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S Lory

Publications and source records attributed to S Lory.

At least 73 records · Page 4Linked to original sources
PubMed1991

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.

Amino Acid Sequence↗
PubMed1991

Interaction of Pseudomonas aeruginosa with A549 pneumocyte cells.

The interaction of Pseudomonas aeruginosa with a human lung pneumocyte cell line (A549) was studied. Wild-type strain PAK adhered efficiently to the A549 cells, while an isogenic mutant, carrying a mutation in the pilin structural gene, adhered at 10 to 20% of the wild-type levels. Another nonpiliated mutant of P. aeruginosa PAK, defective in the pleiotropic regulatory gene rpoN, did not adhere to A549 cells, suggesting the presence of a second, RpoN-controlled adhesin on the bacterial surface. Endocytosis of wild-type P. aeruginosa PAK by A549 cells was also demonstrated. A significant fraction of the internalized bacteria were recovered in a viable form after several hours of residence within the A549 cells. When examined by electron microscopy, intracellular bacteria were located in membranous vesicles, and no evidence of killing by lysosomal mechanisms was observed. These studies raise the possibility that during chronic respiratory tract infections in immunocompromised patients, P. aeruginosa may persist in intracellular compartments and therefore be protected from the defense mechanisms of the host.

Adhesins, Bacterial↗
PubMed1991

Adhesion of Pseudomonas aeruginosa pilin-deficient mutants to mucin.

Attachment of Pseudomonas aeruginosa to epithelial cells or tracheobronchial mucin is mediated by surface adhesins. Pili, composed of monomeric pilin subunits, make up one such class of adhesins. The formation of pili and flagella in P. aeruginosa is under the control of the alternative sigma factor rpoN. Isogenic mutant strains with insertionally inactivated rpoN genes were constructed with strains PAK, 1244, and CF613 and were tested for their ability to adhere to respiratory mucin. All rpoN mutants showed significant reduction of adherence to mucin relative to that of their wild-type parents. In contrast, the adherence of pilin structural gene mutants was similar to the adherence of wild types. These results provide suggestive evidence that P. aeruginosa also binds to mucin via adhesins that are distinct from pilin and are still under the genetic control of rpoN. Unlike the laboratory strain PAK, the clinical strains 1244 and CF613 are capable of agglutinating erythrocytes. The rpoN mutation had a minimal effect on the interaction of bacteria with erythrocytes, indicating that the transcription of a gene(s) specifying the agglutination phenomenon does not utilize rpoN. These findings collectively indicate the existence of several classes of adhesins on the surface of P. aeruginosa that may play an important role in colonization of the human respiratory tract.

Bacterial Adhesion↗
PubMed1991

Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the genes pilB, pilC, and pilD encode proteins necessary for posttranslational modification and assembly of pilin monomers into pilus organelles (D. Nunn, S. Bergman, and S. Lory, J. Bacteriol. 172:2911-2919, 1990). We show that PilD, encoding a putative pilin-specific leader peptidase, also controls export of alkaline phosphatase, phospholipase C, elastase, and exotoxin A. pilD mutants accumulate these proteins in the periplasmic space, while secretion of periplasmic and outer membrane proteins appears to be normal. The periplasmic form of exotoxin A was fully mature in size, contained all cysteines in disulfide bonds, and was toxic in a tissue culture cytotoxicity assay, suggesting that in pilD mutants, exotoxin A was folded into its native conformation. The function of the other two accessory proteins, PilB and PilC, appears to be restricted to pilus biogenesis, and strains carrying mutations in their respective genes do not show an export defect. These studies show that in addition to cleaving the leader sequence from prepilin, PilD has an additional role in secretion of proteins that are released from P. aeruginosa into the surrounding media. PilD most likely functions as a protease that is involved in processing and assembly of one or more components of the membrane machinery necessary for the later stages of protein extracellular localization.

ADP Ribose Transferases↗
PubMed1990

The effect of piliation and exoproduct expression on the adherence of Pseudomonas aeruginosa to respiratory epithelial monolayers.

The adherence properties of Pseudomonas aeruginosa strains with known pilin DNA sequences were studied. A polar pilus clearly contributed to adherence, as 35S-labeled pilus-positive (Pil+) strains bound significantly more to bovine trachea epithelial monolayers than did pilus-negative (Pil-) mutants (P less than .05) and minimally more than hyperpiliated strains. A pil- mutant PAK/NP, constructed by gene replacement, demonstrated low levels of attachment (3.8% of the inoculum adherent compared with 7% for the wild-type strain PAK), suggesting that other adhesins are functional. The Pil-, flagellum-negative strain PAO1150.1 bound the least (0.3% of the inoculum adherent), confirming the importance of motility in the binding process. The pilin sequences of strains P1 and PA1244 were virtually identical, although P1 bound threefold more than did PA1244. P1 produced 10-fold more proteinase than did PA1244, and a proteinase-negative mutant of P1, isolated by transposon mutagenesis, had binding equivalent to that of PA1244. The adherence of PAO1 was increased 60% in the presence of bacterial supernatants from phosphate-limited cultures and correlated with phospholipase C activity in the supernatant. Thus, the expression of several Pseudomonas genes may be required to promote efficient binding to epithelial surfaces.

