusions and encapsulated Tfp fibers seen in the parental strain (data not shown). The suppressor variants were next analyzed by immunoblotting with a pilin-specific monoclonal antibody and, in three cases, no pilin antigen was detected, while in all others, the pilin subunit was proteolytically degraded (Figure 4A). In addition, the relative migration of both intact and degraded pilin polypeptides in these strains was altered from that seen in the pilQ background. Since pilin can undergo mutation and combinatorial diversification by genetic conversion events involving pilE and multiple, partial donor alleles (Zhang et al., 1992), we determined the nucleotide sequences of the pilE alleles within these strains (Figure 4B). A deletion mutation encompassing the 5′ end of the expression locus (strain MW19) and frameshift mutations (strains MW16 and MW22) accounted for the complete absence of pilin antigen, while, in the other cases, multiple nucleotide substitutions had occurred within the variable regions of the pilE open reading frame (ORF; Figure 4B). These alterations were characteristic of the recombination events demonstrated to be responsible for pilin antigenic variation, with diversification arising by gene conversion with both different donor alleles and differing stretches of the same donor allele (Swanson and Koomey, 1989). Figure 4.Variants that suppress the growth defect associated with the lack of PilQ and PilT show alterations in the expression, degradation and structure of the pilin subunit. (A) Immunoblot analysis of pilin subunits expressed by variants derived from the pilQ/pilT mutant (strain MW11, pilQ::mTncm21, pilTind) that no longer show a growth defect. Pilin-specific mAb MC02 was used to probe whole-cell lysates. Lane 1, wild-type (N400, recA6); lane 2, pilQ/pilT (MW11, pilQ::mTncm21, pilTind); lanes 3–14, variants isolated from MW11 (strains MW12–MW23, respectively). (B) Variants that suppress the pilQ/pilT-associated growth defect show multiple changes in the primary structure of pilin. The predicted amino acid sequence of pilin, expressed from pilE (designated pilEwt) of the parental pilQ/pilT strain is shown. Changes in the predicted primary structure of variant pilins, based on DNA sequence, are indicated. Periods (·) denote identical residues and dashes (–) represent gaps in the sequence. N-terminal sequences are not shown, as no changes were detected. No sequence is presented for MW19 as pilE was deleted in this strain. Download figure Download PowerPoint pilE alleles which suppress pilQ/T phenotypes encode fiber subunits intrinsically defective in Tfp biogenesis The pilin subunits encoded by the suppressor alleles had no distinctive, structural features that might define the basis for their shared defects in fiber formation or susceptibility to proteolytic degradation. To examine the basis for this phenomenon, we introduced the suppressor pilE alleles into an otherwise wild-type background and assessed Tfp expression. To this end, a strain was constructed in which expression of the endogenous pilE allele was placed under the control of an inducible promoter (Figure 5A). In the absence of derepression, this strain was no longer competent for natural transformation (data not shown), nor did it express detectable pilin antigen or purifiable Tfp (Figure 5B, lane 2). This strain was transformed with a cloned copy of each of the full-length pilE suppressor alleles (including upstream promoter elements) linked to a selectable antibiotic resistance marker. Recombination of the suppressor pilE alleles onto the gonococcal chromosome at an ectopic site within the iga locus was selected for by acquisition of antibiotic resistance (Figure 5A). Strains expressing pilin produced exclusively from the ectopic alleles were examined for pilin expression by immunoblotting, and for Tfp expression by electron microscopy and yields of purified pili. As a control, the wild-type pilE gene (pilEwt) from the parental strain was expressed ectopically and found to confer levels of Tfp expression and associated phenotypes identical to those of the original wild-type parental strain (Figure 5B, lane 3). In contrast, strains carrying the suppressor pilE alleles uniformly expressed a significant amount of pilin in degraded form (Figure 5B, upper panel). Of the nine suppressor alleles studied in this manner, four were completely defective for Tfp expression as assessed by TEM (data not shown) and yields of purifiable pilus filaments (Figure 5B, lower panel, lanes 4, 5, 8 and 10). The remainder had levels of purifiable pilus filaments that were <5% of that found for the