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In this study we report on a novel structural phenotype in Escherichia coil biofilms: cellular chain formation. Biofilm chaining in E. coli K-12 was found to occur primarily by clonal expansion, but was not due to filamentous growth. Rather, chain formation was the result of intercellular interactions facilitated by antigen 43 (Ag43), a self-associating autotransporter (SAAT) protein, which has previously been implicated in auto-aggregation and biofilm formation. Immunofluorescence microscopy suggested that Ag43 was concentrated at or near the cell poles, although when the antigen was highly overexpressed, a much more uniform distribution was seen. Immunofluorescence microscopy also indicated that other parameters, including dimensional constraints (flow, growth alongside a surface), may also affect the final biofilm architecture. Moreover, chain formation was affected by other surface structures; type I fimbriae expression significantly reduced cellular chain formation, presumably by steric hindrance. Cellular chain formation did not appear to be specific to E. coli K-12. Although many urinary tract infection (UTI) isolates were found to form rather homogeneous, flat biofilms, three isolates, including the prototypic asymptomatic bacteriuria strain, 83972, formed highly elaborate cellular chains during biofilm growth in human urine. Combined, these results illustrate the diversity of biofilm architectures that can be observed even within a single microbial species.

Urinary tract infection (UTI) is the most common infection in patients with indwelling urinary catheters, and bacterial biofilm formation is a major problem in this type of infection. Escherichia coil is responsible for the large majority of UTIs. Free iron is strictly limited in the human urinary tract and there is fierce competition between the host and infectious bacteria for this essential metal. Urinary tract infectious E. coil have highly efficient mechanisms of iron acquisition, one of which is the yersiniabactin system. The fyuA gene, encoding the yersiniabactin receptor, is one of the most upregulated genes in biofilm; it was upregulated 63-fold in the E. coil UTI strain VR50. FyuA was found to be highly important for biofilm formation in iron-poor environments such as human urine. Mutants in fyuA show aberrant biofilm formation and the cells become filamentous; a VR50fyuA mutant showed a 92 % reduction in biofilm formation in urine flow-cell chambers compared with the wild-type. The FyuA/yersiniabactin system is known to be important for virulence. Here we demonstrate a direct link between FyuA and biofilm formation in iron-poor environments. We also show that the availability of iron greatly influences UTI strains' ability to form biofilm.

Many bacterial infections are associated with biofilm formation. In the urinary tract bacterial biofilms develop on both living surfaces and artificial implants, producing chronic and often intractable infections. Escherichia coli is the most common organism associated with urinary tract infections. In contrast to uropathogenic E. coli (UPEC), which cause symptomatic urinary tract infection, asymptomatic bacteriuria (ABU) strains are associated with essentially symptom-free infections. Here the biofilm-forming capacity on abiotic surfaces of selected E. coli ABU strains and UPEC strains in human urine was investigated. It was found that there is a strong bias for biofilm formation by the ABU strains. Not only were the ABU strains significantly better biofilm formers than UPEC strains, they were also able to out-compete UPEC strains as well as uropathogenic strains of Klebsiella spp. during biofilm formation. The results support the notion of bacterial prophylaxis employing selected ABU strains to eliminate UPEC strains and other pathogens in patients prone to recalcitrant infections.

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infection (UTI), very little is known about the mechanisms by which these strains colonize the urinary tract. Bacterial adhesion conferred by specific surface-associated adhesins is normally considered as a prerequisite for colonization of the urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. This study characterized the molecular status of one of the primary adhesion factors known to be associated with UTI, namely F1C fimbriae, encoded by the foc gene cluster. F1C fimbriae recognize receptors present in the human kidney and bladder. Expression of the foc genes was found to be up-regulated in human urine. It was also shown that although strain 83972 contains a seemingly intact foc gene cluster, F1C fimbriae are not expressed. Sequencing and genetic complementation revealed that the focD gene, encoding a component of the F1C transport and assembly system, was non-functional, explaining the inability of strain 83972 to express this adhesin. The data imply that E. coli 83972 has lost its ability to express this important colonization factor as a result of host-driven evolution. The ancestor of the strain seems to have been a pyelonephritis strain of phylogenetic group B2. Strain 83972 therefore represents an example of bacterial adaptation from pathogenicity to commensalism through virulence factor loss.

Glycosylation is a common modulation of protein function in eukaryotes and is biologically important. However, in bacteria protein glycosylation is rare, and relatively few bacterial glycoproteins are known. In Escherichia coli only two glycoproteins have been described to date. Here we introduce a novel member to this exclusive group, namely, antigen 43 (Ag43), a self-recognizing autotransporter protein. By mass spectrometry Ag43 was demonstrated to be glycosylated by addition of heptose residues at several positions in the passenger domain. Glycosylation of Ag43 by the action of the Aah and TibC glycosyltransferases was observed in laboratory strains. Importantly, Ag43 was also found to be glycusylated in a wild-type strain, suggesting that Ag43-glycosylation may be a widespread phenomenon. Glycosylation of Ag43 does not seem to interfere with its self-associating properties. However, the glycosylated form of Ag43 enhances bacterial binding to human cell lines, whereas the nonglycosylated version of Ag43 does not to confer this property.

