The role of pilin glycan in neisserial pathogenesis

The pilus of pathogenic Neisseria is a polymer composed mainly of the glycoprotein, pilin. Recent investigations significantly enhanced characterization of pilin glycan (Pg) from N. gonorrhoeae (gonococcus, GC) and N. meningitidis (meningococcus, MC). Several pilin glycosylation genes were discovered recently from these bacteria and some of these genes transfer sugars previously unknown to be present in neisserial pili. Due to these findings, glycans of GC and MC pilin are now considered more complex. Furthermore, various Pg can be expressed by different strains and variants of GC, as well as MC. Intra-species variation of Pg between different groups of GC or MC can partly be due to polymorphisms of glycosylation genes. In pilus of pathogenic Neisseria, alternative glycoforms are also produced due to phase-variation (Pv) of pilin glycosylation genes. Most remarkably, the pgtA (pilin glycosyl transferase A) gene of GC can either posses or lack the ability of Pv. Many GC strains carry the phase-variable (Pv+) pgtA, whereas others carry the allele lacking Pv (Pv–). Mostly, the GC isolates from disseminated gonococcal infection (DGI) carry Pv+ pgtA but organisms from uncomplicated gonorrhea (UG) contain the Pv– allele. This data suggests that Pv of pgtA facilitates DGI, whereas constitutive expression of the Pv– pgtA may promote UG. Additional implications of Pg in various physiological and pathogenic mechanisms of Neisseria can also be envisaged based on various recent data.     

Role of Glycosylation at Ser63 in Production of Soluble Pilin in Pathogenic Neisseria

Pilus-mediated adhesion is essential in the pathogenesis of Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC). Pili are assembled from a protein subunit called pilin. Pilin is a glycoprotein, and pilin antigenic variation has been shown to be responsible for intrastrain variability with respect to the degree of adhesion in both MC and GC. In MC, high-adhesion pilins are responsible for the formation of bundles of pili which bind bacteria and cause them to grow as colonies on infected monolayers. In this work, we selected MC and GC pilin variants responsible for high and low adhesiveness and introduced them into the other species. Our results demonstrated that a given pilin variant expressed an identical phenotype in either GC or MC with respect to bundling and adhesiveness to epithelial cells. However, the production of truncated soluble pilin (S pilin) was consistently more abundant in GC than in MC. In the latter species, the glycosylation of pilin at Ser63 was shown to be required for the production of a truncated monomer of S pilin. In order to determine whether the same was true for GC, we engineered various pilin derivatives with an altered Ser63 glycosylation site. The results of these experiments demonstrated that the production of S pilin in GC was indeed more abundant when pilin was posttranslationally modified at Ser63. However, nonglycosylated variants remained capable of producing large amounts of S pilin. These data demonstrated that for GC, unlike for MC, glycosylation at Ser63 is not required for S-pilin production, suggesting that the mechanisms leading to the production of S pilin in GC and MC are different.     

Structure of the Vibrio cholerae Type IVb Pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus

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.     

Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion

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.     

Molecular models accounting for the gene conversion reactions mediating gonococcal pilin antigenic variation

The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high-frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high-frequency gene conversions that mediate pilin Av.     

Structural Characterization of Novel Pseudomonas aeruginosa Type IV Pilins

Pseudomonas aeruginosa type IV pili, composed of PilA subunits, are used for attachment and twitching motility on surfaces. P. aeruginosa strains express one of five phylogenetically distinct PilA proteins, four of which are associated with accessory proteins that are involved either in pilin posttranslational modification or in modulation of pilus retraction dynamics. Full understanding of pilin diversity is crucial for the development of a broadly protective pilus-based vaccine. Here, we report the 1.6-Å X-ray crystal structure of an N-terminally truncated form of the novel PilA from strain Pa110594 (group V), which represents the first non-group II pilin structure solved. Although it maintains the typical T4a pilin fold, with a long N-terminal α-helix and four-stranded antiparallel β-sheet connected to the C-terminus by a disulfide-bonded loop, the presence of an extra helix in the αβ-loop and a disulfide-bonded loop with helical character gives the structure T4b pilin characteristics. Despite the presence of T4b features, the structure of PilA from strain Pa110594 is most similar to the Neisseria gonorrhoeae pilin and is also predicted to assemble into a fiber similar to the GC pilus, based on our comparative pilus modeling. Interactions between surface-exposed areas of the pilin are suggested to contribute to pilus fiber stability. The non-synonymous sequence changes between group III and V pilins are clustered in the same surface-exposed areas, possibly having an effect on accessory protein interactions. However, based on our high-confidence model of group III PilA_PA14, compensatory changes allow for maintenance of a similar shape.     

