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1.
Comparative Biochemical Studies on F and EDP208 Conjugative Pili   总被引:11,自引:9,他引:2       下载免费PDF全文
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.  相似文献   

2.
Characterization and sequence analysis of pilin from F-like plasmids.   总被引:20,自引:11,他引:9       下载免费PDF全文
Conjugative pili are expressed by derepressed plasmids and initiate cell-to-cell contact during bacterial conjugation. They are also the site of attachment for pilus-specific phages (f1, f2, and QB). In this study, the number of pili per cell and their ability to retract in the presence of cyanide was estimated for 13 derepressed plasmids. Selected pilus types were further characterized for reactivity with anti-F and anti-ColB2 pilus antisera as well as two F pilus-specific monoclonal antibodies, one of which is specific for a sequence common to most F-like pilin types (JEL92) and one which is specific for the amino terminus of F pilin (JEL93). The pilin genes from eight of these plasmids were cloned and sequenced, and the results were compared with information on F, ColB2, and pED208 pilin. Six pilus groups were defined: I, was F-like [F, pED202(R386), ColV2-K94, and ColVBtrp]; IIA was ColB2-like in sequence but had a lowered sensitivity to f1 phage due to its decreased ability for pilus retraction [pED236(ColB2) and pED203(ColB4)]; IIB was ColB2-like but retained f1 sensitivity [pED200(R124) and pED207(R538-1)]; III contained R1-19, which had a ColB2-like amino terminus but had an additional lysine residue at its carboxy terminus which may affect its phage sensitivity pattern and its antigenicity; IV was R100-1-like [R100-1 and presumably pED241(R136) and pED204(R6)] which had a unique amino-terminal sequence combined with a carboxy terminus similar to that of F. pED208(Folac) formed group V, which was multipiliated and exhibited poor pilus retraction although it retained full sensitivity to f1 phage. The pED208 pilin gene could not be cloned at this time since it shared no homology with the pilin gene of the F plasmid.  相似文献   

3.
Major antigenic determinants of F and ColB2 pili.   总被引:9,自引:7,他引:2       下载免费PDF全文
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.  相似文献   

4.
N-terminal amino acid sequencing of EDP208 conjugative pili.   总被引:7,自引:6,他引:1       下载免费PDF全文
EDP208 conjugative pili contain a single polypeptide subunit of 11,500 daltons with a blocked N-terminus. This N-terminal blocking moiety was identified as an N-acetyl group by 1H nuclear magnetic resonance analysis of an N-terminal tripeptide isolated from pronase digests of EDP208 pilin. Limited acid hydrolysis of the tripeptide allowed its sequence to be determined as acetyl-NH-Thr-Asp-Leu. Trypsin digestion of EDP208 pilin resulted in the quantitative release of a fragment containing 12 residues from the N-terminus of the protein. The sequence of this dodecapeptide was determined to be acetyl-NH-Thr-Asp-Leu-Leu-Ala-Gly-Gly-Lys-Asp-Val-Asp-Lys.  相似文献   

5.
Location of the antigenic determinants of conjugative F-like pili   总被引:4,自引:3,他引:1       下载免费PDF全文
The amino terminus of the pilin protein constitutes the major epitope of F-like conjugative pili studied to date (F, ColB2, R1-19, R100-1, and pED208). Anti-pED208 pilus antibodies were passed through a CNBr-Sepharose affinity column linked to bovine serum albumin which was conjugated to a synthetic peptide, AcP(1-12), containing the major epitope at the amino terminus of pED208 pilin. This allowed the separation of two classes of antibodies; one was specific for the amino terminus and bound to the column, while the other, which recognizes a second epitope on the pilus, did not bind to the column. In addition, antibodies were raised against two amino-terminal peptide-bovine serum albumin conjugates [AcP(1-8) and AcP(1-12)] to ensure a source of pure, high-titer antibodies directed against the amino terminus. The location of these antibodies on intact pili was assayed by immunoelectron microscopy with a protein A-gold technique. The amino terminus-specific antibodies did not bind to the sides of the pili but appeared to be associated with the pilus tip. In addition, these antibodies were found to bind to the vesicle-like structure at the base of the pilus. The anti-pilus antibodies not specific for the amino terminus (unbound immunoglobulin G) were found to bind to the sides of the pilus. Anti-F and anti-ColB2 pilus antibodies bound to the sides of F, ColB2, and R1-19 pili, which have only their secondary epitope in common. The carboxyl-terminal lysine of R1-19 pilin prevents the absorption of anti-F plus antiserum but not anti-ColB2 pilus antiserum to the sides of the pilus, presumably by interfering with the recognition of this secondary epitope.  相似文献   

