Genetic mapping of the structural gene for phospholipase C of Pseudomonas aeruginosa PAO.

An insertion mutation constructed by gene replacement methods was used to map the gene corresponding to the hemolytic phospholipase C (plcS gene) in Pseudomonas aeruginosa PAO1 by R68.45-mediated conjugation. plcS mapped approximately at 67 min on the 75-min chromosomal map (B. W. Holloway, K. O'Hoy, and H. Matsumoto, p. 213-221, in S. J. O'Brien, ed., Genetic Maps 1987, vol. 4, 1987), between the markers pur-67 and pru-375 and considerably distal to the regulatory genes plcA and plcB, which are located at approximately 12 min.     

A Pseudomonas aeruginosa mutant non-derepressible for orthophosphate-regulated proteins.

Using a rapid screening assay based on the hydrolysis of p-nitrophenylphosphorylcholine, we isolated several mutants of Pseudomonas aeruginosa deficient in the production of phospholipase C. One, designated strain A50N, was also markedly deficient in the synthesis of alkaline phosphatase and several unidentified extracellular proteins. Because strain A50N produces these proteins under conditions of derepression at levels equal to those produced by the parental strain PAO1 grown in medium containing excess phosphate, it appears to have a mutation in a genetic element involved in the derepression of phosphate-repressible proteins.     

Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO.

Two independently derived, exotoxin A-deficient (Tox- phenotype), nitroso-guanidine-induced mutants of Pseudomonas aeruginosa PAO1 were isolated by using sensitive immunological assays. One mutant, designated PAOT10, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT10 and the parental strain. The mutation in PAOT10 (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.     

The autotransporter esterase EstA of Pseudomonas aeruginosa is required for rhamnolipid production, cell motility, and biofilm formation

Pseudomonas aeruginosa PAO1 produces the biodetergent rhamnolipid and secretes it into the extracellular environment. The role of rhamnolipids in the life cycle and pathogenicity of P. aeruginosa has not been completely understood, but they are known to affect outer membrane composition, cell motility, and biofilm formation. This report is focused on the influence of the outer membrane-bound esterase EstA of P. aeruginosa PAO1 on rhamnolipid production. EstA is an autotransporter protein which exposes its catalytically active esterase domain on the cell surface. Here we report that the overexpression of EstA in the wild-type background of P. aeruginosa PAO1 results in an increased production of rhamnolipids whereas an estA deletion mutant produced only marginal amounts of rhamnolipids. Also the known rhamnolipid-dependent cellular motility and biofilm formation were affected. Although only a dependence of swarming motility on rhamnolipids has been known so far, the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation. In order to demonstrate that EstA enzyme activity is responsible for these effects, inactive variant EstA* was constructed by replacement of the active serine by alanine. None of the mutant phenotypes could be complemented by expression of EstA*, demonstrating that the phenotypes affected by the estA mutation depend on the enzymatically active protein.     

Mapping of the arginine deiminase gene in Pseudomonas aeruginosa

A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB.     

Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa

Summary In order to demonstrate a role for anion-specific protein P channels in phosphate transport in Pseudomonas aeruginosa PAO, we wished to isolate a transposon insertion mutant deficient in protein P. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMT1000 (Tsuda et al. 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the isolation of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was deficient in high-affinity phosphate transport exhibiting a Km for uptake (3.60±0.64 M) almost ten times greater than that of the wild type strain (Km=0.39 M). There was, however, no change in the V_max for high-affinity phosphate transport as a result of the loss of protein P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium resulting in an 18%–35% decrease in growth when compared with the wild type.     

PssO, a unique extracellular protein important for exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii

Synthesis and secretion of polysaccharides by Gram-negative bacteria are a result of a concerted action of enzymatic and channel-forming proteins localized in different compartments of the cell. The presented work comprises functional characterization of PssO protein encoded within the previously identified, chromosomal exopolysaccharide (EPS) biosynthesis region (Pss-I) of symbiotic bacterium Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). pssO gene localization between pssN and pssP genes encoding proteins engaged in exopolysaccharide synthesis and transport, suggested its role in EPS synthesis and/or secretion. RtTA1 pssO deletion mutant and the PssO protein overproducing strains were constructed. The mutant strain was EPS-deficient, however, this mutation was not complemented. The PssO-overproducing strain was characterized by increase in EPS secretion. Subcellular fractionation, pssO-phoA/lacZ translational fusion analyses and immunolocalisation of PssO on RtTA1 cell surface by electron microscopy demonstrated that PssO is secreted to the extracellular medium and remains attached to the cell. Western blotting analysis revealed the presence of immunologically related proteins within the species R. leguminosarum bv. trifolii, bv. viciae and Rhizobium etli. The secondary structure of PssO-His(6), as determined by FTIR spectroscopy, consists of at least 32% alpha-helical and 12% beta-sheet structures. A putative function of PssO in EPS synthesis and/or transport is discussed in the context of its cellular localization and the phenotypes of the deletion mutant and pssO-overexpressing strain.     