Animals↗
PubMed1990

The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene.

The product of the rpoN gene is an alternative sigma factor of RNA polymerase which is required for transcription of a number of genes in members of the family Enterobacteriaceae, including those that specify enzymes of nitrogen assimilation, amino acid uptake, and degradation of a variety of organic molecules. We have previously shown that transcription of the pilin gene of Pseudomonas aeruginosa also requires RpoN (K. S. Ishimoto and S. Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and have undertaken a more extensive survey of genes under RpoN control. Strains of P. aeruginosa that carry an insertionally inactivated rpoN gene were constructed and shown to be nonmotile because of the inability of these mutants to synthesize flagellin. The mutation in rpoN had no effect on expression of extracellular polypeptides, outer membrane proteins, and the alginate capsule. However, the rpoN mutants were glutamine auxotrophs and were defective in glutamine synthetase, indicating defects in nitrogen assimilation. In addition, the P. aeruginosa rpoN mutants were defective in urease activity. These findings indicate that the sigma factor encoded by the rpoN gene is used by P. aeruginosa for transcription of a diverse set of genes that specify biosynthetic enzymes, degradative enzymes, and surface components. These rpoN-controlled genes include pili and flagella which are required for full virulence of the organism.

Bacterial Outer Membrane Proteins↗
PubMed1990

Role of the far-upstream sites of the algD promoter and the algR and rpoN genes in environmental modulation of mucoidy in Pseudomonas aeruginosa.

The role of several regulatory elements in environmental modulation of mucoidy in Pseudomonas aeruginosa was studied. Transcriptional activation of algD, necessary for the mucoid phenotype, was found to depend on FUS, the newly identified far-upstream sites of the algD promoter. The FUS were delimited to a region spanning nucleotides -432 to -332 relative to the algD mRNA start site. Insertional inactivation of algR in PAO568 abolished the algD promoter response to nitrogen availability and greatly diminished but did not completely eliminate reactivity to changes in salt concentration. Insertional inactivation of rpoN (ntrA) in PAO568 did not affect algR and algD transcription.

Base Sequence↗
PubMed1990

Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK.

Flagella in procaryotes are complex structures requiring the coordinate expression of over 50 genes, including flagellin, the major repeating structural protein. We have previously shown that a functional RpoN gene product is required for expression of flagellin in Pseudomonas aeruginosa PAK (P. A. Totten and S. Lory, J. Bacteriol. 172:389-396, 1990) and have now cloned, sequenced, and determined the transcriptional start site of the structural gene for this flagellin. The clones containing this gene produced a protein that reacted on Western immunoblots with polyclonal and four different monoclonal antibodies to purified flagella. However, this flagellin protein in Escherichia coli was slightly smaller (41 kDa) than flagellin protein produced in P. aeruginosa PAK (45 kDa), indicating degradation in E. coli or modification in P. aeruginosa. Comparison of the deduced amino acid sequence of this gene with the amino acid sequences of other flagellins revealed a conservation in the N- and C-terminal domains, suggesting conservation of secretion or assembly signals between these organisms. The sequence 5' of the structural gene contained potential RpoN-specific promoters as well as a promoter sequence recognized by RpoF (sigma 28), the alternative sigma factor required for expression of flagellin genes in E. coli (and Bacillus subtilis). Deletion analysis of the promoter region as well as transcriptional start site mapping implicated the RpoF, and not the RpoN, consensus sequences as the functional promoter for the flagellin gene. Models for the involvement of both RpoN and RpoF in the expression of flagellin in P. aeruginosa are presented.

Amino Acid Sequence↗
PubMed1990

Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili.