6.
Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.  相似文献   

7.
ColB2 is a colicin-producing, 96-kilobase plasmid which encodes a conjugative system that is similar, but not identical, to F. A restriction map of this plasmid was generated, and DNA homology studies between F and ColB2 plasmids revealed homology only between their transfer operons. The locations of the ColB2 transfer operon and ColB2 pilin gene were localized on this restriction map. The gene encoding ColB2 pilin, traA, was cloned and sequenced. The pilin protein of ColB2 is identical to F, except at the amino terminus, where ala-gln of ColB2 pilin corresponds to Ala-Gly-Ser-Ser of F pilin. This is due to a 6-base-pair deletion in the ColB2 pilin gene. Biochemical studies on tryptic peptides derived from ColB2 pilin demonstrate the location of this gene to be correct. There is a putative signal peptidase cleavage site after the sequence Ala-Met-Ala, giving a signal peptide of 51 amino acids and a mature pilin protein of 68 amino acids (7,000 daltons). The amino terminus is blocked, probably with an acetyl group. A chimera containing the ColB2 pilin gene was able to complement an F traA mutant, demonstrating that the pilus assembly proteins of F can utilize the ColB2 pilin protein to form a pilus.  相似文献   

8.
The preparation and properties of gonococcal pili.   总被引:8,自引:0,他引:8  
Pili have been isolated from Neisseria gonorrhoeae by controlled homogenization followed by selective disaggregation in sucrose and purification by CsCl density gradient centrifugation. Pili from six gonococcal strains had buoyant densities of 1-30 to 1-31 g ml-1 on CsCl. The pili were immunologically distinct when tested with rabbit antisera to purified pili. The amino acid composition of pilin from strains P9 and 201 was very similar, consisting of 208 and 212 amino acid residues respectively giving molecular weights of 22 600 and 22352. The pili contained a high proportion (46%) of non-polar amino acids. Further analysis of strain P9 pili revealed the presence of 1 to 2 phosphate groups and 1 to 2 hexose groups per pilin subunit; no amino sugars were detected. Pili from strain P9 were resolved into two bands by equilibrium density gradient centrifugation or column isoelectric focusing, suggesting the presence of more than one kind of pilus.  相似文献   

9.
Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO.   总被引:17,自引:0,他引:17  
Pseudomonas aeruginosa strains PAO and PAK bear polar pili which are flexible filaments having a diameter of 6 nm and an average length of 2500 nm. Both types of pili are retractile and promote infection by a number of bacteriophages. The present communication describes the partial biochemical characterization of PAO pili isolated from a multipiliated nonretractile mutant of PAO. The observed properties are compared to those of PAK pili which were characterized previously. PAO pili were found to contain a single polypeptide subunit of 18 700 daltons. This is similar to PAK pili which contain a single polypeptide of 18 100 daltons. The amino acid composition of PAO pilin was also similar to that of PAK pilin. Neither protein contained phosphate or carbohydrate residues and both were found to contain N-methylphenylalanine at the amino terminus. Sequencing of 20 amino acid residues at the amino terminal end of PAO pilin revealed the sequence to be identical with that of PAK pilin, while tryptic peptide analyses of PAO and PAK pilin indicated that the two proteins probably contain a number of homologous regions within the polypeptide. It was concluded that PAO and PAK pili were closely related structures.  相似文献   

10.
Phage Folac: an Folac plasmid-dependent bacteriophage   总被引:2,自引:0,他引:2  
By enriching sewage with Escherichia coli or Salmonella typhimurium strains harbouring the plasmid EDP208, a constitutive pilus-producing derivative of plasmid F olac, a phage was isolated which plated on these two organisms but not on isogenic strains without the plasmid. The phage was named F olac; it has a hexagonal outline with a diameter of 28 nm, contained RNA, was resistant to chloroform, and probably adsorbed preferentially to the sides of EDP208 pili very near the tip. Phage multiplication could be demonstrated on E. coli or S. typhimurium strains carrying the plasmid F olac, but an increase in titre did not occur on E. coli strains carrying plasmids of the F complex. Results of phage multiplication experiments on strains carrying the depressed pilus-producing plasmids R71 or TP224-Tc, which determine pili serologically related to those of EDP208, were inconclusive. Phage F olac was found to be serologically related to phage UA-6, another isolate specific for EDP208.  相似文献   

