Loss of CD28 expression on T lymphocytes: a marker of replicative senescence

A massive accumulation of neutrophils, mainly due to enhanced interleukin-8 (IL-8) levels, is believed to contribute to the deleterious effects of Pseudomonas aeruginosa lung infection, e.g., in cystic fibrosis (CF). Antibodies to phospholipase C, an exoenzyme of P. aeruginosa, are detected early and at high levels in CF patients. However, P. aeruginosa produces at least two types of phospholipase C (PLC), one haemolytic (PLC-H) and the other non-haemolytic (PLC-N), both with mol.wts of c. 77 kDa. Experiments were performed to evaluate the potential contribution of P. aeruginosa PLC to neutrophil accumulation during infection. Therefore, P. aeruginosa PLC-H and PLC-N were compared with regard to IL-8 generation from human monocytes. Purified PLC-H as well as culture supernates (mol.wt > 50 kDa) of a P. aeruginosa strain capable of producing both PLC-H and PLC-N, and mutant strains deficient in the production of one or other phospholipase, or both, were examined. Purified PLC-H (only at low concentrations up to 1 unit/4 x 10(5) monocytes), induced a dose-dependent increase in IL-8 release and IL-8-specific mRNA expression over that of unstimulated cells (at 4-, 12- and 24-h incubation times). Higher concentrations of PLC-H led to a decrease in IL-8 release and IL-8-specific mRNA expression. These findings were confirmed by the results obtained with the supernates of cultures of mutant strains of P. aeruginosa PAO1 that produced either a PLC-H or PLC-N or neither. Stimulation and inhibition of IL-8 release and mRNA expression were associated with a culture supernate fraction of mol. wt > 50 kDa and containing PLC-H. These results contribute to the understanding of the role of both P. aeruginosa PLC in IL-8 generation during their interaction with human monocytes.     

Pyoverdin is essential for virulence of Pseudomonas aeruginosa

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.     

High-affinity transport of choline-O-sulfate and its use as a compatible solute in Bacillus subtilis

We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a Ki of approximately 4 microM. Uptake studies with [1, 2-dimethyl-14C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a Km value of 4 +/- 1 microM and a maximum rate of transport (Vmax) of 54 +/- 3 nmol/min. mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance 13C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and gamma-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83-90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.     

Hepatic betaine-homocysteine methyltransferase activity in the chicken is influenced by dietary intake of sulfur amino acids, choline and betaine

There is much interest in the metabolism of homocysteine, because elevated plasma homocysteine [hyperhomocyst(e)inemia] is an independent risk factor for the development of cardiovascular disease. Four chick assays were conducted to determine the effects of varying dietary sulfur amino acids, choline and betaine on the activity of hepatic betaine-homocysteine methyltransferase (BHMT), an enzyme likely to be important in modulating plasma homocysteine. In Experiment 1, chicks were fed a purified crystalline amino acid diet containing adequate sulfur amino acids and choline. Excess dietary methionine, or the combination of excess cystine with choline or betaine, caused a small increase (P < 0.05) in BHMT activity. In Experiment 2, use of a methionine-deficient purified diet resulted in a threefold increase (P < 0.05) in BHMT activity, and addition of choline or betaine further increased (P < 0.05) BHMT activity. In Experiment 3, use of a methionine-deficient corn-peanut meal diet increased BHMT (P < 0.05) relative to that of chicks supplemented with adequate methionine, and addition of surfeit choline to the methionine-deficient basal diet caused a further increase (P < 0.05). In Experiment 4, addition of both surfeit choline and surfeit betaine to the methionine-deficient corn-peanut meal diet caused an increase (P < 0.05) in BHMT activity relative to that observed in chicks fed the methionine-deficient basal diet. These assays show that large increases in BHMT activity can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine.     

Betaine aldehyde dehydrogenase in sorghum

The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa.     

Osmoprotective compounds in the Plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance

