Biomedical subjects

A E Sage

Publications and source records attributed to A E Sage.

4 recordsLinked to original sources

PubMed1997

Molecular characterization of mutants affected in the osmoprotectant-dependent induction of phospholipase C in Pseudomonas aeruginosa PAO1.

Production of the two phospholipases C (PLCs) in Pseudomonas aeruginosa PAO1 is induced under conditions of phosphate limitation, or by the osmoprotectants choline or glycine betaine. Tn5 mutagenesis was performed on strain PAO1 to isolate mutants deficient in choline-dependent induction of PLC. Two mutants, Tn5T1 and Tn5G19, were identified which produce decreased levels of PLC in phosphate-replete media supplemented with choline. A total of 136 and 496 bp of flanking DNA from Tn5G19 and Tn5T1 was cloned by an inverse polymerase chain reaction (PCR) and sequenced. The DNA flanking the Tn5T1 insertion contains an open reading frame predicted to encode a peptide that is approx. 60% identical to the N-terminus of a previously identified protein (P35) of unknown function from Escherichia coli. The P35 gene, which is located in the nusA-infB operon in E. coll, was designated orp (osmoprotectant regulator of PLC). Haemolytic titres, total PlcH protein and beta-galactosidase activity expressed from a chromosomally inserted plcH-lacZ operon fusion were reduced in strain Tn5T1 in comparison with the parental strain (PAO1) carrying the same fusion. However, this mutant expressed several-fold higher levels of plcH message than strain PAO1 in the presence of choline, while the phosphate-starvation-dependent transcript of plcH could not be detected in this mutant. The defects in Tn5T1 are complemented by a DNA fragment, isolated from a genomic library of PAO1, that carries the orp gene. The deduced amino acid sequence of the DNA fragment cloned from Tn5G19 exhibits 84% identity with the betB gene product of E. coli that has betaine aldehyde dehydrogenase activity. This enzyme catalyses the conversion of betaine aldehyde to glycine-betaine. Unlike the parental strain, the Tn5G19 mutant could not utilize choline as a sole carbon, nitrogen and energy source, and it was deficient in betaine aldehyde dehydrogenase activity. Also, consistent with a disruption of betB in Tn5G19, choline inhibited growth of this strain in media containing 0.7 M NaCl, while glycine-betaine restores growth to wild-type levels. The defects in Tn5G19 are complemented by a DNA fragment from PAO1 that carries the betB gene. The orp gene is located between 0.6 to 6.6 min while betB is located between 10.5 to 12.5 min on the chromosome of PAO1.

Amino Acid Sequence↗

PubMed1997

Osmoprotectant-dependent expression of plcH, encoding the hemolytic phospholipase C, is subject to novel catabolite repression control in Pseudomonas aeruginosa PAO1.

Expression of the hemolytic phospholipase C (PlcH) of Pseudomonas aeruginosa is induced under phosphate starvation conditions or in the presence of the osmoprotectants choline and glycine betaine. Because choline and glycine betaine may serve as carbon and energy sources in addition to conferring osmoprotection to P. aeruginosa, it seemed possible that induction of plcH is subject to catabolite repression control (CRC) by tricarboxylic cycle intermediates such as succinate. Total phospholipase (PLC) activity in osmoprotectant-induced cultures of P. aeruginosa PAO1 supplemented with 20 mM succinate was three- to fourfold lower than the levels in cultures supplemented with the non-catabolite-repressive substrate lactate. Analyses of osmoprotectant-dependent plcH expression in a derivative of strain PAO1 containing a plcH::lacZ operon fusion showed that (i) succinate prevented induction of plcH expression by osmoprotectants; and (ii) addition of succinate reduced or shut down further expression of plcH in osmoprotectant-induced bacteria, while cultures supplemented with lactate had little or no change in plcH expression. RNase protection analysis confirmed that repression of plcH occurs at the transcriptional level. However, a P. aeruginosa mutant decoupled in CRC exhibited a phenotype similar to that of the wild-type strain (PAO1) with respect to succinate-dependent repression of plcH expression. Osmoprotectant-induced total PLC activities, levels of expression of plcH measured with the same plcH::lacZ fusion, and levels of plcH transcription in a CRC-deficient strain reflected those seen in strain PAO1. This indicates that CRC of plcH functions by a distinct mechanism which differs from that regulating the glucose or mannitol catabolic pathway. A strain carrying a mutation in vfr, which encodes the Escherichia coli Crp homolog in P. aeruginosa, still exhibited a wild-type phenotype with respect to osmoprotectant-dependent expression and CRC of plcH. These data indicate that there is a novel CRC system that regulates the expression of plcH in P. aeruginosa.

Bacterial Proteins↗

PubMed1996

Role of Pseudomonas aeruginosa lipase in inflammatory mediator release from human inflammatory effector cells (platelets, granulocytes, and monocytes.

Previously, we have shown that Pseudomonas aeruginosa lipase and phospholipase C (PLC), two extracellular lipolytic enzymes, interact with each other during 12-hydroxyeicosatetraenoic acid (HETE) generation from human platelets. In this regard. the addition of purified P. aeruginosa lipase to PLC-containing crude P. aeruginosa culture supernatants enhances the generation of the chemotactically active 12-HETE from human platelets. Therefore, we analyzed the interaction of purified P. aeruginosa lipase and purified hemolytic P. aeruginosa PLC with regard to inflammatory mediator release from human platelets, neutrophilic and basophilic granulocytes, and monocytes. Purified P. aeruginosa PLC, but not purified lipase by itself, induced 12-HETE generation from human platelets, the generation of leukotriene B4 (LTB4) and oxygen metabolites, enzyme release from human neutrophils, and histamine release from basophils but diminished interleukin-8 (IL-8) release from human monocytes in a dose-dependent manner. The addition of purified lipase enhanced PLC-induced 12-HETE and LTB4 generation, did not influence enzyme, histamine, or IL-8 release, but diminished the PLC-induced chemiluminescent response. Similar results were obtained when the hemolytic PLC from Clostridium perfringens was used instead of P. aeruginosa PLC. For further comparison, we used the well-defined calcium ionophore A23187 and phorbol-12-myristate-13-acetate (PMA) as stimuli. Lipase enhanced calcium ionophore-induced LTB4 generation and beta-glucuronidase release but reduced calcium ionophore-induced and PMA-induced chemiluminescence. In parallel, we analyzed the role of lipase in a crude P. aeruginosa culture supernatant containing PLC and lipase. Lipase activity in the P. aeruginosa culture supernatant was inhibited by treatment with the lipase-specific inhibitor hexadecylsulfonyl fluoride, leaving the activity of PLC unaffected. The capacity of "lipase-inactivated culture supernatant" to induce 12-HETE and LTB4 generation was diminished by 50 to 100%. Our results suggest that the simultaneous secretion of lipase and PLC by P. aeruginosa residing in an infected host may result in severe pathological effects which cannot be explained by the sole action of the individual virulence factor on inflammatory effector cells.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1996

A two-component response regulator, gltR, is required for glucose transport activity in Pseudomonas aeruginosa PAO1.

A 729-bp open reading frame (gltR) was identified in Pseudomonas aeruginosa PAO1 that encodes a product homologous to the two-component response regulator family of proteins. Disruption of gltR caused loss of glucose transport activity. Restoration of gltR resulted in wild-type levels of glucose transport. These findings indicate that gltR is required for expression of the glucose transport system in P. aeruginosa.

Amino Acid Sequence↗