DNA sequence of the F traALE region that includes the gene for F pilin

The complete sequence of a 1.4-kilobase PstI fragment containing the F transfer genes traA, -L, and -E is presented. The traA reading frame has been located both genetically and by comparing the primary structure of F pilin (the traA product) predicted by the DNA sequence to the amino acid composition and sequence of N- and C-terminal peptides isolated from purified F pilin. Taken together, these data show that there is a leader peptide of 51 amino acids and that F pilin contains 70 amino acids, giving molecular weights of 13,200 for F propilin and 7,200 for mature F pilin. Secondary structure predictions for F pilin revealed a reverse turn that precedes the sequence Ala-Met-Ala51, a classic signal peptidase cleavage site. The N-terminal alanine residue is blocked by an acetyl group as determined by 1H-nuclear magnetic resonance spectroscopy. The traL and traE genes encode proteins of molecular weights 10,350 and 21,200, respectively. According to DNA sequence predictions, these proteins do not contain signal peptide leader sequences. Secondary structure predictions for these proteins are in accord with traLp and traEp being membrane proteins in which hydrophobic regions capable of spanning the membrane are linked by sequences that form turns and carry positively charged residues capable of interacting with the membrane surface.     

Loss of both Holliday junction processing pathways is synthetically lethal in the presence of gonococcal pilin antigenic variation

The obligate human pathogen Neisseria gonorrhoeae (Gc) has co-opted conserved recombination pathways to achieve immune evasion by way of antigenic variation (Av). We show that both the RuvABC and RecG Holliday junction (HJ) processing pathways are required for recombinational repair, each can act during genetic transfer, and both are required for pilin Av. Analysis of double mutants shows that either the RecG or RuvAB HJ processing pathway must be functional for normal growth of Gc when RecA is expressed. HJ processing-deficient survivors of RecA expression are enriched for non-piliated bacteria that carry large deletions of the pilE gene. Mutations that prevent pilin variation such as recO, recQ, and a cis-acting pilE transposon insertion all rescue the RecA-dependent growth inhibition of a HJ processing-deficient strain. These results show that pilin Av produces a recombination intermediate that must be processed by either one of the HJ pathways to retain viability, but requires both HJ processing pathways to yield pilin variants. The need for diversity generation through frequent recombination reactions creates a situation where the HJ processing machinery is essential for growth and presents a possible target for novel antimicrobials against gonorrhoea.     

Sortases and pilin elements involved in pilus assembly of Corynebacterium diphtheriae

Corynebacterium diphtheriae SpaA pili are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals, and SpaC seems to be positioned at the pilus tip. Pilus assembly in C. diphtheriae requires the pilin motif and the C-terminal sorting signal of SpaA, and is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side-chain amino groups of pilin motif sequences to generate covalent linkages between pilin subunits. We show here that two elements of SpaA pilin precursor, the pilin motif and the sorting signal, are together sufficient to promote the polymerization of an otherwise secreted protein by a process requiring the function of the sortase A gene (srtA). Five other sortase genes are dispensable for SpaA pilus assembly. Further, the incorporation of SpaB into SpaA pili requires a glutamic acid residue within the E box motif of SpaA, a feature that is found to be conserved in other Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs. When the main fimbrial subunit of Actinomyces naeslundii type I fimbriae, FimA, is expressed in corynebacteria, C. diphtheriae strain NCTC13129 polymerized FimA to form short fibres. Although C. diphtheriae does not depend on other actinomycetal genes for FimA polymerization, this process involves the pilin motif and the sorting signal of FimA as well as corynebacterial sortase D (SrtD). Thus, pilus assembly in Gram-positive bacteria seems to occur by a universal mechanism of ordered cross-linking of precursor proteins, the multiple conserved features of which are recognized by designated sortase enzymes.     

Salmonella typhimurium pgtB mutants conferring constitutive expression of phosphoglycerate transporter pgtP independent of pgtC.

