A Single Amino Acid Substitution Changes the Self-Assembly Status of a Type IV Piliation Secretin.

Secretins form large multimeric complexes in the outer membranes of many Gram-negative bacteria, where they function as dedicated gateways that allow proteins to access the extracellular environment. Despite their overall relatedness, different secretins use different specific and general mechanisms for their targeting, assembly, and membrane insertion. We report that all tested secretins from several type II secretion systems and from the filamentous bacteriophage f1 can spontaneously multimerize and insert into liposomes in an in vitro transcription-translation system. Phylogenetic analyses indicate that these secretins form a group distinct from the secretins of the type IV piliation and type III secretion systems, which do not autoassemble in vitro. A mutation causing a proline-to-leucine substitution allowed PilQ secretins from two different type IV piliation systems to assemble in vitro, albeit with very low efficiency, suggesting that autoassembly is an inherent property of all secretins.

Identification of the gate regions in the primary structure of the secretin pIV.

Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function ('leaky' mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C-terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N-terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24-residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non-functional.

Genome comparison in silico in Neisseria suggests integration of filamentous bacteriophages by their own transposase.

We have identified filamentous prophages, Nf (Neisserial filamentous phages), during an in silico genome comparison in Neisseria. Comparison of three genomes of Neisseria meningitidis and one of Neisseria gonorrhoeae revealed four subtypes of Nf. Eleven intact copies are located at different loci in the four genomes. Each intact copy of Nf is flanked by duplication of 5'-CT and, at its right end, carries a transposase homologue (pivNM/irg) of RNaseH/Retroviral integrase superfamily. The phylogeny of these putative transposases and that of phage-related proteins on Nfs are congruent. Following circularization of Nfs, a promoter-like sequence forms. The sequence at the junction of these predicted circular forms (5'-atCTtatat) was found in a related plasmid (pMU1) at a corresponding locus. Several structural variants of Nfs--partially inverted, internally deleted and truncated--were also identified. The partial inversion seems to be a product of site-specific recombination between two 5'-CTtat sequences that are in inverse orientation, one at its end and the other upstream of pivNM/irg. Formation of internally deleted variants probably proceeded through replicative transposition that also involved two 5'-CTtat sequences. We concluded that the PivNM/Irg transposase on Nfs integrated their circular forms into the chromosomal 5'-CT-containing sequences and probably mediated the above rearrangements.

Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd.

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the beta-subunit is covalently linked and the alpha-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.

Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX.

Previous studies have shown that Shigella flexneri bacteriophage X (SfX) encodes a glucosyltransferase (GtrX, formerly Gtr), which is involved in O antigen modification (serotype Y to serotype X). However, GtrX alone can only mediate a partial conversion. More recently, a three-gene cluster has been identified next to the attachment site in the genome of two other S. flexneri bacteriophages (i.e. SfV and SfII). This gene cluster was postulated to be responsible for a full O antigen conversion. Here it is reported that besides the gtrX gene, the other two genes in the gtr locus of SfX were also involved in the O antigen modification process. The first gene in the cluster (gtrA) encodes a small highly hydrophobic protein which appears to be involved in the translocation of lipid-linked glucose across the cytoplasmic membrane. The second gene in the cluster (gtrB) encodes an enzyme catalysing the transfer of the glucose residue from UDP-glucose to a lipid carrier. The third gene (gtrX) encodes a bacteriophage-specific glucosyltransferase which is largely responsible for the final step, i.e. attaching the glucosyl molecules onto the correct sugar residue of the O antigen repeating unit. A three-step model for the glucosylation of bacterial O antigen has been proposed.

Factors limiting display of foreign peptides on the major coat protein of filamentous bacteriophage capsids and a potential role for leader peptidase.

Many small peptides can be displayed on every copy of the major coat protein in recombinant filamentous bacteriophages but larger peptides can only be accommodated in hybrid virions mixed with wild-type protein subunits. A peptide insert of 12 residues capable of display at high copy number in a hybrid virion was found to be incapable of supporting recombinant virion assembly, a defect that could not be overcome by over-expressing leader peptidase in the same Escherichia coli cell. In contrast, over-expressing leader peptidase did increase the copy number of two 9-residue peptides that were poorly incorporated into hybrid virions. The factors that limit peptide display are varied and not restricted to the early stages of viral assembly.

Accessibility of peptides displayed on filamentous bacteriophage virions: susceptibility to proteinases.

The genome of the filamentous bacteriophage fd has been engineered so that small peptides can be inserted into the exposed N-terminal segment of pVIII, the major protein of the virus capsid. Most small peptides can be displayed on all 2700 copies of pVIII (a recombinant virion), but larger peptides can be displayed only in virions in which modified and wild-type proteins are intermingled (hybrid virions). Peptides displayed in this way are highly immunogenic and capable of interacting with receptors and other ligands. The physical accessibility of the displayed peptides was tested by examining their susceptibility to digestion with proteinases. Potential cleavage sites in peptides displayed on recombinant or hybrid virions were in general found to be accessible to trypsin and chymotrypsin; and the density of incorporation of peptides in the virion had no effect on the susceptibility to cleavage. However, peptide bonds towards the C-terminal end of an insert, located approximately 47 residues or fewer from the C-terminus of the coat protein, were protected from digestion, presumably because of their proximity to the bulk viral surface. These results have important implications for the design and optimization of peptide display systems using filamentous bacteriophages.

Selection of beta-lactamase on filamentous bacteriophage by catalytic activity.

Recently the display of repertoires of peptides and proteins on the surface of filamentous phage, and selection of the phage by binding to a ligand, has allowed the isolation of peptides and proteins with rare binding activities. Furthermore, phages displaying enzymes (phage enzymes) have been selected by affinity of binding to inhibitors. Here we show, using a suicide inhibitor, that phage enzymes can also be selected by their catalytic activity. Two phage enzymes were constructed by fusion to the minor coat protein of the phage (g3p), displaying either an active beta-lactamase or a catalytically inactive mutant in which the essential serine of the active site was mutated to alanine. The phages were then incubated with a beta-lactamase suicide inhibitor connected by a spacer to a biotin moiety. The active (but not the inactive) phages were labelled, and the active phages selected from mixtures with inactive phages by binding and elution from streptavidin-coated beads. The selection ratio for active versus inactive phages (about ten on elution of the phages by reduction of an S-S bond in the spacer between the warhead and biotin) could be improved to about 50 on elution by proteolytic cleavage of beta-lactamase from g3p at an intervening factor X site. Selection of phage-enzymes by catalysis may provide a means of creating new enzymes and refining their catalytic properties.