Processing and maturation of the pilin of the type IV secretion system encoded within the gonococcal genetic island

The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili.     

F Plasmid TraF and TraH Are Components of an Outer Membrane Complex Involved in Conjugation

F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.The F plasmid (99,159 bp) of Escherichia coli is a model system for the study of the horizontal gene transfer among prokaryotes via conjugation (3, 10, 27). F encodes a 33.3-kb transfer region that is responsible for the formation of mating junctions between donor and recipient cells prior to DNA transfer and establishment in the recipient. The hallmark of F conjugation is the formation of extracellular filaments, F pili, that initiate contact between mating cells and retract, bringing the donor and recipient cells together (5, 19). Synthesis of the F pilus is not well understood, despite the morphological simplicity of this organelle (7, 15, 28). The F transfer region consists of nearly 40 tra genes, with 18 being involved in construction of the transferosome, which is involved in pilus synthesis, mating pair stabilization, and DNA transfer (9). Eight of the encoded Tra proteins (TraA, -B, -C, -E, -G [the N-terminal domain], -K, -L, and -V) correspond to widely conserved members of type IV secretion systems (T4SS), whereas another 9 (TraF, -G [C-terminal domain], -H, -N, -U, and -W and TrbB, -C, and -I) are involved in the F-specific T4SS (4, 18). Two other proteins (TraQ and -X) are specific to the F plasmid itself. The roles of the F-specific proteins that are involved in pilus assembly and DNA transfer are intriguing, since other conjugative T4SS appear to function efficiently without them (18). These tra proteins do not affect F pilin levels, and hence, they have been assigned functions in pilus assembly/retraction and mating pair stabilization, which are characteristics of F-like transfer systems (18). TraF, -H, and -W and TrbC are required for F pilus assembly (9), and mutations in traU reduce the number and the mean length of pili but do not abolish pilus outgrowth (24). TraU is required for DNA transfer and has been tentatively grouped with TraN and -G as proteins involved in mating pair stabilization (18). TrbI is thought to play a role in pilus retraction, since trbI mutants have unusually long pili (21). TrbB contains the thioredoxin-like domain with a C-X-X-C motif and appears to be a periplasmic disulfide bond isomerase (6). Previously, we hypothesized that TrbB and TraF, the latter of which also has the thioredoxin-like domain but lacks the C-X-X-C motif, might have chaperone-like activity. These proteins might help F T4SS proteins such as TraH, -U, and -N, which have 6, 10, and 22 conserved cysteines, respectively, achieve the correct conformation for assembly into the transferosome complex (6). Interestingly, yeast two-hybrid (Y2H) analysis demonstrated that TraF, -H, -U, and -W and TrbB and -I form an interaction group, with TraH directly linked to TraF, TraU, and TrbI (14). TraH is the only one of the three cysteine-rich proteins required for pilus assembly; it is the largest protein (458 amino acids [aa]; 50.2-kDa precursor, processed to 47.8 kDa) in the interaction group and contains a C-terminal coiled-coil domain that can contribute to its oligomerization and interaction with other T4SS proteins (18). Y2H analysis also showed that the C-terminal region of TraH is critical for its interaction with TraF (28 kDa, processed to 25.9 kDa) and TraU (36.8 kDa, processed to 34.3 kDa) and that a deletion within the N-terminal region of TraH enhanced its interaction with TrbI (14.1 kDa) (14).Mutations in traH affect pilus outgrowth but not pilus tip formation at the cell surface, since traH mutants are sensitive to the M13K07 transducing phage, which binds to the pilus tip (1). Membrane fractionation studies of cells containing subclones of the F transfer region originally suggested that TraH fractionates with the inner membrane (IM) (22). TraH contains three N-terminal hydrophobic domains of approximately 20 aa each, which supports this model. In contrast, Ham et al. predicted TraH to be a soluble periplasmic protein (12). Sucrose density gradient sedimentation studies suggested that FLAG-tagged TraH, in the presence of F lac traH80, is in the outer membrane (OM) (23). Since TraH is extracted from membrane preparations with guanidine-HCl or urea but not Triton X-100, Manwaring concluded that TraH is a peripherally associated outer membrane protein (23). By use of subclones of the F transfer region, TraF, -U, -W, and TrbB were localized to the periplasm, whereas TrbI was thought to be an inner membrane protein (21, 24, 29, 30). Using the F plasmid derivative pOX38-Tc (2), which carries the entire F transfer region, we reassessed the localization of TraH as well as TraF, TraU, and TraW (23.6 kDa, processed to 21.7 kDa).E. coli strains were grown at 37°C in Luria-Bertani (LB) broth (1% tryptone [Difco], 0.5% yeast extract [Difco], 1% NaCl [BDH]) with shaking to mid-exponential phase (optical density at 600 nm [OD₆₀₀] of ca. 0.5) with appropriate antibiotics at the following concentrations: 50 μg/ml ampicillin (Ap), 20 μg/ml chloramphenicol (Cm), 25 μg/ml kanamycin (Km), 200 μg/ml streptomycin (Sm), 100 μg/ml spectinomycin (Sp), and 10 μg/ml tetracycline (Tc). Sucrose density flotation studies of cell membrane fractions and immunoblot analysis were performed as previously described (17). Cell pellets corresponding to 0.1 OD₆₀₀ equivalents were used in all immunoblot assays. Samples were boiled in sodium dodecyl sulfate (SDS) sample buffer for 5 min and were analyzed by resolving SDS-15% polyacrylamide gel by using the Bio-Rad Minigel system. The positions of the inner and outer membrane fractions were determined using polyclonal antibodies to the C-terminal region of OmpA, the major outer membrane porin, and CpxA, the inner membrane sensor of the CpxAR two-component system (25). Anti-CpxA, anti-TraE, anti-TraF, anti-TraH, anti-TraU, anti-TraW, and anti-TrbB polyclonal antisera (raised in rabbits) were diluted 1:7,000, 1:5,000, 1:2,000, 1:1,000; 1:500, 1:20,000 and 1:10,000, respectively, in blocking solution and were incubated with the blots at room temperature for 1 h. Anti-OmpA antibodies were used at a 10⁻⁵ dilution in 5% bovine serum albumin (BSA; Roche) to avoid heavy background. Unfortunately, TrbI protein could not be overproduced and specific antibodies could not be raised.Log-phase cultures of E. coli MC4100 (Sm^r) (17) containing pOX38-Tc (2) were separated into periplasmic, cytoplasmic, and membrane fractions according to a previously described method (26). The fractions were tested for the presence of TraH, TraF, TraU, and TraW by SDS-PAGE, followed by immunoblot analysis. All four proteins were found associated with the membrane fraction and not the periplasmic fraction (Fig. ​(Fig.1A).1A). TrbB was found in the periplasmic fraction, in agreement with its proposed role in disulfide bond isomerization (6; data not shown).Open in a separate windowFIG. 1.(A) The F-specific proteins TraH, -F, -U, and -W were detected in the membrane fraction when expressed from MC4100/pOX38-Tc. Proteins were detected by immunoblotting using antisera specific for each protein as described in the text. (B) TraF localization was tested in pOX38-Km and pOX38 ΔtraV::cat. The cells were fractionated into cytoplasmic (C), periplasmic (P), and total membrane (M) fractions, and TraF was detected by immunoblotting with anti-TraF antibodies. TraV was complemented by pRS29 (pRS31 acted as a negative control). The following abbreviations are used: WT, wild type; ΔV, pOX38 ΔtraV::cat; ΔH, pOX38-Tc ΔtraH::cat; and ΔF, pOX38 ΔtraF::kan. The positions of the proteins are indicated by arrows on the right of each panel. The asterisk indicates a band that reacts nonspecifically with anti-F antiserum.Sucrose density flotation gradients of the membrane preparations of MC4100 (Sm^r) cells harboring pOX38-Tc (2), pOX38-Tc ΔtraF::kan (6) and pOX38-Tc ΔtraH::cat were performed to distinguish between OM and IM proteins according to reference 17. pOX38-Tc ΔtraH::cat was constructed according to the method described by Elton et al. (6) by inserting a chloramphenicol acetyltransferase cassette into traH. Gradients were fractionated, and a subset of the fractions (fractions 26 to 54, renamed 1 to 29) that contained the proteins of interest were subjected to SDS-PAGE and immunoblot analyses (Fig. ​(Fig.2).2). OmpA and CpxA were controls for the outer and inner membrane fractions and helped define the subset of fractions examined (Fig. ​(Fig.2,2, panels 1 and 2, respectively). The TraE pilus assembly protein of the F plasmid was used as an IM marker for the F transfer system (Fig. ​(Fig.2,2, panel 9) (9). TraH fractionated as an OM protein in MC4100/pOX38-Tc (Fig. ​(Fig.2,2, panel 3), as did TraF, TraU, and TraW (Fig. ​(Fig.2,2, panels 5, 7, and 8, respectively). TraH did not appear to be required for TraF localization, which was unaffected in a traH mutant (Fig. ​(Fig.2,2, panel 6). In addition, TraF did not appear to be required for TraH localization, although its absence caused a reduction in the levels of TraH (Fig. ​(Fig.2,2, panel 4; see below).Open in a separate windowFIG. 2.The cellular localizations of TraE, TraF, TraH, TraU, and TraW in subcellular fractions of E. coli MC4100/pOX38-Tc and its derivatives. Flotation sucrose density gradients were performed with subsequent immunodetection of tra proteins in a subset of gradient fractions (fractions 26 to 54, renumbered 1 to 29). The positions of the IM and OM fractions are shown above the gels, and the identities of the samples are indicated on the left. The panel numbers are indicated on the right.TraF, -H, -U, and -W appear to be periplasmic proteins that associate with the outer membrane when in the context of the complete transfer apparatus. TrbC, which is fused to TraW in the F-like R27 T4SS, might also be part of this complex (18). Therefore, an as yet unidentified transfer protein should act as an anchor in the outer membrane, directing these proteins to this location. Of the 18 transferosome proteins, only TraV and TraN are known to be located in the OM, with TraV being the only OM protein involved in pilus assembly. Preliminary localization studies using TraF as a test case and a traV insertion mutant, pOX38 ΔtraV::cat (this study, constructed as described above for pOX38-Tc ΔtraH::cat), demonstrated that the levels of TraF decreased dramatically. However, the remaining TraF was found in the periplasm (Fig. ​(Fig.1B).1B). Complementation of the traV mutation with pRS29, but not pRS31 (1), restored TraF localization to the outer membrane. Thus, TraV is probably the anchor protein for both the F-specific transferosome proteins (TraF, -H, -U, and -W) as well as the TraV, -K, and -B complex (13).MC4100 (Sm^r) cells bearing pOX38-Tc (2) or insertion mutant pOX38-Tc ΔtraH::cat, pOX38-Tc ΔtraF::kan (6), pOX38-Tc ΔtrbB::cat (6), pOX38-Tc ΔtraW::cat (this study), pOX38 traU347 (Km^r) (24), or pOX38-trbI472 (Km^r) (21) were used in subsequent experiments. pOX38-Tc ΔtraW::cat was constructed according to the method described by Elton et al. (6) by inserting a chloramphenicol acetyltransferase gene within traW. Mating efficiencies of these mutants were determined according to previously described methods using E. coli ED24 (Sp^r) as the recipient (20). Transconjugants were selected based on double resistance toward chloramphenicol or kanamycin and spectinomycin (Fig. ​(Fig.2).2). Observed mating efficiencies were in agreement with the data obtained previously, as were the results of complementation assays using subclones carrying the appropriate transfer gene (1, 6, 21, 24, 29, 30). These subclones were pK184TraH (Km^r) (this study), pFTraF and pFTrbB (Ap^r) (6), and pKI175 (Ap^r; traWU) (30) (Fig. ​(Fig.2).2). pK184TraH is based on the vector pK184 (Km^r) and contains the traH gene plus its ribosome binding site cloned into the EcoRI and HindIII