Specific protein synthesis in cellular differentiation: V. A secretory defect of chorion formation in the Grcol mutant of Bombyx mori

When homozygous, the Gr^col mutation of Bombyx mori causes the production of an eggshell in which most proteins are underrepresented to varying degrees. Neither the relative rates nor the timing of chorion protein synthesis appear to be affected; instead, the mutant phenotype results from the post-translational loss of normally synthesized proteins. The extent of loss of each protein correlates with its developmental timing, being maximal at early to middle stages. At the same stages, secretion appears to be deficient: chorion proteins overaccumulate within mutant cells, and slowly disappear. A preliminary electron microscopic examination has revealed the presence of mutant-specific cytoplasmic vesicles. The deficient complement of secreted proteins fails to form the highly ordered structure characteristic of normal chorion.     

Molecular defects in Drosophila rhodopsin mutants

Four well characterized Drosophila rhodopsin (ninaE) mutants possess low levels of rhodopsin in their major class of photoreceptors. The molecular defect present in each strain was determined by isolating and sequencing the mutant genes. Two missense mutants encode proteins which have arginine residues positioned within membrane-spanning domains. The third missense mutant eliminates a proline found near an extracellular domain/membrane-spanning domain interface. Thus, the low levels of rhodopsin protein found in these mutants result directly from changes in protein structure which likely affect the positioning and stability of membrane-spanning domains. The fourth and most severe mutation is a nonsense mutation.     

Alpha-thrombin-induced tyrosine phosphorylation of 43,000- and 41,000-Mr proteins is independent of cytoplasmic alkalinization in quiescent fibroblasts.

Incubation of quiescent Chinese-hamster fibroblasts (CCL39) with alpha-thrombin, a potent mitogen for the cells, was found to stimulate the rapid phosphorylation of two 43,000-Mr and two 41,000-Mr proteins at tyrosine, threonine and/or serine, and two 63,000-Mr proteins at serine. Insulin, 12-O-tetradecanoylphorbol 13-acetate (TPA) and epidermal growth factor (EGF) are weak mitogens for cells; insulin and TPA did not stimulate the phosphorylation of those proteins significantly, whereas EGF stimulated their phosphorylation to the same extent as did alpha-thrombin. We analysed alpha-thrombin-induced protein phosphorylation at different external pH values in CCL39 and in the mutant derivative PS120, which lacks Na+/H+-antiport activity. We showed that cytoplasmic alkalinization, a common and early response to mitogens, is not required to trigger phosphorylation of 63,000-, 43,000- and 41,000-Mr proteins, either at tyrosine or serine and threonine residues. This finding contrasts with the phosphorylation of ribosomal protein S6, which takes place only at permissive pH for reinitiation of DNA synthesis. These results, demonstrating that phosphorylation of 63,000-, 43,000- and 41,000-Mr proteins and cytoplasmic alkalinization are not coupled, reinforce the idea that the site of action of intracellular pH controlling the commitment of G0/G1-phase-arrested cells to DNA synthesis might be restricted to mitogen-stimulated S6 phosphorylation.     

Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H₂O₂) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H₂O₂ and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H₂O₂ was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H₂O₂ mediated cell lysis.     

Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters

Phenylarsine oxide (PAO) has been shown to exert a biphasic effect on glucose transport in 3T3-L1 adipocytes. At 10 microM, PAO activates transport threefold, but at higher concentrations an inhibition of transport is observed. In this paper we report a procedure for the subcellular fractionation of these cells which we use to examine the distribution of glucose transporters following PAO challenge. Quantitative immunoblotting showed that the glucose transporter content of the plasma membrane fraction increased with increasing PAO concentrations; a parallel increase in another insulin-responsive protein, the transferrin receptor, also occurred. However, cell-surface labeling procedures for the glucose transporter and transferrin receptor showed that PAO actually decreased the cell-surface concentrations of these proteins; the basis of this discrepancy may be that in the presence of PAO, intracellular vesicles containing these proteins associate with the plasma membrane, but do not fuse with it. The possibility that PAO modulated transport by direct interaction with the glucose transporter was investigated by examining the effects of PAO on transport in both erythrocytes and a reconstituted system of purified erythrocyte transporter in lipid vesicles. PAO was without effect on the rate of transport in these systems. The hypothesis that the stimulatory effect of PAO on transport might be due to the activation of the insulin receptor kinase activity was examined by assessing the phosphotyrosine content of the receptor and other proteins using anti-phosphotyrosine antibodies. PAO alone caused no detectable increase in receptor phosphotyrosine content. However, the combination of PAO and insulin led to the tyrosine phosphorylation of two proteins of Mr 68,000 and 57,000 which were not detected in cells treated with either PAO or insulin, and an increased phosphotyrosine content of proteins of Mr 95,000 and 165,000 when compared to cells treated with insulin alone.