Selective distribution of Pseudomonas aeruginosa O-antigen among strains producing group I pilin.

Strains of Pseudomonas aeruginosa that produce type IVa pili categorized as group I have the potential to covalently attach an O-antigen repeating unit to the pilin C-terminal residue. PCR, employing primers targeting a conserved region of a group-I-specific gene, was used to provide evidence that 110 of 206 clinical isolates studied had the capability of producing this type of pilus. The potential of P.

The group I pilin glycan affects type IVa pilus hydrophobicity and twitching motility in Pseudomonas aeruginosa 1244.

The group I pilin category is the most common type of type IVa pilus produced by Pseudomonas aeruginosa. The lateral surfaces of these pili are characterized by the presence of closely spaced, covalently attached O-antigen repeating units. The current work was conducted to investigate the pilin glycan's effect on pilus solubility and function.

Glycosylation of pilin and nonpilin protein constructs by Pseudomonas aeruginosa 1244.

PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation of Pseudomonas aeruginosa 1244 pilin by adding a single O-antigen repeating unit to the β carbon of the C-terminal residue (a serine). While PilO has an absolute requirement for Ser/Thr at this position, it is unclear if this enzyme must recognize other pilin features. To test this, pilin constructs containing peptide extensions terminating with serine were tested for the ability to support glycosylation.

Immunization with a Pseudomonas aeruginosa 1244 pilin provides O-antigen-specific protection.

The O antigen is both a major structural outer membrane component and the dominant epitope of most gram-negative bacteria. Pseudomonas aeruginosa 1244 produces a type IV pilus and covalently links an O-antigen repeating unit to each pilin monomer. Here we show that immunization of mice with pure pilin from strain 1244 by use of either the mouse respiratory model or the thermal injury model resulted in protection from challenge with a pilus-null O-antigen-producing 1244 mutant.

PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment.

PilO of Pseudomonas aeruginosa 1244 catalyses the attachment of an O-antigen repeating unit to the beta-carbon of the pilin C-terminal residue, a serine. The present study was conducted to locate the regions of this enzyme important in catalysis and to establish the cellular location of the pilin glycosylation reaction. While PilO was not detectable in extracts of P.

Pseudomonas aeruginosa 1244 pilin glycosylation: glycan substrate recognition.

The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction.

Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance.

Glycosylation substrate specificity of Pseudomonas aeruginosa 1244 pilin.

The beta-carbon of the Pseudomonas aeruginosa 1244 pilin C-terminal Ser is a site of glycosylation. The present study was conducted to determine the pilin structures necessary for glycosylation. It was found that although Thr could be tolerated at the pilin C terminus, the blocking of the Ser carboxyl group with the addition of an Ala prevented glycosylation.

Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity.

The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin.

Identification of the Pseudomonas aeruginosa 1244 pilin glycosylation site.

Previous work (P. Castric, F. J.

Structural characterization of the Pseudomonas aeruginosa 1244 pilin glycan.

An antigenic similarity between lipopolysaccharide (LPS) and glycosylated pilin of Pseudomonas aeruginosa 1244 was noted. We purified a glycan-containing molecule from proteolytically digested pili and showed it to be composed of three sugars and serine. This glycan competed with pure pili and LPS for reaction with an LPS-specific monoclonal antibody, which also inhibited twitching motility by P.

Peptide epitope mapping in vaccine development: introduction.

Protection from infectious disease by the host immune response requires specific molecular recognition of unique antigenic determinants of a given pathogen. An epitope is an antigenic determinant which: 1) specifically stimulates the immune response (either B or T cell mediated); and 2) is acted upon by the products of these protective mechanisms. In B cell immunity, antibodies produced from stimulation by specific epitopes recognize and bind to these same antigenic structures.

pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin.

Nucleotide sequencing of a region downstream from the Pseudomonas aeruginosa 1244 pilin structural gene, pilA, revealed an ORF potentially able to code for a protein of M(r) 50,862. This ORF, called pilO, was flanked by a tRNAthr gene, which was followed by a transcriptional termination sequence. The tRNAthr gene and the termination sequence were nearly identical to sequences found immediately adjacent to the pilA gene of several P.

