The gene encoding porin protein F of Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed as the predominant outer membrane protein in a porin-deficient E. coli background and was clearly visible on one-dimensional sodium dodecyl sulfate-polyacrylamide gels in a porin-sufficient background. The identity of the protein F from the E. coli clone and native P. aeruginosa protein F was demonstrated by their identical mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, 2-mercaptoethanol modifiabilities, and reactivities with monoclonal antibodies specific of two separate epitopes of protein F. In the course of gene subcloning, a 2-kilobase DNA fragment was isolated, with an apparent truncation of the part of the gene encoding the carboxy terminus of protein F. This subclone produced a 24,000-molecular-weight, outer membrane-associated, truncated protein F derivative which was not 2-mercaptoethanol modifiable and which reacted with only one of the two classes of protein F-specific monoclonal antibodies. The 2-kilobase fragment was used in Southern blot hybridizations to construct a restriction map of the cloned and subcloned fragments and to demonstrate with restriction digests of whole P. aeruginosa DNA that only one copy of the protein F gene was present in the P. aeruginosa chromosome. The protein F produced by the original cosmid clone in a porin-deficient E. coli background was purified. To demonstrate retention of porin function after cloning, the protein F from the E. coli clone was incorporated into black lipid bilayer membranes. Two major classes of channels were revealed. The predominant class of channels had an average conductance of 0.36 nS in 1 M KCl, whereas larger channels (4 to 7 nS) were seen at a lower frequency. Similar channel sizes were observed for porin protein F purified by the same method from P. aeruginosa outer membranes.

Examination of the distribution of cell-associated and soluble hemagglutinins (HA) produced by Vibrio cholerae revealed the existence offour different HAs. A cell-associated mannose-sensitive HA (MSHA) was produced only by the El Tor biotype. This was evident with all El Tor strains examined. It appears to be responsible for the HA biotyping differentiation of El Tor from classical biotype V. cholerae. The MSHA had no apparent divalent ion requirement; it was inhibited by D-mannose and D-fructose; and it was active on all human (A, B, 0) and all chicken erythrocytes tested. Spontaneous MSHAmutants of El Tor strains were selected by cosedimentation of MSHA+ parent bacteria with erythrocytes. An L-fucose-sensitive HA was detected transiently in early logphase growth with two of the four classical strains examined and with MSHAmutants of El Tor biotype strains 3083, 26-3, and 17. MSHAmutants also expressed another cell-associated HA in late log-phase cultures. A "soluble" HA was detected in late log-phase cultures of all strains tested. This HA was not inhibitable by any sugars tested; it required CA2+ ions for maximum activity; and it was active on some chicken erythrocytes but not others.

The soluble hemagglutinin (HA) (cholera lectin) produced by Vibrio cholerae strain CA401 was purified to apparent homogeneity by a sequence of ammonium sulfate fractionation, gel filtration, and preparative isoelectric focusing. Soluble HA activity was found to focus at three different pls, 6.3, 5.3, and 4.7. Each of the factors migrated as a large-molecular-weight protein in sodium dodecyl sulfatepolyacrylamide gel electrophoresis under normal conditions, and each, upon heating in sodium dodecyl sulfate was found to dissociate into 32,000-molecularweight subunits. Treating the samples with a reducing agent did not affect their mobility. Each gave a reaction of immunological identity with antiserum prepared against the others. Thus, there are apparently three distinct pH isotypes of soluble HA which exist as noncovalently associated polymers of 32,000-molecular-weight subunits. Electron microscopy of purified preparations revealed long filamentous polymers. The molecule does not stain as a glycoprotein; it is hydrophobic; it is inactivated during incubation at 25, 37, or 60'C; and it has significant protease activity. The protease activity likewise focused at pH values of 6.3 and 5.3 to 4.7, and it was inhibited by antiserum against the HA. However, whereas the HA is active at 4TC, the protease is not. The soluble HA is, therefore, a bifunctional molecule capable of mediating hemagglutination and proteolysis. Its amino acid composition is reported. Fab fragments of antibody against the purified HA inhibited attachment of heterologous serotype-biotype V. cholerae to infant rabbit small bowel. MATERIALS AND METHODS Bacterial strains. V. cholerae strain CA401 is a classical biotype, Inaba serotype originally isolated in Calcutta in 1953 by C. E. Lankford. It and other V. cholerae strains used have been described previously (4, 7, 9). HA assay. The techniques for microtiter quantitation of HA activity were as described (7). Responsive and nonresponsive chicken erythrocytes (7) used for monitoring HA activity throughout the purification scheme, were obtained from individual chickens from an inbred line of White Leghorn chickens at the University of Missouri Department of Poultry Husbandry. Purification of soluble HA. V. cholerae CA401 was grown confluently on four petri plates of meat extract agar overnight at 37°C. Growth was harvested with sterile tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) and inoculated into 9.5 liters of TSB in each of four fermentor jars. Fermentors (New Brunswick Scientific, Inc., Edison, N.J.), to which were added 3 ml of antifoam (antifoam B,