wild-type control strain (Figure 5B, lower panel, lanes 6, 7, 9, 11 and 12) and, when analyzed by TEM, these strains showed a comparable reduction in piliation (data not shown). These results indicated that pilE alleles which arise in association with suppression of the expression of intracellular Tfp by the pilQ/T mutant and its associated growth defects encode subunits intrinsically defective in organelle biogenesis. Figure 5.Variant pilE alleles that arise in association with suppression of the pilQ/pilT growth defect are intrinsically defective in Tfp biogenesis. (A) Schematic diagram outlining the approach used to analyze variant pilE alleles. Strain MW24 was constructed such that expression from endogenous pilE was placed under the control of a regulated promoter. The chromosomal organization for this strain is shown at the top. The expression of altered pilE alleles arising in the pilQ/pilT background was analyzed following cloning and recombination into an ectopic site within the gonococcal iga locus of strain MW24, as depicted at the bottom. In the absence of induction, pilin is produced solely from the ectopically expressed allele. (B) Upper panel: immunoblot analysis of pilin, expressed from the altered pilE alleles, using the pilin-specific mAb MC02. Lower panel: Coomassie Blue-stained SDS–polyacrylamide gel showing the relative amounts of pilin subunit in purified pilus preparations. Lane 1, wild-type (N400, recA6); lane 2, pilE (MW24, pilEind); lanes 3–12, designated pilE alleles expressed ectopically in the pilEind background (strains MW25–MW34, respectively). Download figure Download PowerPoint Tfp fibers and growth defects are not seen in the context of pilD/T or pilF/T mutants It was of obvious interest to ask what effect loss of PilT might have in two other well characterized classes of Tfp biogenesis mutants; those carrying defective pilD and pilF alleles (Freitag et al., 1995). The gonococcal PilD protein, the prepilin peptidase, is localized to the inner membrane and is responsible for removing a short signal sequence from the prepilin (Strom and Lory, 1992). Consistent with previous observations (Freitag et al., 1995), the pilD mutant examined expressed unprocessed as well as degraded pilin (Figure 6A, lane 3) and failed to express Tfp (data not shown). In contrast to the results with other pilT double mutants, the pilD/T strain was phenotypically indistinguishable from the pilD mutant, having no Tfp fibers seen by electron microscopy, no discernible growth defect and no alteration in levels of degraded pilin (Figure 6A, lane 4). Figure 6.PilT does not influence PilE degradation in pilD and pilF mutants or double mutants simultaneously lacking PilQ. PilE degradation was assessed by immunoblotting of whole-cell lysates using the pilin-specific mAb MC02. Prepilin denotes the migration of the unprocessed PilE, while S-pilin denotes the migration of the truncated species of PilE lacking the first 39 residues present in the mature molecule. (A) PilT does not alter PilE stability in pilD and pilF mutants. Lane 1, wild-type (N400, recA6); lane 2, pilT (MW4, pilTind); lane 3, pilD (GDClaI–XhoI, pilDfs); lane 4, pilD/pilT (MW37, pilDfs, pilTind); lane 5, pilF (GF2, pilF::mTnerm2); lane 6, pilF/pilT (MW38, pilF::mTnerm2, pilTind). (B) Epistastic relationships of pilD, pilF and pilQ with regard to PilT-dependent PilE degradation. Lane 1, wild-type (N400, recA6); lane 2, pilQ/pilT (MW39, pilQind, pilTind); lane 3, pilQ/pilD (MW40, pilQind, pilDfs); lane 4, pilQ/pilD/pilT (MW41, pilQind, pilDfs, pilTind); lane 5, pilQ/pilF (MW42, pilQind, pilF::mTnerm2); lane 6, pilQ/pilF/pilT (MW43, pilQind, pilF::mTnerm2, pilTind). (C) Inferred order of action of components in the Tfp biogenesis pathway. Shown is a diagrammatic pathway summarizing the activities of Tfp biogenesis components in fiber formation, stabilization and surface localization as determined from this study and prior results (Wolfgang et al., 1998b). Note that although PilT is shown to impact antagonistically on the pathway downstream of PilD and PilF, this does not relate directly to its actual physical localization in the cell or the site where it may function. Download figure Download PowerPoint The PilF Tfp biogenesis component is related structurally to PilT as well as to other members of the large family of cytoplasmically localized proteins bearing consensus nucleotide-binding motifs (Freitag et al., 1995). As in the case of PilD, a strain lacking both PilF and PilT was identical to an isogenic pilF mutant, with no Tfp fibers seen, no growth defect observed and pilin being degraded (
Referência(s)