In iron-limited environments, the plant-growth-stimulating Pseudomonas putida WCS358 produces a yellow-green fluorescent siderophore called pseudobactin 358. The transcriptional organization and the iron-regulated expression of a major gene cluster involved in the biosynthesis and transport of pseudobactin 358 were analyzed in detail. The cluster comprises a region with a minimum length of 33.5 kilobases and contains at least five transcriptional units, of which some are relatively large. The directions of transcription of four transcriptional units were determined by RNA-RNA hybridization and by analysis in Escherichia coli minicells. The latter also demonstrated that large polypeptides were encoded by these transcriptional units. The results allowed us to localize several promoter regions on the DNA. The iron-dependent expression of at least two genes within this cluster appears to be regulated at the transcriptional level.

The P fimbrial gene clusters encoding the serologically different F71, F72, F9, and Fll fimbriae were compared functionally. The results show that these gene clusters are closely related.

was studied. Deletion of two regions that code for hypervariable parts of the P fimbrillin resulted in strong reduction or total absence of fimbria production. Replacement of deleted amino acids by other amino acid residues restored the formation of fimbriae. The hypervariable regions may be important for biogenesis of fimbriae by imposing correct spacing between conserved regions of the protein.The potential for substituting amino acids in the P-fimbrial subunit opens interesting possibilities for use of fimbriae as carriers of foreign antigenic determinants. An antigenic determinant of foot-and-mouth disease virus (FMDV) was incorporated in the F11 fimbrial subunit. Hybrid fimbriae, recognized by an FMDV-specific neutralizing monoclonal antibody directed against FMDV, were formed.Fimbriae are long filamentous appendages that occur on many bacteria and consist of about 1,000 protein subunits (for a review, see reference 14). Many uropathogenic Escherichia coli strains expose on their cell surfaces P fimbriae that mediate adherence of the pathogen to the uroepithelium (11,31,32). P fimbriae recognize the a-D-galactose-(1,4)-P-D-galactose moiety of antigens of the P blood group (13,18). Among P fimbriae, the serotypes F7 through F13 have been distinguished (27). From uropathogenic E. coli, the genes responsible for synthesis of various serologically different P fimbriae, i.e., F71, F72, F8, F9, Fll, and F13, have been cloned (5,6,10,12,28,36,37). It has been shown that these fimbrial gene clusters are very similar in general organization (25,36,38), which suggests that the biogenesis of P fimbriae may follow a general concept.The composition of P fimbriae is very complex. The major subunit, or P fimbrillin, is predominant and determines the antigenic properties (35). Several minor components, among them the adhesin protein, are also present in the fimbrial structure (19,20,29 MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. E. coli K-12 HB101 (2), a strain deficient in the production of type 1 fimbriae, was used as the host strain in all experiments unless otherwise indicated. JM101 was used as the host strain for M13mp8 derivatives (24). HB2154 (3) was used as the host strain for M13mpl8 (26) derivatives in the localized mutagenesis experiments. The mutant plasmids described were derived from pPIL110-75, carrying thefso gene cluster, and pPIL291-15, carrying the fel (F-eleven) gene cluster (6,29,36

Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes. Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively. Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors. Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids. The inserted peptides appeared to be exposed in the fimbrial filament. One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice.

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The gene cluster coding for expression of F71 fimbriae of the uropathogenic Escherichia coli strain AD110 has been cloned by a cosmid‐cloning procedure. A positive clone was further subcloned to a plasmid of 17.5 kilobases (kb), pPIL110‐75. Analysis of pPIL110‐75 showed that at least six genes are present encoding proteins with apparent Mrs of 75 000, 36 000, 23 000, 20 000, 17 000 and 14 000. The 20‐kDa protein, encoding the F71 fimbrial subunit is dispensable for expression of the MRHA phenotype. Complementation experiments of mutants in the F72 gene cluster by gene products of the F71 gene cluster show that the two gene clusters are related.

Construction and analysis of mutations in the hypervariable regions of the F71, F9 and F11 fimbrillin genes are described. The results show that mutations in the hypervariable regions of the fimbrillin can be made without abolishing the ability of these fimbrillins to assemble into filaments. The mutant fimbriae show binding patterns with specific monoclonal antibodies that differed from those of the corresponding wild‐type fimbriae. These results confirm that antigenic determinants of the P‐fimbriae are, at least partly, located in the hypervariable regions, and suggest that more than one antigenic determinant is involved in the F71 and F11 specificity.