sites in pK184 (16). Immunoblot analyses revealed that traF, traU, or trbB, but not traW, insertion mutants had slightly reduced levels of TraH in MC4100 cells whereas the trbI insertion mutant had undetectable levels of TraH (Fig. ​(Fig.3).3). Since TraH interacts directly with TrbI, TraF, and TraU in Y2H assays (14), the absence of these proteins would be expected to destabilize TraH. TraH is thought to interact indirectly with TraW via TraU (14); its levels were unaffected in a traW mutant. TraH was destabilized in a dsbA mutant and was undetectable by immunoblotting (data not shown) and decreased slightly in a trbB mutant, suggesting that disulfide bond formation (DsbA) and isomerization (TrbB) are important for TraH.Open in a separate windowFIG. 3.Immunoblot analysis of the levels of TraH in the absence of other members of Y2H interaction group by using pOX38-Tc and its derivatives containing insertion mutations in traH, traF, trbB, traW, traU, or trbI. A loading control is shown in the lower panel, and the mating efficiency (ME) expressed as a percentage of transconjugants relative to donor cells is given below the gels. n.d., not detected; n.a., not applicable. The last line of data are the complementation data (percent complementation mating efficiency [CME]) obtained by use of clones as described in the text. Previously, TraH was found to interact with TraF, TraU, and TrbI, and TraU interacts with TraW (14).The absence of TrbI appeared to have the most profound effect on the level of TraH, although there was only a 20-fold decrease in mating efficiency, suggesting that enough TraH was present to support mating (Fig. ​(Fig.3).3). Complementation assays performed with pOX38-trbI472 and pBAD24TrbI plasmids (this study) restored the levels of TraH, possibly by stabilizing it (Fig. ​(Fig.3).3). pBAD24TrbI is based on the vector pBAD24 (Ap^r) and contains the trbI gene cloned into the EcoRI site in pBAD24 (11). However, complementation with pBAD24TrbI did not restore mating efficiency to wild-type levels, confirming that the insertion mutation within pOX38-trbI472 has a weak polar effect on downstream genes in the tra operon (21). Alternatively, overexpression of TrbI from pBAD24TrbI affected mating efficiency.Y2H analysis revealed two regions within TraH that appeared to be important for TraH-TrbI interactions (14). The deletion of 50 N-terminal amino acids (aa 25 to 75) from the mature TraH gave a 40-fold increase in TraH-TrbI interaction in the Y2H assay (14). This region of TraH also contains the highly conserved residues N31, T44, G60, and R65 (numeration includes the 25-aa signal peptide) (Fig. ​(Fig.4A).4A). Site-directed mutagenesis was performed on plasmid pK184TraH by using the QuikChange kit (Stratagene). The mating abilities of MC4100/pOX38-Tc ΔtraH::cat/pK184TraH and derivatives with amino acid substitutions N31A, T44A, G60A, and R65A were determined according to previously described methods using ED24 (Sp^r) as the recipient (20). Transconjugants were selected based on double resistance toward tetracycline and spectinomycin. TraH levels within the donor cells were monitored by immunoblot analysis. The N31A and T44A substitutions did not affect mating efficiency and did not change the level of TraH within donor cells (Fig. ​(Fig.5).5). The G60A and R65A substitutions decreased mating efficiency to undetectable levels. TraH levels remained unchanged in both mutants (Fig. ​(Fig.5).5). MC4100/pOX38-Tc ΔtraH::cat cells with pK184TraHG60A or pK184TraHR65A were also resistant toward pilus-specific phage f1, suggesting that the pilus was not assembled.Open in a separate windowFIG. 