PgtC is one of the three components of the atypical "two-component" pgt regulatory system. To investigate whether functional PgtC required for the induction of pgtP expression could be bypassed in the signal transduction process, we sought, and succeeded in isolating, intergenic suppressors arising in the low-copy mini-F plasmid, pSJ11, bearing the entire pgt system except for a 168-bp deletion near the end of the pgtC gene. By transport assays, these suppressors were found to confer constitutive pgtP expression. Intriguingly, all five mutations reside near the 5' end of the pgtB gene, at codons 19 and 21. One mutation alters Arg-19 to Gln, two alter Ala-21 to Thr, one alters Ala-21 to Val, and one alters Ala-21 to Ile. Appropriate strains in which the pgtP promoter was fused to lacZ and which bore the pgtB mutations with and without mutations in pgtC and pgtA genes were constructed, and the epistatic relationships of the wild-type pgtC allele, a mutant pgtA allele, and an essentially total deletion of pgtC to the constitutive pgtB mutations were determined. In the mutant strains bearing the Ala-21 --> Ile and Ala-21 --> Val substitutions, the level of constitutive pgtP-lacZ reporter expression was not affected by the presence of the wild-type pgtC allele, nor was it affected by the total absence of PgtC in the case of the Ala-21 --> Val alteration examined; however, in the mutant strains bearing the Ala-21 --> Thr and the Arg-19 --> Gln substitutions, the extent of constitutive pgtP-lacZ reporter expression was markedly enhanced by the presence of wild-type pgtC allele and, in the case of the Arg-19 -->Gln change examined, by the total absence of PgtC as well. These results indicate that PgtC contains no domain necessary for the kinase activity; that PgtB can be activated in the absence of PgtC mutational alterations of the protein itself; and that PgtB and PgtC interact in the signaling process, with PgtC functioning to activate and modulate the kinase activity of Pgtb. In all strains, the replacement of the wild type pgtA allele with a mutant pgtA allele completely abolished expression of the pgtP-lacZ reporter, indicating that functional pgtA is essential for the constitutivity. His-457 of PgtB, a potential site of autophosphorylation, is also required for the constitutivity because its change to Val drastically reduced pgtP-lacZ reporter expression. The structural basis for the activation of the altered PgtB is discussed in terms of putative structure of PgtB in the membrane.     

Glycosylation substrate specificity of Pseudomonas aeruginosa 1244 pilin

The beta-carbon of the Pseudomonas aeruginosa 1244 pilin C-terminal Ser is a site of glycosylation. The present study was conducted to determine the pilin structures necessary for glycosylation. It was found that although Thr could be tolerated at the pilin C terminus, the blocking of the Ser carboxyl group with the addition of an Ala prevented glycosylation. Pilin from strain PA103 was not glycosylated by P. aeruginosa 1244, even when the C-terminal residue was converted to Ser. Substituting the disulfide loop region of strain PA103 pilin with that of strain 1244 allowed glycosylation to take place. Neither conversion of 1244 pilin disulfide loop Cys residues to Ala nor the deletion of segments of this structure prevented glycosylation. It was noted that the PA103 pilin disulfide loop environment was electronegative, whereas that of strain 1244 pilin had an overall positive charge. Insertion of a positive charge into the PA103 pilin disulfide loop of a mutant containing Ser at the C terminus allowed glycosylation to take place. Extending the "tail" region of the PA103 mutant pilin containing Ser at its terminus resulted in robust glycosylation. These results suggest that the terminal Ser is the major pilin glycosylation recognition feature and that this residue cannot be substituted at its carboxyl group. Although no other specific recognition features are present, the pilin surface must be compatible with the reaction apparatus for glycosylation to occur.     

A genetic screen identifies genes and sites involved in pilin antigenic variation in Neisseria gonorrhoeae

It has previously been shown that the frequency of pilin antigenic variation in Neisseria gonorrhoeae (the gonococcus, Gc) is regulated by iron availability. To identify factors involved in pilin variation in an iron-dependent or an iron-independent manner, we conducted a genetic screen of transposon-mutated gonococci using a pilus-dependent colony morphology phenotype to detect antigenic variation deficient mutants. Forty-six total mutants representing insertions in 30 different genes were shown to have reduced colony morphology changes resulting from impaired pilin variation. Five mutants exhibited an iron-dependent decrease in pilin variation, while the remaining 41 displayed an iron-independent decrease in pilin variation. Based on the levels of antigenic variation impairment, we defined the genes as being essential for, important for, or involved in antigenic variation. DNA repair and DNA transformation frequencies of each mutant were measured to determine whether other recombination-based processes were also affected in the mutants. Each mutant was placed into one of six classes based on their pilin variation, DNA repair and DNA transformation phenotypes. Among the many genes identified, recR is shown to be an additional member of the gonococcal RecF-like recombination pathway. In addition, recG and ruvA represent the first evidence that the processing of Holliday junctions is required for pilin antigenic variation. Moreover, two independent insertions in a non-coding region upstream of the pilE gene suggest that cis-acting sequences important for pilin variation are found in that region. Finally, insertions that effect expression of the thrB and thrC genes suggest that molecules in the threonine biosynthetic pathway are important for pilin variation. Many of the other genes identified in this genetic screen do not have an obvious role in pilin variation, DNA repair, or DNA transformation.     

Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.     

Identification and nucleotide sequence of the activator gene of the externally induced phosphoglycerate transport system of Salmonella typhimurium

A recent study from this laboratory (G-q. Yu, D. Goldrick, H.R. Kaback and J-s. Hong, in preparation) indicates that the externally induced phosphoglycerate transport system (pgt) of Salmonella typhimurium is positively regulated by the activator gene, pgtA, and that the pgtA is localized in the SalI-PstI restriction fragment 3.0 kb from the permease gene, pgtP. In this paper, we describe the identification of the activator gene and its gene product and the determination of the complete nucleotide (nt) sequence of the activator gene as well as of a downstream gene not required for pgtP expression. The amino acid sequence of the activator based on the nt sequence shows an N-terminal signal-like sequence which is apparently not cleaved and three potential transmembrane sequences in the C-terminal half of the protein based on the hydropathy analysis.     

Interesting sequence differences between the pilin gene inversion regions of Moraxella lacunata ATCC 17956 and Moraxella bovis Epp63.

The bacterium Moraxella lacunata is a causative agent of human conjunctivitis and keratitis. We have previously reported construction of plasmid pMxL1, which includes a 5.9-kb fragment on which the pilin gene inversion region of M. lacunata resides. The inversion region of pMxL1 was shown to invert when pMxL1 was in an Escherichia coli host cell. In this report, we present Western immunoblot analysis using Moraxella bovis Epp63 anti-I and anti-Q pilin sera which demonstrate that pMxL1 makes pilin only when in orientation 1. The sequence of the pMxL1 plasmid containing the invertible region contains a perfect tandem repeat of 19 bp in the orientation 1 nonexpressed pilin gene at the middle of the recombination junction site. This 19-bp insert causes a frameshift and disrupts the pilin gene. The predicted amino acid sequence of this nonfunctional pilin gene (with the 19-bp repeat subtracted) bears closest resemblance to M. bovis Epp63 Q pilin sequence, although the other (functional) M. lacunata pilin encoded by pMxL1 shows slightly higher homology to Q pilin. Comparison of the pMxL1 sequence with that of the M. bovis Epp63 sequence shows two other particularly interesting differences. One is a 15-bp sequence addition found in pMxL1 at the 60-bp region previously reported as a possible M. bovis recombinational enhancer. The second is an AT deletion in pMxL1 compared with Epp63 within an open reading frame (tfpB) which results in the pMxL1 tfpB open reading frame being one-third shorter than in Epp63. The DNA sequences in these three altered regions from the M. lacunata strain from which pMxL1 was derived were amplified by polymerase chain reaction and sequenced. The parent strain was found to contain the differences seen in pMxL1. Comparison of the M.bovis and M. lacunata pilin gene amino acid sequences is also presented.     

Nucleotide sequence of the gene encoding the two-subunit pilin of Bacteroides nodosus 265.

The nucleotide sequence of the gene encoding pilin from Bacteroides nodosus 265 has been determined. The pilin is encoded by a single-copy gene, from which can be predicted a prepilin comprising a single protein chain of Mr 16,637. The prepilin sequence differs in several respects from the mature protein sequence. Seven additional N-terminal amino acid residues are present in prepilin, whereas residue 8, phenylalanine, undergoes posttranslational modification to become the N-methylated amino-terminal residue of mature pilin. In addition, further processing occurs through internal cleavage to produce two noncovalently linked subunits characteristic of pilins from serogroup H of B. nodosus, of which strain 265 is a member. The position of cleavage has been identified between alanine residues at positions 72 and 73 of the mature 149-residue pilin protein. The predicted pilin sequence of B. nodosus 265 shows extensive N-terminal amino acid sequence homology with other pilins of the N-methylphenylalanine type. In addition this sequence also shows homology with these N-methylphenylalanine-type pilins in the C-terminal region of the molecule, especially with pilin from Pseudomonas aeruginosa PAK.     