The polar pili of Pseudomonas aeruginosa are composed of monomers of the pilin structural subunits. The biogenesis of pili involves the synthesis of pilin precursor, cleavage of a six-amino-acid leader peptide, membrane translocation, and assembly of monomers into a filamentous structure extending from the bacterial surface. This report describes three novel genes necessary for the formation of pili. DNA sequences adjacent to pilA, the pilin structural gene, were cloned and mutagenized with transposon Tn5. Each of the insertions were introduced into the chromosome of P. aeruginosa PAK by gene replacement. The effect of the Tn5 insertions in the bacterial chromosome on pilus assembly was assessed by electron microscopy and sensitivity of mutants to a pilus-specific bacteriophage. The resultant mutants were also tested for synthesis and membrane localization of the pilin antigen in order to define the genes required for maturation, export, and assembly of pilin. A 4.0-kilobase-pair region of DNA adjacent to the pilin structural gene was found to be essential for formation of pili. This region was sequenced and found to contain three open reading frames coding for 62-, 38- to 45-, and 28- to 32-kilodalton proteins (pilB, pilC, and pilD, respectively). Three proteins of similar molecular weight were expressed in Escherichia coli from the 4.0-kilobase-pair fragment flanking pilA with use of a T7 promoter-polymerase expression system. The results of the analyses of the three genes and the implications for pilin assembly and maturation are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1989

Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase.

The promoter region of the Pseudomonas aeruginosa pilin gene has a high degree of similarity to the nitrogen-regulated promoters of enteric bacteria. These promoters are recognized by the alternative sigma factor of RNA polymerase, termed RpoN (NtrA or GlnF). This observation suggested that the P. aeruginosa pilin gene may be transcribed by the RpoN-containing RNA polymerase. We, therefore, cloned the RpoN gene from P. aeruginosa into Escherichia coli (where it formed a functional product) and used that cloned gene to construct a mutant of P. aeruginosa that was insertionally inactivated in its RpoN gene. This mutant failed to synthesize pilin, indicating that the RpoN sigma factor is required for transcription of the pilin gene.

Bacterial Outer Membrane Proteins↗
PubMed1988

Expression and secretion of the cloned Pseudomonas aeruginosa exotoxin A by Escherichia coli.

The exotoxin A gene from Pseudomonas aeruginosa PAK was expressed in Escherichia coli from recombinant plasmids when transcription was initiated from a promoter in the cloning vector. The exotoxin A polypeptide synthesized was found to have an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of 66,000 daltons, identical in size to the mature exotoxin A made by P. aeruginosa. Analysis of the location of exotoxin A in various bacterial compartments by immunoblotting revealed that exotoxin A was exported by E. coli into its periplasmic space. Several functional assays, including analyses of disulfide bond formation, potentiation of ADP-ribosyltransferase activity, and HeLa cell cytotoxicity, were used to establish that the conformation of exotoxin A isolated from the E. coli periplasmic space is identical to that of exotoxin exported by P. aeruginosa to its extracellular space. Previous studies with recombinant plasmids expressing exotoxin A from P. aeruginosa PA103 (G. D. Gray, D. Smith, J. Baldridge, R. Markins, M. Vasil, E. Chen, and M. Heyneker, Proc. Natl. Acad. Sci. USA 81:2645-2649, 1984) showed a complete lack of processing and export of pre-exotoxin A in E. coli, differing from results reported here. These discrepancies may be explained by observed differences in the sequence of signal peptides encoded by the exotoxin A genes of PAK and PA103 strains of P. aeruginosa.

ADP Ribose Transferases↗
PubMed1988

Biotinylated DNA probes for exotoxin A and pilin genes in the differentiation of Pseudomonas aeruginosa strains.

Biotin-labeled DNA probes derived from Pseudomonas aeruginosa exotoxin A and pilin genes were tested for their ability to distinguish strains among a selected group of P. aeruginosa isolates. Probing of Southern blots of restriction digests of DNA from test strains with the exotoxin A probe demonstrated a unique hybridization pattern for each independently isolated strain containing the exotoxin A gene. Two phenotypically distinct strains isolated from the same patient were found to be identical in their DNA hybridization patterns. By using a pilin gene probe, similar distinction was made between independent strains, while strains from the same source were confirmed to be identical. Furthermore, DNA from a strain of P. aeruginosa lacking the exotoxin A gene yielded a unique pattern of restriction fragments which hybridized to the pilin gene probe. The exotoxin A and the pilin probes may together prove to be useful tools in epidemiological surveys during outbreaks of P. aeruginosa infection.

ADP Ribose Transferases↗
PubMed1987

Characterization of Pseudomonas aeruginosa mutants with altered piliation.

The pilus-specific Pseudomonas aeruginosa bacteriophage P04 was used to select spontaneous mutants of strain PAK which have altered piliation. The largest class of phage-resistant mutants synthesized the pilin polypeptide, but did not assemble pili. These mutants are likely to contain mutations in genes required for pilus assembly and not mutations in the pilin structural gene, as they could not be complemented by a normal copy of the pilin gene. In addition, two alterations in pilin gene transcription were found among the mutants--hyperpiliated mutants which overproduce pilin mRNA, and a mutant with temperature-sensitive pilin gene transcription. We also present a model for the regulation of pilin gene transcription by a feedback mechanism sensitive to the relative rates of pilus assembly and disassembly.