Differentiation of Pseudomonas aeruginosa pili based on sequence and B-cell epitope analyses.

The nucleotide sequences of three previously undescribed Pseudomonas aeruginosa pilin structural genes are presented. Comparisons of deduced pilin primary structure and flanking DNA sequence allowed placement of these and six previously published sequences into one of two groups. Epitope mapping, using overlapping immobilized peptides representing the pilin primary structure, with antipilin monoclonal antibodies revealed several B-cell determinants grouped near the carboxyl terminus of P.

Cloning and sequencing of the Pseudomonas aeruginosa 1244 pilin structural gene.

The pilin structural gene of Pseudomonas aeruginosa 1244 was cloned in both cosmids and lambda. Expression of the cloned gene was detected in P. aeruginosa strains PAO2003, PA103, and 653A by an immunoblot reaction utilizing monoclonal antibodies.

Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels.

Hydrogen cyanide production by Pseudomonas aeruginosa growing in a synthetic medium required aerobosis but operated efficiently at low dissolved oxygen concentration. Half maximum levels of cyanogenesis occurred at 0.015 microM oxygen; maximum cyanogenesis occurred over a wide range, 0.

Method for rapid detection of cyanogenic bacteria.

An agar plate method is described in which the production of hydrogen cyanide by as many as 50 microbial isolates per plate may be detected. Cyanide produced by the organisms reacts with copper(II) ethylacetoacetate and 4,4'-methylenebis-(N,N-dimethylaniline) in a paper disk suspended above the microbial colonies. Cell growth occurs in depressions in the agar surface, which allows separation of colonies and enhances sensitivity of hydrogen cyanide detection.

In vitro susceptibility of Pseudomonas aeruginosa to carbenicillin, glycine, and ethylenediaminetetraacetic acid combinations.

Striking bacterial activity against Pseudomonas aeruginosa 9D-2 was achieved by glycine-carbenicillin, ethylenediaminetetraacetic acid-carbenicillin, and glycine-ethylenediaminetetraacetic acid combinations, whereas none of the agents used alone was capable of the same degree of bactericidal activity. Studies using a microtiter modification of the checkerboard technique were performed to evaluate the comparative activity of these antimicrobial combinations. Isobolograms showed synergistic effects with carbenicillin-glycine, carbenicillin-ethylene-diaminetetraacetic acid, and glycine-ethylenediaminetetraacetic acid combinations.

The effect of inorganic phosphate on cyanogenesis by Pseudomonas aeruginosa.

The biosynthesis of hydrogen cyanide (HCN) by a strain of Pseudomonas aeruginosa is found to be significantly influenced by inorganic phosphate. Optimum HCN production occurs when the phosphate concentration is between 1 and 10 mM. Above and below this concentration the amount of HCN produced decreases sharply and at 0.

Glycine metabolism by Pseudomonas aeruginosa: hydrogen cyanide biosynthesis.

Hydrogen cyanide (HCN) production by Pseudomonas aeruginosa in a synthetic medium is stimulated by the presence of glycine. Methionine enhances this stimulation but will not substitute for glycine as a stimulator of cyanogenesis. Threonine and phenylalanine are effective substitutes for glycine in the stimulation of HCN production.

Purification and properties of the urea amidolyase from Candida utilis.

Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis.

Urea amidolyase of Candida utilis. Characterization of the urea cleavage reactions.

Evidence is presented that the enzymes catalyzing the three reactions involved in urea cleavage in Candida utilis, biotin carboxylation, urea carboxylation, and allophanate hydrolysis occur as a complex of enzymes. The allophanate-hydrolyzing activity could not be separated from the urea-cleaving activity using common methods of protein purification. Further, urea cleavage and allophanate hydrolysis activities are induced coordinately in cells grown on various nitrogen sources.

Hydrogen cyanide, a secondary metabolite of Pseudomonas aeruginosa.

Seventy-four of 110 strains of Pseudomonas aeruginosa tested produced detectable amounts of HCN from growth in 2% peptone or nutrient agar. Of the 25 species of12 bacterial and fungal genera tested, other than P. aeruginosa, only P.