11.
Pili from Bacteroides nodosus were purified to greater than 99% homogeneity by precipitation at pH 4.0 and in MgCl2 followed by chromatography on BioGel A150. The pili were composed entirely of one type of polypeptide subunit, pilin. No carbohydrates, nucleic acid, lipid, lipopolysaccharide or phosphate could be detected in purified pili preparations. The molecular weight of pilin from B. nodosus strains 91B and 198 was 18,400 and from strain 80 was 19,300. The isoelectric points of pili from B. nodosus strains 91B and 80 were both 4.5. The buoyant densities of pili from strains 91B, 80 and 198 were 1.287, 1.284 and 1.286 g ml-1, respectively. The three strains of B. nodosus did not cross-react in K-agglutination tests and produced pili which did not cross-react in immunodiffusion tests. Antiserum to highly purified pili caused a characteristic K-type agglutination reaction. It was concluded that pili are the K-agglutinogen.  相似文献   

12.
Pili, which are assembled from protein subunits called pilin, are indispensable for the adhesion of capsulated Neisseria meningitidis (MC) to eukaryotic cells. Both MC and Neisseria gonorrhoeae (GC) pilins are glycosylated, but the effect of this modification is unknown. In GC, a galactose α-1,3-N-acetyl glucosamine is O-linked to Ser-63, whereas in MC, an O-linked trisaccharide is present between residues 45 and 73 of pilin. As Ser-63 was found to be conserved in pilin variants from different strains, it was replaced by Ala in two MC variants to test the possible role of this residue in pilin glycosylation and modulation of pili function. The mutated alleles were stably expressed in MC, and the proteins they encoded migrated more quickly than the normal protein during SDS–PAGE. As controls, neighbouring Asn-61 and Ser-62 were replaced by an Ala with no effect on electrophoretic mobility. Silver staining of purified pilin obtained from MC after oxidation with periodic acid confirmed the loss of glycosylation in the Ser-63→Ala pilin variants. Mass spectrometry of HPLC-purified trypsin-digested peptides of pilin and Ser-63→Ala pilin confirmed that peptide 45–73 has the molecular size of a glycopeptide in the wild type. In strains producing non-glycosylated pilin variants, we observed that (i) no truncated S pilin monomer was produced; (ii) piliation was slightly increased; and (iii) presumably as a consequence, adhesiveness for epithelial cells was increased 1.6- to twofold in these derivatives. In addition, pilin monomers and/or individual pilus fibres, obtained after solubilization of a crude pili preparation in a high pH buffer, were reassociated into insoluble aggregates of pili more completely with non-glycosylated variants than with the normal pilin. Taken together, these data eliminate a major role for pilin glycosylation in piliation and subsequent pilus-mediated adhesion, but they demonstrate that glycosylation facilitates solubilization of pilin monomers and/or individual pilus fibres.  相似文献   

13.
Dissociation and reassembly of Escherichia coli type 1 pili.   总被引:41,自引:12,他引:29       下载免费PDF全文
Escherichia coli type 1 pili, which mediate the mannose-sensitive adherence of the bacterium to eucaryotic cells, are comprised of very stable arrays of pilin protein subunits (molecular weight, approximately 17,000). Previous methods for the dissociation of pili caused their irreversible denaturation. We have found that incubation of pili in saturated guanidine hydrochloride at 37 degrees C led to their complete dissociation, as evidenced by nephelometry and electron microscopy. Gel chromatography of the dissociated pili on a Sepharose CL-6B column in the presence of saturated guanidine hydrochloride yielded a single protein peak with a molecular weight corresponding to that of pilin. Dialysis of this peak against 5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) and rechromatography in the same buffer afforded a major protein peak, probably consisting of pilin dimers. About 25% of the protein in this peak bound to a mannan-sepharose column and could be eluted with methyl alpha-D-mannoside. The pilin dimer gave a single protein band upon polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (molecular weight, 16,600) or 10 M urea and penetrated completely into 7% gels in the absence of denaturants. Reassembly of the pilin dimers into pili was achieved upon dialysis against the tris(hydroxymethyl)aminomethane buffer containing 5 mM MgCl2, as observed by electron microscopy. Thus, the conditions used allow renaturation of the dissociated subunits and may aid in further studies of the structure-function relationship of pili.  相似文献   

14.
The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe–2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na+-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na+-NQR contains approximately 1.7 mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na+-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na+-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).  相似文献   

15.
An isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter was constructed in Myxococcus xanthus. The single-copy pilA gene encodes pilin, the monomer unit of M. xanthus type IV pili. To vary the level of pilA expression, we cloned its promoter in front of the lac operator, and a plasmid containing the construct was inserted into the chromosome of a DeltapilA strain. Induction of pilin expression increased smoothly as the dose of IPTG added to the culture was increased. IPTG-induced pilin rescued S motility of the DeltapilA strain to wild-type levels. The rate of S-motile swarming was found to be proportional to the number of pili (shear-sensitive pilin) produced rather than to the level of total pilin. In fact, S motility was not rescued until the total level of pilin was more than 50% of the wild-type level. This observation implies that a threshold concentration of pilin must be exceeded before the shear-sensitive material (pili) is polymerized in M. xanthus.  相似文献   