4.Multiple sequence alignment of F-like TraH proteins. (A) Alignment of the N-terminal regions. (B) Alignment of the putative TraH-TrbI interaction region. The leader peptide is cleaved after A24 in F TraH, which is marked by an arrow. The degrees of identity are indicated by black and gray boxes above the sequences, with the tallest black boxes representing conservation over all 7 sequences. Positions with 5 or more different amino acids are marked with the shortest black boxes. The gray boxes in the residue number line indicate gaps in some of the sequences. The putative nucleotide triphosphate (NTP) binding site (aa 193 to 200) and the conserved sequence (aa 220 to 226) are underlined. The regions thought to interact with TrbI are bracketed. Asterisks refer to amino acids selected for mutational analysis. GenBank protein accession numbers for the sequences are as follows: for F, {"type":"entrez-protein","attrs":{"text":"BAA97968","term_id":"8918921","term_text":"BAA97968"}}BAA97968; for SXT, {"type":"entrez-protein","attrs":{"text":"AAL59676","term_id":"21885270","term_text":"AAL59676"}}AAL59676; for R391, {"type":"entrez-protein","attrs":{"text":"AAM08008","term_id":"20095142","term_text":"AAM08008"}}AAM08008; for pNL1, {"type":"entrez-protein","attrs":{"text":"NP_049152","term_id":"10956932","term_text":"NP_049152"}}NP_049152; for RTS1, {"type":"entrez-protein","attrs":{"text":"NP_640201","term_id":"21233903","term_text":"NP_640201"}}NP_640201; for pED208, {"type":"entrez-protein","attrs":{"text":"AAM90722","term_id":"22539465","term_text":"AAM90722"}}AAM90722; and for R27, {"type":"entrez-protein","attrs":{"text":"NP_058340","term_id":"10957316","term_text":"NP_058340"}}NP_058340. Sequence alignment was performed with DNAStar software (LazerGene), using the ClustalW algorithm. Highly conserved amino acids as well as a consensus sequence are given above the residue number line.Open in a separate windowFIG. 5.Immunoblot analysis of intracellular levels of TraH in MC4100/pOX38-Tc ΔtraH::cat complemented with different pK184TraH plasmids. C represents the vector control pK184, WT is the wild-type pK184TraH plasmid, and an asterisk refers to the nonspecific band used as the loading control. Mating efficiency (ME), expressed as a percentage of transconjugants relative to donor cells, is given below the gels. n.d., not detected; MW, molecular mass.Sequence analysis also showed the presence of conserved residues N(L/I/Y)X(W/Y)XX(F/L) (N₂₂₀IMWNAL₂₂₆ in F TraH) within the putative TrbI interaction domain (aa 193 to 226) (Fig. ​(Fig.4B)4B) (14). Substitution of N220 with alanine (N220A) did not change the levels of TraH protein in pOX38-Tc ΔtraH::cat/pK184TraHN220A but decreased the mating ability to undetectable levels. The W223A mutation in TraH decreased the level of TraH within donor cells and reduced the mating efficiency 1,000-fold compared to the wild-type level (Fig. ​(Fig.5).5). The N220A and W223A mutants were resistant to f1 phage and could not assemble functional pili. Thus, mutations in N220 and W223 could affect TraH-TrbI interaction, or they may act independently to block TraH function. If TrbI is in the IM as previously reported (21), then the TrbI:TraH pair could be part of a second envelope-spanning structure analogous to the TraV:TraK:TraB scaffold (8, 13).Primary sequence analysis also revealed the presence of a putative Walker A motif within aa 193 to 226 of TraH (G₁₉₃CTVGGKS₂₀₀) (9). Comparison of seven TraH orthologs revealed that this motif is not conserved among TraH-like sequences (Fig. ​(Fig.4B).4B). To confirm whether this sequence might be important in the F plasmid, a triple mutant (G193A/K199A/S200A) was constructed. It reduced mating efficiency 20-fold but did not change the levels of TraH within donor cells (data not shown). Single substitutions (G193A, K199A, or S200A) did not change the mating efficiency or the level of TraH (data not shown). Thus, TraH, a peripheral OM protein, is probably not an NTPase, nor does it bind nucleotides.Our data also revealed that several conserved amino acid residues are critical for TraH function and structure and that TraH stability is dependent on TrbI as well as DsbA and TrbB, which affect disulfide bond formation and isomerization, respectively. Thus, TrbI, in which mutations have only a minor effect on mating ability, plays a more important role than previously thought (21).     

