Novel Zn(2+)-chelating peptides selected from a fimbria-displayed random peptide library.

The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces by virtue of the FimH adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the E. coli cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of approximately 40 million individual clones, was screened for peptide sequences that conferred on recombinant cells the ability to bind Zn(2+). By serial selection, sequences that exhibited various degrees of binding affinity and specificity toward Zn(2+) were enriched. None of the isolated sequences showed similarity to known Zn(2+)-binding proteins, indicating that completely novel Zn(2+)-binding peptide sequences had been isolated. By changing the protein scaffold system, we demonstrated that the Zn(2+)-binding seems to be uniquely mediated by the peptide insert and to be independent of the sequence of the carrier protein. These findings might be applied in the design of biomatrices for bioremediation purposes or in the development of sensors for detection of heavy metals.

Valency conversion in the type 1 fimbrial adhesin of Escherichia coli.

FimH protein is a lectin-like adhesive subunit of type 1, or mannose-sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose-specific (i.e. multivalent) binding. Here, we demonstrate that replacement of residues 185-279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct monomannose-specific (i.e. monovalent) binding capability. Bacteria expressing the monovalent hybrid adhesins were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeast or bind human buccal cells -- functions readily accomplished by the E. coli-expressing mannotriose-specific FimH variants. Based on the relative potency of inhibiting compounds of different structures, the receptor binding site within monovalent FimH-FocH adhesin has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. The monomannose-only specific phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor-binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting the FimH quaternary structure.

Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens.

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of E. coli cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants.

Sequestration of zinc oxide by fimbrial designer chelators.

Type 1 fimbriae are surface organelles of Escherichia coli. By engineering a structural component of the fimbriae, FimH, to display a random peptide library, we were able to isolate metal-chelating bacteria. A library consisting of 4 x 10(7) independent clones was screened for binding to ZnO. Sequences responsible for ZnO adherence were identified, and distinct binding motifs were characterized. The sequences selected exhibited various degrees of affinity and specificity towards ZnO. Competitive binding experiments revealed that the sequences recognized only the oxide form of Zn. Interestingly, one of the inserts exhibited significant homology to a specific sequence in a putative zinc-containing helicase, which suggests that searches such as this one may aid in identifying binding motifs in nature. The zinc-binding bacteria might have a use in detoxification of metal-polluted water.

Antigen 43 facilitates formation of multispecies biofilms.

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation of E. coli cells in static cultures. Here, we show that Ag43 can be expressed in a functional form on the surface of Pseudomonas fluorescens. Ag43 expression dramatically enhances the biofilm-forming potential of both E. coli and P. fluorescens to abiotic surfaces in simple microtitre well assays and in flow chambers. Importantly, Ag43-expressing E. coli and P. fluorescens cells tagged with Gfp and Rfp were shown to form interwoven biofilms in flow chambers. The three-dimensional structures of the biofilms were analysed by laser-confocal microscopy. Heterogeneous expression of Ag43 induced interspecies cell-to-cell contact that generated multispecies biofilm formation. Our data indicate that this versatile molecular tool can be used for the rational design of multispecies biofilms. More specifically, this novel technology offers opportunities for the design of multispecies consortia in which the concerted action of several bacterial species is required, e.g. waste treatment and degradation of pollutants.

Bioaccumulation of heavy metals by fimbrial designer adhesins.

Naturally occurring adhesins bind to specific molecular targets in a lock-and-key fashion due to the composition of the binding domain of the adhesin. By introduction of random peptide libraries in a suitable surface exposed carrier protein it is possible to create and select designer adhesins with novel binding affinities. Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose sensitive binding to different host surfaces through the FimH adhesin, an integral part of these organelles. We have studied the ability of the FimH adhesin to display random peptide sequences. By serial selection and enrichment procedures specific sequences were identified which conferred the ability on recombinant cells to adhere to various metal oxides (PbO2, CoO, MnO2, Cr2O3). The properties inherent in these sequences permitted the distinct recognition of metals to varying degrees, indicating that this system allow for the isolation of peptide sequences with a variety of binding avidities. These studies demonstrate the potential and versatility of the FimH display system for presenting random peptide sequences. In addition, the possibility exists for the construction of microorganisms for the bioaccumulation of heavy metals from the environment.

[Baby boom in Christiania]. / Babyboom på Christiania.

PIP: The "Free State of Christiania" was established in 1971 in a former army camp in Copenhagen. The several hundred inhabitants of Christiania participate in a live-in social experiment in which drugs are openly used and in which the normal standards of Danish society are little observed. Until about 1988 only about 10 children/year were born in Christiania but since then the number of births has more than doubled. There has not been much increase in the total number of inhabitants and the cause appears to be simply that the people who live there, as is the case in the rest of Denmark, are deciding to have children. Most of the children are born at home with the aid of a midwife either from one of the Copenhagen municipal hospitals or from a group of midwife either from on of the Copenhagen municipal hospitals or from a group of midwives who operate a home birth agency in Copenhagen. In spite of the typically primitive living conditions in Christiania, the children are not sick more often than other Danish children. Health workers have characterized the inhabitants of Christiania as very sensible parents who dress their children carefully if not always in the latest style. Christiania has its own child care facilities, which operate cooperatively, and care for both children and adults who are in need of regular day care. There are about 20 children on the waiting list for day care and plans are underway to establish a second center. Many of Christiania's citizens work in small shops and craft studios, which decreases the need for institutional day care. The baby boom has been the cause of a certain amount of internal disagreement in Christiania. Families with younger children say that children make the free state more livable, but many of the old timers have come to Christiania to avoid structure and stability and the baby boom, they say, raises the question of who Christiania is really for.^ieng