16.
Type IV pili are multifunctional filaments displayed on many bacterial pathogens. Members of the Type IVa pilus subclass are found on a diverse group of human pathogens, whereas Type IVb pili are found almost exclusively on enteric bacteria. The Type IVa and IVb subclasses are distinguished by differences in the pilin subunits, including the fold of the globular domain. To understand the implications of the distinct pilin folds, we compared the stabilities of pilin subunits and pilus filaments for the Type IVa GC pilus from Neisseria gonorrhoeae and the Type IVb toxin-coregulated pilus (TCP) from Vibrio cholerae. We show that while recombinant TCP pilin is more stable than GC pilin, the GC pili are more resistant to proteolysis, heat and chemical denaturation than TCP, remaining intact in 8?M urea. To understand these differences, we determined the TCP structure by electron microscopy and three-dimensional image reconstruction. TCP have an architecture similar to that of GC pili, with subunits arranged in a right-handed 1-start helix and related by an 8.4-? axial rise and a 96.8° azimuthal rotation. However, the TCP subunits are not as tightly packed as GC pilins, and the distinct Type IVb pilin fold exposes a segment of the α-helical core of TCP. Hydrophobic interactions dominate for both pilus subtypes, but base stacking by aromatic residues conserved among the Type IVa pilins may contribute to GC pilus stability. The extraordinary stability of GC pili may represent an adaptation of the Type IVa pili to harsh environments and the need to retract against external forces.  相似文献   

17.
Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilA(PAK) pilin subunit in N. gonorrhoeae. We show here that, although PilA(PAK) pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilA(PAK) pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilA(PAK) pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilA(PAK) pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilA(PAK) pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.  相似文献   

18.
Pili or fimbriae, which are filamentous structures present on the surface of bacteria, were purified from a periodontal pathogen, Porphyromonas gingivalis, in 1980s. The protein component of pili (stalk pilin), which is its major component, was named FimA; it has a molecular weight of approximately 41 kDa. Because the molecular weight of the pilin from P. gingivalis is twice that of pilins from other bacterial pili, the P. gingivalis Fim pili were suggested to be formed via a novel mechanism. In earlier studies, we reported that the FimA pilin is secreted on the cell surface as a lipoprotein precursor, and the subsequent N-terminal processing of the FimA precursor by arginine-specific proteases is necessary for Fim pili formation. The crystal structures of FimA and its related proteins were determined recently, which show that Fim pili are formed by a protease-mediated strand-exchange mechanism. The most recent study conducted by us, wherein we performed cryoelectron microscopy of the pilus structure, provided evidence in support of this mechanism. As the P. gingivalis Fim pili are formed through novel transport and assembly mechanisms, such pili are now designated as Type V pili. Surface lipoproteins, including the anchor pilin FimB of Fim pili that are present on the outer membrane, have been detected in certain Gram-negative bacteria. Here, we describe the assembly mechanisms of pili, including those of Type V and other pili, as well as the lipoprotein transport mechanisms.  相似文献   

19.
The pili of the stalked bacterium Caulobacter crescentus are assembled at a specific time in the life cycle at one pole of the cell and are composed of the monomer protein, pilin. A previous study demonstrated that the onset of pilin synthesis occurs well before pili appear on the surface, suggesting that pilin accumulates within the cell. In the present study, an electron microscope immunocytochemistry assay was used to determine the subcellular location of this unassembled pilin and its fate during pilus assembly and cell division. Populations of synchronously growing cells were embedded in epoxy resin at selected times during the cell cycle. Ultrathin sections were treated with pilin-specific antibody, followed by protein A coupled to colloidal gold. It was determined that the cellular location for unassembled pilin was the cell cytoplasm. All cell membranes and regions of nuclear material were poorly labeled. Quantitation demonstrated that label density increased during the period of pilin synthesis and declined during the period of pilus assembly and maintenance. The pilin pool was not unequally segregated at division; e.g., to the daughter cell that is elaborating pili. Mutants which have simultaneously lost the ability to produce flagella, pili, and other polar organelles, possibly due to alterations in the specialized region of polar organelle assembly, were also examined by the immunocytochemistry technique. There was no significant difference in the pilin pool size relative to the wild type, indicating that pilin synthesis continues in the absence of a functioning assembly site. This pattern of synthesis and assembly for the pilus is significantly different from that of the polar flagellum which is produced at the same time and location on the cell surface. These findings are discussed in relation to the hypothesized organization center at the cell pole which may have a major role in directing the assembly of all the polar structures.  相似文献   

20.
A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.  相似文献   

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