Vaccinia Virus Protein F12 Associates with Intracellular Enveloped Virions through an Interaction with A36

Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.     

Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI.

The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid. We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141. Studies of traW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing. Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein. Electron microscopy also showed that F lac traW546 hosts do not express F pili, confirming that TraW is required for F-pilus assembly. Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW. The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product. Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein. Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages. Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency. Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance. The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed.     

F-like type IV secretion systems encode proteins with thioredoxin folds that are putative DsbC homologues

F and R27 are conjugative plasmids of enteric bacteria belonging to the IncF and IncHI1 plasmid incompatibility groups, respectively. Based on sequence analysis, two genes of the F transfer region, traF and trbB, and three genes of the R27 transfer region, trhF, dsbC, and htdT, are predicted to encode periplasmic proteins containing a C-terminal thioredoxin fold. The C-X-X-C active-site motif of thioredoxins is present in all of these proteins except TraF(F). Escherichia coli carrying a dsbA mutation, which is deficient in disulfide bond formation, cannot synthesize pili and exhibits hypersensitivity to dithiothreitol (DTT) as monitored by mating ability. Overproduction of the E. coli disulfide bond isomerase DsbC, TrbB(F), DsbC(R27), or HtdT(R27), but not TraF(F) or TrhF(R27), reverses this hypersensitivity to DTT. Site-directed mutagenesis established that the C-X-X-C motif was necessary for this activity. Secretion into the periplasm of the C-terminal regions of TrbB(F) and DsbC(R27), containing putative thioredoxin folds, but not TrhF(R27), partially complemented the host dsbA mutation. A trbB(F) deletion mutant showed a 10-fold-lower mating efficiency in an E. coli dsbC null strain but had no phenotype in wild-type E. coli, suggesting redundancy in function between TrbB(F) and E. coli DsbC. Our results indicate that TrbB(F), DsbC(R27), and HtdT(R27) are putative disulfide bond isomerases for their respective transfer systems. TraF(F) is essential for conjugation but appears to have a function other than disulfide bond chemistry.     

RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation

The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.     

Identification and characterisation of regions in the cellular protein LaXp180 and the<Emphasis Type="Italic"> Listeria monocytogenes</Emphasis> surface protein ActA necessary for the interaction of the two proteins

The Listeria monocytogenes surface protein ActA is an important virulence factor that plays an essential role in intracellular movement of Listeria cells by inducing actin polymerisation. The ActA protein is known to interact with several mammalian proteins including the phosphoprotein VASP, actin and the Arp2/3 complex. In a search for additional ActA-binding proteins we recently employed the yeast two-hybrid system to search for proteins that interact with ActA, and identified, among others, the mammalian protein LaXp180 as a binding partner. In the present study the interaction of the two proteins was investigated in more detail. A number of variants were tested in the yeast two-hybrid system for their ability to interact. On the basis of these assays, the 14 C-terminal amino acids of LaXp180 were identified as being necessary for the interaction with ActA. The proline-rich repeat (PRR) region of ActA was found to be necessary for the interaction with LaXp180, but upstream or downstream sequences are also required to enhance the specificity of the interaction. The second and third repeats in ActA are especially important, and the minimal sequence of ActA capable of interacting with LaXp180 was a proline- and glutamate-rich stretch of PRR3 fused to part of the N-terminal sequence of ActA. Further analysis using site-specific mutations located in either the C-terminal region of LaXp180 or the proline-rich motif of PRR3 of ActA showed that three positively charged amino acids in LaXp180 and two negatively charged amino acids in ActA are critical for the interaction of the two proteins.     

KIF2Abeta: A kinesin family member enriched in mouse male germ cells, interacts with translin associated factor-X (TRAX)

Translin associated factor X (TRAX) is a binding partner of TB-RBP/Translin. A cDNA encoding the 260 C-terminal amino acids of KIF2Abeta was isolated from mouse testis cDNAs in a yeast two-hybrid library screen for specific TRAX-interacting proteins. KIF2Abeta was expressed predominantly in the mouse testis and enriched in germ cells. The interaction of full-length KIF2Abeta or its C-terminus with TRAX was verified using in vitro synthesized fusion proteins. Deletion mapping of the TRAX-binding region of KIF2Abeta indicated that amino acids 514-659 were necessary and sufficient for the interaction in vivo. Confocal microscopy studies using GFP-fusion proteins demonstrated that KIF2Abeta colocalizes with TRAX in a perinuclear location. KIF2Abeta does not interact with TB-RBP, suggesting that either TRAX can function as an adaptor molecule for motor proteins and TB-RBP, or that this interaction reveals an undescribed role for TRAX in germ cells. The interaction with KIF2Abeta suggests a role for TRAX in microtubule-based functions during spermatogenesis.     