A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.

Twelve mutants of Pseudomonas aeruginosa PAO defective in pyoverdin production were isolated (after chemical and transposon mutagenesis) that were nonfluorescent and unable to grow on medium containing 400 ,uM ethylenediaminedi(o-hydroxyphenylacetic acid). Four mutants were unable to produce hydroxamate, six were hydroxamate positive, one was temperature sensitive for pyoverdin production, and another was unable to synthesize pyoverdin on succinate minimal medium but was capable of synthesizing pyoverdin when grown on Casamino Acids medium (Difco Laboratories, Detroit, Mich.). The mutations were mapped on the PAO chromosome. All the mutations affecting pyoverdin production were located at 65 to 70 min, between catAl and mtu-9002.Pathogenic bacteria require iron for growth in a mammalian host (6). To obtain iron from the host, bacteria must effectively compete with the iron-sequestering proteins transferrin and lactoferrin (1). Many microbes possess iron acquisition systems mediated by siderophores, lowmolecular-weight products capable of binding and delivering iron to the cell via high-affinity transport systems (31).Pseudomonas aeruginosa is a major cause of nosocomial infections which result in high mortality. P. aeruginosa is known to produce two siderophores, pyochelin (9, 10, 25) and pyoverdin (8,42). Pyochelin is a phenolic siderophore that has two sulfur-containing heterocyclic rings (9, 10). Pyoverdin, previously termed bacterial fluorescein (A. Turfreijer, Ph.D. thesis, University of Amsterdam, Amsterdam, The Netherlands, 1941), has long been thought to be involved in iron metabolism because of its hydroxamate character (16) and the inhibition of its production by iron (14,17,38). Only recently has the siderophore activity of pyoverdin been demonstrated (8) and its molecular structure elucidated (42). This fluorescent siderophore produced by P. aeruginosa is a complex peptide containing two hydroxamate groups and a dihydroxyquinoline derivative (42) as its theorized chelating moieties. Yellow-green fluorescent peptides produced by other fluorescent Pseudomonas species and Azotobacter vinelandii (5,15,28,32,33,37,44) have similar spectral characteristics and extensive structural homology with pyoverdin.Recent investigations have suggested that iron acquisition by P. aeruginosa may play a role in its pathogenesis. The concentration of iron in culture medium has a significant effect on the production of the extracellular proteins, toxin A, alkaline protease, and elastase (3, 4). Pyochelin has been shown to increase the lethality of P. aeruginosa during infections in mice (7). A mutant unable to synthesize pyoverdin had an extremely depressed growth rate compared with that of wild-type strains when grown in human serum and transferrin (2).Advances in P. aeruginosa genetics have allowed the map positions of several reported virulence factors to be determined. Use of the chromosome-mobilizing plasmid R68.45 * Corresponding author.(19), R-prime plasmids (21), and transposon-facilitated recombination (24) has m...