Diarrhea-causing Escherichia coli strains are responsible for numerous cases of gastrointestinal disease and constitute a serious health problem throughout the world. The ability to recognize and attach to host intestinal surfaces is an essential step in the pathogenesis of such strains. AIDA is a potent bacterial adhesin associated with some diarrheagenic E. coli strains. AIDA mediates bacterial attachment to a broad variety of human and other mammalian cells. It is a surface-displayed autotransporter protein and belongs to the selected group of bacterial glycoproteins; only the glycosylated form binds to mammalian cells. Here, we show that AIDA possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrate that intercellular AIDA-AIDA interaction is responsible for bacterial autoaggregation. Interestingly, AIDA-expressing cells can interact with antigen 43 (Ag43)-expressing cells, which is indicative of an intercellular AIDA-Ag43 interaction. Additionally, AIDA expression dramatically enhances biofilm formation by E. coli on abiotic surfaces in flow chambers.

Bacterial surface structures such as capsules and adhesins are generally regarded as important virulence factors. Here we demonstrate that capsules block the function of the self-recognizing protein antigen 43 through physical shielding. The phenomenon is not restricted to Escherichia coli but can occur in other gram-negative bacteria. Likewise, we show that other short adhesins exemplified by the AIDA-I protein are blocked by the presence of a capsule. The results support the notion that capsule polysaccharides sterically prevent receptor-target recognition of short bacterial adhesins. This negative interference has important biological consequences, such as affecting the ability of bacteria to form biofilms.

Results reveal that the alpha module of the self-recognizing outer membrane protein of Escherichia coli, antigen 43, can accommodate and display correctly folded proteins containing entire functional domains in gram-negativve baceria including Salmonella strains. Implications of the research for vaccine development is discussed.

Researchers have identified novel zinc-binding peptide sequences in a random peptide library based on the FimH adhesion on the tip of Escherichia coli type 1 fimbriae. These peptides could be expressed in E. coli and used for heavy metal bioremediation or as a chemical sensor for detecting heavy metals in the environment.

Research demonstrates that the surface-displayed autotransporter protein antigen 43 of Escherichia coli can be expressed in Pseudomonas fluorescens strain SBW25. The protein's characteristic cell aggregation and frizzy colony morphology properties render them useful in waste treatment and pollutant degradation.

Research has been conducted on colony morphology of Eschericiia coli K-12. The effect of antigen 43 and type 1 fimbriae on colony morphology has been investigated.

A strain of E. coli that can bind zinc could be used to remove heavy metals from soil or water. The type 1 fimbriae on the bacterial surface bind zinc oxide and the protein appears to be a zinc-containing helicase.

Research concerning the ability of Escherichia coli Nissle 1917 to express type 1 fimbriae is presented. Results demonstrate the effect of liquid shaking conditions on type 1 fimbriae expression.

Antigen 43 (Ag43), the product of the flu gene, is a surface-displayed autotransporter protein of Escherichia coli. Ag43 plays a role in the autoaggregation and flocculation of static liquid cultures of many E. coli strains. It was shown through the application of well-defined mutants, that fimbriation eliminates Ag43-mediated autoaggregation but does not affect Ag43 expression. Autoaggregation was found to require and intercellular Ag43-Ag3 interaction, and the physical presence of fimbriae on the cells appears to revoke this interaction.

Fimbriae-displayed FimH-FocH hybrids were utilized to investigate the receptor recognition site of the FimH adhesin. Results revealed that erythrocytes and yeast cells agglutinated following the fusion of the N-terminus of FimH to the complementary sector of FocH. The receptor binding capacity of some of the fusions disappeared. Moreover, the existence of a D-mannose-receptor-binding core domain in FimH was confirmed.

A heterobifunctional adhesin was engineered based on the Escherichia coli FimH adhesin. The primary binding domain of FimH recognizes D-mannoside residues. The addition of histidine clusters created a secondary binding site which recognizes metals. Transformed bacteria expressing the recombinant adhesin are able to bind saccharides and metal. Further, the binding of the two domains are independently regulated.

The effects of insert position and host background on the cholera toxin B fimA gene were determined by utilizing a reporter epitope mimicking a neutralizing epitope of the cholera toxin B. Results reveal that insertion of the wild-type fim gene mediated the production and surface exposure of chimeric FimA proteins in contrast with the mutated MS7 strain. This is secondary to the co-assembly of wild-type and chimeric FimA proteins into the fimbrial organeles.

The preS2 hepatitis B protein segment and the cholera toxin epitope inserted into the Escherichia coli FimH proteins are effective as a surface display system for heterologous sequences. The conformation of the inserted DNA segments is similar to the sequences in the wild-type proteins. The inserted protein segments contain almost 56 amino acids and has no effect on FimH production or exposure to the cell surface. The adhesive activity of FimH reduces slightly.

Variations in fimH genes are responsible for the functional heterogeneity of type 1 fimbriae. This is revealed by distinct adhesion to yeast mannans, human plasma fibronectin and fibronectin derivatives evident for the type 1-fimbriated Escherichia coli strains CSH-50 and HB101 which are K-12 derivatives. Functional heterogeneity among clinical isolates is studied. The role of variations in explaining difference in receptor specificity is also examined.