Comparative Biochemical Studies on F and EDP208 Conjugative Pili

EDP208 pili are encoded by a derepressed derivative of a naturally occurring lac plasmid, F(0)lac (incompatibility group FV), originally isolated from Salmonella typhi. EDP208 pili are serologically unrelated to F pili and do not promote infection by F-specific ribonucleic acid bacteriophages. However, they do confer sensitivity to the F-specific filamentous deoxyribonucleic acid phages. EDP208-containing cells are multi-piliated and contain approximately 20 pili per cell. These pili contain a single polypeptide subunit of 11,500 daltons. EDP208-specific RNA phages were readily isolated from local sewage. These phages were somewhat smaller in diameter than the F-specific ribonucleic acid phages and absorbed relatively weakly to EDP208 pili. Comparing EDP208 pilin to F, it was found that both contain the equivalent of two to three hexose units per subunit as well as blocked N-termini. EDP208 pilin contains one covalently linked phosphate residue per subunit, whereas the F pilin subunit contains two such residues. Although notable differences were found in the case of three or four amino acids, the overall amino acid compositions of F and EDP208 were very similar. Moreover, the tryptic peptide maps of the two proteins contained seven peptides with similar mobilities, suggesting considerable homology in their amino acid sequences. Substantial similarities were also noted in the secondary structures of F and EDP208 pilin on the basis of circular dichroism studies. The alpha-helix content of both proteins was calculated to be 65 to 70%. X-ray fiber diffraction studies have indicated that the arrangements of subunits in F and EDP208 pili are also similar. It was concluded that F and EDP208 pili are closely related structures.     

Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system.

Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling. Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined. In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor. We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells. We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site. Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay. These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved. Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay. Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells. The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction.     

Major antigenic determinants of F and ColB2 pili.

F-like conjugative pili are expressed by plasmids with closely related transfer systems. They are tubular filaments that are composed of repeating pilin subunits arranged in a helical array. Both F and ColB2 pilin have nearly identical protein sequences, and both contain an acetylated amino-terminal alanine residue. However, they differ by a few amino acid residues at their amino termini. Rabbit antisera raised against purified F and ColB2 pili are immunologically cross-reactive by only 25%, as measured by a competition enzyme-linked immunosorbent assay (ELISA). A tryptic peptide corresponding to the first 15 amino acid residues of ColB2 pilin was isolated and found to remove nearly 80% of ColB2 pilus-directed rabbit antibodies. The corresponding tryptic peptide from F pilin, which reacted with anti-F pilus antibodies to remove 80%, was less than 20% reactive with anti-ColB2 pilus antiserum. Cleavage of these peptides with cyanogen bromide (at a methionine residue approximately in the middle of the peptide) did not affect the antigenicity of these peptides. Synthetic N alpha-acetylated peptides corresponding to the first eight amino acids of F pilin (Ac-Ala-Gly-Ser-Ser-Gly-Gln-Asp-Leu-COOH) and the first six amino acids of ColB2 pilin (Ac-Ala-Gln-Gly-Gln-Asp-Leu-COOH) were prepared and tested by competition ELISA with homologous and heterologous anti-pilus antisera. The F peptide F(1-8) inhibited the interaction of F pili and anti-F pilus antiserum to 80%, while the ColB2 peptide ColB2(1-6) inhibited anti-ColB2 pilus antiserum reacting with ColB2 pili by greater than 60%. The two peptides F(1-8) and ColB2(1-6) were inactive by competition ELISAs with heterologous antisera. These results suggest that the major antigenic determinant of both F and ColB2 pili is at the amino terminus of the pilin subunit and that 80% of antibodies raised against these pili are specific for this region of the pilin molecule.     

Structure and antigenic properties of the tip-located P pilus proteins of uropathogenic Escherichia coli.