Caulobacter crescentus pili: analysis of production during development

The pili of Caulobacter crescentus are structures whose appearance is regulated during the development of the swarmer cell pole. Pili are assembled during the predivisional and swarmer cell stages, at the same time as the flagellum, and disappear as the swarmer cell differentiates into a stalked cell. Pilin is the protein which polymerizes to form the pilus. An immune precipitation assay, developed to examine the periodicity of pilin synthesis during the cell cycle, demonstrated that pilin synthesis begins in the early stalked cell and is probably completed before cell division. Thus, the entire period of synthesis occurs before the pili are clearly visible at the differentiated cell pole. Likewise, the functional stability of the pilin mRNA is relatively short, further suggesting that the protein monomers accumulate prior to assembly. Unlike the case of the flagellins, experiments with the DNA replication inhibitor hydroxyurea did not establish a correlation between the DNA replication and the onset of pilin synthesis. In addition to pilin, several other developmentally regulated proteins, including the flagellins, are reproducibly precipitated in the pilin immunoassay. Their presence in the precipitate is a specific consequence of the antipilin antibody. Analysis of the antibody preparations yielded conflicting results; electron microscopic studies with ferritin-coupled antibody and double diffusion analysis indicated no binding activity to any cell components other than pilin. However, an assay based on filter transferred preparations of electrophoresed cell proteins indicated that at least one additional class of proteins in the immune precipitate may bind pilin antibody. These results are discussed in the context of the possible formation of a discrete membrane complex in the polar region of the cell which may be involved in the regulation of spatial development in Caulobacter.     

The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli

Previous work has demonstrated the expression of the cloned pilin gene of Pseudomonas aeruginosa PAK within Escherichia coli and has pinpointed this protein's localization exclusively to the cytoplasmic membrane (Finlay et al., 1986). To define regions of the pilin subunit necessary for its stability and transport within E. coli, we constructed six mutants of the pilin gene and studied their expression and localization using a T7 promoter system. Two of the mutants have either a 4- or 8-amino-acid deletion at the N-terminus and both were stably expressed and transported primarily to the cytoplasmic membrane of E. coli. The other four mutants are C-terminal truncations having between 36 and 56 amino acids of the N-terminal region of the unprocessed pilin. Studies with these truncated mutants revealed that only the first 36 residues of the unprocessed pilin subunit were required for insertion into the E. coli membrane.     

Nature of the Carbohydrate and Phosphate Associated with ColB2 and EDP208 Pilin

Studies were carried out to elucidate the nature of phosphate and sugar linkages to F-like conjugative pili encoded by the ColB2Fdr (compatibility group FII) and EDP208 (compatibility group FV) plasmids. Both types of pili, when in the intact undissociated state, were found to contain approximately 3 mol of phosphate and 3 mol of sugar per mol of pilin. However, further purification of the two types of pilin by gel filtration chromatography in the presence of sodium dodecyl sulfate (SDS) removed all of the carbohydrate from EDP208 pilin and approximately 65% of the carbohydrate from ColB2 pilin. Approximately 0.8 to 1.0 mol of glucose per mol of protein remained associated with ColB2 pilin after SDS gel filtration chromatography, but it was not possible to determine whether this was covalently linked to the pilin, or tightly associated in an SDS-resistant manner. SDS-gel chromatography did not remove phosphate from either ColB2 or EDP208 pilins. ³¹P nuclear magnetic resonance studies indicated that the pilin-associated phosphate is involved in a phosphodiester linkage. Acetone precipitation or chloroform-methanol extraction of the purified pilin material reduced the phosphate associated with EDP208 pilin to less than 0.04 molecule per pilin monomer. ColB2 pilin, under the same conditions, retained approximately 0.5 phosphate per pilin monomer. The extracted phosphate-containing moieties were identified as phosphatidylglycerol and phosphatidylethanolamine by thin-layer chromatography. Since the ³¹P nuclear magnetic resonance spectra for both ColB2 and EDP208 were identical and no signal other than that of a phosphodiester was detected in the ColB2 spectrum, the phosphate remaining with the ColB2 pilin after chloroform-methanol extraction is most likely due to a tightly bound noncovalent residue.