Bacterial Outer Membrane Proteins↗
PubMed1987

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.

Alkaline Phosphatase↗
PubMed1986

Nucleotide sequence and transcriptional initiation site of two Pseudomonas aeruginosa pilin genes.

The complete nucleotide sequences of the 1.2-kilobase HindIII fragments which contain the pilin genes of two independently isolated strains of Pseudomonas aeruginosa (PAK and PA103) have been determined and compared to that of strain PA01 (Sastry, P. A., Finlay, B. B., Pasloske, B. L., Paranchych, W., Pearlstone, J. R., and Smollier, L. B. (1985) J. Bacteriol. 164, 571-577). The fragments share extensive regions of homology, including the 5'- and 3'-flanking sequences as well as the 5' end of the pilin gene. The most highly diverged segments of the pilin genes are those which encode the variable carboxyl-terminal region of the pilin polypeptides. The pilin polypeptides each contain a 6-amino acid amino-terminal leader peptide (Met-Lys-Ala-Gln-Lys-Gly) and are nearly identical in the following 60 amino acids. The carboxyl-terminal portion of the pilin polypeptides contain extensive regions of divergence in their amino acid sequences, although hydropathicity analysis of the pilin polypeptides indicated that they are structurally similar. The transcriptional initiation site of the PAK pilin gene has been determined by S1 nuclease mapping. The promoter region at -10 and -35 base pairs from the transcriptional initiation site shows no significant homology to the consensus Escherichia coli promoter, but the -12 and -24 regions show a high degree of homology to promoters which require the ntrA gene product for transcription. Several other Pseudomonas promoters and the promoters of the homologous pilin genes from other bacterial species also share homology to this sequence.

Amino Acid Sequence↗
PubMed1986

Cloning and expression of the pilin gene of Pseudomonas aeruginosa PAK in Escherichia coli.

Many strains of Pseudomonas aeruginosa possess pili which have been implicated in the pathogenesis of the organism. This report presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the pili of P. aeruginosa PAK. Total DNA from this strain was partially digested with Sau3A and inserted into the cloning vector pUC18. Recombinant E. coli clones were screened with oligonucleotide probes prepared from the constant region of the previously published amino acid sequence of the mature pilin subunit. Several positive clones were identified, and restriction maps were generated. Each clone contained an identical 1.1-kilobase HindIII fragment which hybridized to the oligonucleotide probes. Western blot analysis showed that all of the clones expressed small amounts of the P. aeruginosa pilin subunit, which has a molecular mass of ca. 18,000. This expression occurred independently of the orientation of the inserted DNA fragments in the cloning vector, indicating that synthesis was directed from an internal promoter. However, subclones containing the 1.1-kilobase HindIII fragment in a specific orientation produced an order of magnitude more of the pilin subunit. While the expressed pilin antigen was located in both the cytoplasmic and outer membrane fractions of E. coli, none appeared to be polymerized into a pilus structure.

Bacterial Outer Membrane Proteins↗
PubMed1986

Effect of iron on accumulation of exotoxin A-specific mRNA in Pseudomonas aeruginosa.

A DNA probe from an internal fragment of the exotoxin A structural gene was used to study the effects of selected culture conditions on steady-state levels of exotoxin-specific mRNA in Pseudomonas aeruginosa. Cells grown under conditions of iron deprivation began to synthesize and excrete the exotoxin A polypeptide during the late exponential phase of growth and throughout the stationary phase of growth, concomitant with a sharp increase in exotoxin A mRNA pools in P. aeruginosa cells. The addition of iron to the medium resulted in the failure of these cells to synthesize exotoxin A mRNA, despite significantly enhanced growth. The inhibition of the production of exotoxin A and the accumulation of its mRNA by iron was dose dependent, with a half-maximal inhibitory concentration of FeSO4 of 5 to 10 microM. A blockade of the initiation of transcription by rifampin resulted in the decay of exotoxin A mRNA, with a half-life of approximately 8 to 10 min, depending on the media used for growth. The addition of iron to cells actively engaged in exotoxin A synthesis also resulted in a gradual decrease in the amount of this mRNA in bacteria. However, the rate of decline of mRNA induced by iron was relatively slow (half-life, 90 min), with a considerable lag time between the iron addition and the first detectable effect on mRNA. While iron clearly appears to influence the production of exotoxin A at the transcriptional level, the molecular basis of this effect may involve several interacting factors affecting the initiation of transcription and perhaps mRNA turnover.

ADP Ribose Transferases↗