Pyelonephritogenic Escherichia coli frequently expresses pili which bind to Gal alpha (1-4)Gal receptors present on the uroepithelium. Binding of these pili is mediated by a pilus-associated adhesin, PapG, and not by the major subunit which constitutes the bulk of the pilus structure. The adhesin and two pilinlike proteins, PapE and PapF, are present in only a few copies each at the pilus tip. Surface exposure of both PapF and PapG is required to achieve receptor-specific binding. The nucleotide sequences for the genes encoding the tip-associated proteins PapE, PapF, and PapG were determined for two E. coli clones expressing P pili of serotypes F11 and F7(2) and compared with the corresponding sequences established for proteins of F13 pili. Specific antisera were used to study the cross-reactivity between the F13 tip proteins and the equivalent proteins in F11 and F7(2) pili. We present data showing that, like the major pilus subunit, PapE varies its structure and antigenic properties among pili of different serotypes. In contrast, the PapF protein was highly conserved, and PapF-specific antisera raised against serotype F13 cross-reacted with the PapF proteins of both F11 and F7(2) serotypes. The PapG adhesin protein from F11 and F7(2) pili differed by only five amino acids out of 316 residues. However, the F13 adhesin showed only 45% amino acid homology with the other two variants.     

Mapping the interaction site of Rpb4 and Rpb7 subunits of RNA polymerase II in Saccharomyces cerevisiae

Rpb4 and Rpb7, the fourth and the seventh largest subunits of RNA polymerase II, form a heterodimer in Saccharomyces cerevisiae. To identify the site of interaction between these subunits, we constructed truncation mutants of both these proteins and carried out yeast two hybrid analysis. Deletions in the amino and carboxyl terminal domains of Rpb7 abolished its interaction with Rpb4. In comparison, deletion of up to 49 N-terminal amino acids of Rpb4 reduced its interaction with Rpb7. Complete abolishment of interaction between Rpb4 and Rpb7 occurred by truncation of 1-106, 1-142, 108-221, 172-221 or 198-221 amino acids of Rpb4. Use of the yeast two-hybrid analysis in conjunction with computational analysis of the recently reported crystal structure of Rpb4/Rpb7 sub-complex allowed us to identify regions previously not suspected to be involved in the functional interaction of these proteins. Taken together, our results have identified the regions that are involved in interaction between the Rpb4 and Rpb7 subunits of S. cerevisiae RNA polymerase II in vivo.     

Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly.

The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pilus protein binding site of the chaperone. The ability of PapD to bind to fiber-forming pilus subunit proteins to prevent their participation in misassembly interactions depended on the invariant, solvent-exposed arginine-8 (R8) cleft residue. This residue was also essential for the interaction between PapD and a minor pilus adaptor protein. A mutation in the conserved methionine-172 (M172) cleft residue abolished PapD function when this mutant protein was expressed below a critical threshold level. In contrast, radical changes in the variable residue glutamic acid-167 (E167) had little or no effect on PapD function. These studies provide the first molecular details of how a periplasmic pilus chaperone binds to nascently translocated pilus subunits to guide their assembly into adhesive pili.     

The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.     

Autoinhibition of p50 Rho GTPase-activating protein (GAP) is released by prenylated small GTPases

Interaction of p50 Rho GTPase-activating protein (p50RhoGAP) with Rho family small GTPases was investigated in a yeast two-hybrid system, by radioactive GAP assay, and in a Rac-regulated enzymatic reaction, through superoxide production by the phagocytic NADPH oxidase. The yeast two-hybrid system revealed an interaction between the C-terminal GAP domain and the N-terminal part of p50RhoGAP. The first 48 amino acids play a special role both in the stabilization of the intramolecular interaction and in recognition of the prenyl tail of small GTPases. The GAP assay and the NADPH oxidase activity indicate that the GTPase-activating effect of full-length p50RhoGAP is lower on non-prenylated than on prenylated small GTPase. Removal of amino acids 1-48 and 169-197 of p50RhoGAP increases the GAP effect on non-prenylated Rac, whereas prenylated Rac reacts equally well with the full-length and the truncated proteins. We suggest that p50RhoGAP is in an autoinhibited conformation stabilized by the stretches 1-48 and 169-197 and the prenyl group of the small GTPase plays a role in releasing this intramolecular restraint.