In common with other zwitterionic quarternary ammonium compounds (QACs), glycine betaine acts as an osmoprotectant in plants, bacteria, and animals, with its accumulation in the cytoplasm reducing adverse effects of salinity and drought. For this reason, the glycine betaine biosynthesis pathway has become a target for genetic engineering of stress tolerance in crop plants. Besides glycine betaine, several other QAC osmoprotectants have been reported to accumulate among flowering plants, although little is known about their distribution, evolution, or adaptive value. We show here that various taxa of the highly stress-tolerant family Plumbaginaceae have evolved four QACs, which supplement or replace glycine betaine-namely, choline O-sulfate and the betaines of beta-alanine, proline, and hydroxyproline. Evidence from bacterial bioassays demonstrates that these QACs function no better than glycine betaine as osmoprotectants. However, the distribution of QACs among diverse members of the Plumbaginaceae adapted to different types of habitat indicates that different QACs could have selective advantages in particular stress environments. Specifically, choline O-sulfate can function in sulfate detoxification as well as in osmoprotection, beta-alanine betaine may be superior to glycine betaine in hypoxic saline conditions, and proline-derived betaines may be beneficial in chronically dry environments. We conclude that the evolution of osmoprotectant diversity within the Plumbaginaceae suggests additional possibilities to explore in the metabolic engineering of stress tolerance in crops.     

Rapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Betaine aldehyde dehydrogenase (BADH) (EC 1.2.1.8) catalyzes the last, irreversible step in the synthesis of the osmoprotectant glycine betaine from choline. In Pseudomonas aeruginosa this reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. We present here a method for the rapid purification to homogeneity of this enzyme by the use of ion-exchange and affinity chromatographies on 2',5'-ADP-Sepharose, which results in a high yield of pure enzyme with a specific activity at 30 degreesC and pH 7.4 of 74.5 U/mg of protein. Analytical ultracentrifugation, gel filtration, chemical cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that BADH from P. aeruginosa is a homodimer with 61-kDa subunits. The amino acid composition and the N-terminal sequence of 21 amino acid residues showed significant similarity with those of the enzymes from Xanthomonas translucens and Escherichia coli. Neither BADH activity nor BADH protein was found in cell extracts from bacteria grown in the absence of choline. In contrast to other BADHs studied to date, the Pseudomonas enzyme cannot use positively charged aldehydes other than betaine aldehyde as substrates. The oxidation reaction has an activation energy of 39.8 kJ mol-1. The pH dependence of the velocity indicated an optimum at pH 8.0 to 8.5 and the existence of two ionizable groups with macroscopic pK values of 7.0 +/- 0.1 and 9. 7 +/- 0.1 involved in catalysis and/or binding of substrates. The enzyme is inactivated at 40 degreesC, but activity is regained when the heated enzyme is cooled to 30 degreesC or lower. At the optimum pH of 8.0, the enzyme is inactivated by dilution, but it is stable at pH 6.5 even at very low concentrations. Also, P. aeruginosa BADH activity is rapidly lost on removal of K+. In all cases studied, inactivation involves a biphasic process, which was dependent on the enzyme concentration only in the case of inactivation by dilution. NADP+ considerably protected the enzyme against these inactivating conditions.     

Study of the metabolism of choline and phosphatidylcholine in tumors in vivo using phosphonium-choline

The results of an initial study on the feasibility of using the phosphonium analog of choline to follow the metabolism of phosphatidylcholine in tumors in vivo using 31P NMR are reported. C3H/He mice bearing a mammary carcinoma tumor on the foot pad were fed a choline-free diet supplemented with the phosphonium analog of choline. Metabolites of this compound, including the phosphonium analogs of phosphatidylcholine, phosphocholine, glycerophosphocholine, and betaine were observed noninvasively in vivo in tumors by 31P NMR after 2-3 weeks of feeding. Clearance of these phosphonium-labeled metabolites from tumors was measured after a change to a choline-containing diet. Significant decreases were seen in the levels of the analogs of betaine (P < 0.003) and phosphatidylcholine (P < 0.004) by Day 4. A significant increase in the level of authentic phosphocholine (P < 0.003) occurred over the same time period.     

Something's rotten: a nosocomial outbreak of malodorous Pseudomonas aeruginosa

From July 1994 through November 1996, a phenotypically unique strain of Pseudomonas aeruginosa producing a pungent, "rotten-potato" odor and a positive lysine decarboxylase reaction was isolated from 39 patients at UCLA Medical Center (Los Angeles). Most cases (95%) were in intensive care units and had clinical infections (72%). Most isolates (74%) were recovered from cultures of respiratory secretions. To determine risk factors for acquisition of the organism, 23 cases were compared with 23 randomly selected controls matched by service and isolate date. Multivariate analysis revealed that isolation of malodorous P. aeruginosa was associated with mechanical ventilation of > 24 hours' duration (odds ratio [OR] = 9.4; P = .001) and transfer from an outside hospital (OR = 5.7; P = .04). DNA from outbreak strains hybridized to P. aeruginosa-specific toxin A and phospholipase C gene probes and all outbreak isolates tested were found to be identical by use of pulsed-field gel electrophoresis. An unusual phenotypic characteristic of the strain led to the recognition of a nosocomial outbreak of P. aeruginosa infection associated with mechanical ventilation.     

NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a beta-turn

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [McInnes, C., et al. (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al. (1995) Biochemistry 34, 16255-16268] reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.     

Characterization of regulatory mutations causing anaerobic derepression of the sodA gene in Escherichia coli K12: cooperation between cis- and trans-acting regulatory loci

The genetic loci leading to anaerobic derepression of a sodA::lacZ protein fusion in a UV-generated mutant strain (UV14) of Escherichia coli were identified. The mutant (UV14) was found to harbour two altered loci: one is in the trans-regulatory gene fnr (fumarate nitrate reduction) where leucine-129 was changed to glutamine (fnr14), and the second (sodA14) is in the promoter region (cis) of the sodA gene apparently affecting the binding of the Fur (ferric uptake regulation) protein. Introduction of an fnr+ gene into UV14 restored anaerobic repression of sodA::lacZ and restored the ability of the cells to reduce nitrate. However, when either the fnr14 or the sodA14 mutation was introduced into an otherwise wild-type background, only slight anaerobic derepression of sodA was observed. When both the cis- and trans-acting mutations (i.e. sodA14 and fnr14) were combined simultaneously in an otherwise wild-type background, the specific activity of sodA::lacZ expression was comparable to that of the original mutant strain (UV14). Furthermore, a genetically confirmed fur fnr double mutant was also similarly derepressed in anaerobic sodA::lacZ expression. The data presented suggest that the cis-mutation in UV14 (sodA14) affects the Fur-binding site in the sodA promoter, while having no effect on Fnr or Arc mediated repression. Also, a second putative Fnr-binding site that straddles the ribosomal binding-site was identified in the sodA gene.     

Pseudoverdin, a compound related to the pyoverdin chromophore from a Pseudomonas aeruginosa strain incapable to produce pyoverdins

From a genetically manipulated strain of Pseudomonas aeruginosa ATCC 15692 (PAO1 strain) a compound named pseudoverdin, 3-formylamino-6,7-dihydroxycoumarin, was obtained which is related to the typical pyoverdin chromophore and thus allows to shed some light on the biogenesis of the latter.     

Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chain-deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild-type strain PAO1 and derived rough-type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/petroleum-ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of D-glucose (four units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (Hep; two units), 3-deoxy-D-manno-octulosonic acid (Kdo; two units), L-alanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by one-dimensional and two-dimensional (2D) NMR techniques in combination with MS-based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two-dimensional 31P (omega1)-half-filtered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild-type strain. The presence of a 7-O-carbamoyl substituent was observed on the second heptose. The structure of the core region of the O-chain-deficient LPS from P. aeruginosa serotype 05 is as follows: [structure: see text] where R1 is beta-D-Glcp-(1-->2)-alpha-L-Rhap-(1-->6)-alpha-D-Glcp-(1--> and R2 is alpha-D-Glcp-(1-->6)-beta-D-Glcp-(1->. A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.     

Experimental allergic encephalomyelitis raises betaine levels in the spinal cord of strain 13 guinea-pigs

Chronic relapsing experimental allergic encephalomyelitis, an animal model of multiple sclerosis, was induced in Strain 13 guinea-pigs by subcutaneous injection of spinal cord homogenate and Freund's incomplete adjuvant supplemented with Mycobacterium tuberculosis. High resolution 1H NMR spectra of CNS tissue extracts indicated that the levels of choline metabolites, particularly betaine, were elevated in the spinal cord tissue, the principal site of lesion formation in this guinea-pig strain. The spectra also show that N-acetylated compounds are slightly depleted in the disease. The results are discussed in relation to the biochemical interpretation of NMR spectra obtained in vivo from patients with multiple sclerosis.     

Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes: identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP

Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A DeltabetP DeltaputP DeltaproP DeltaectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (Km, 48 microM) to ectoine (Km, 132 microM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (Km, 63 microM) to betaine (Km, 333 microM) and proline (Km, 1,200 microM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.     

Identification of a penicillin-binding protein 3 homolog, PBP3x, in Pseudomonas aeruginosa: gene cloning and growth phase-dependent expression

A homolog of Pseudomonas aeruginosa penicillin-binding protein 3 (PBP3), named PBP3x in this study, was identified by using degenerate primers based on conserved amino acid motifs in the high-molecular-weight PBPs. Analysis of the translated sequence of the pbpC gene encoding this PBP3x revealed that 41 and 48% of its amino acids were identical to those of Escherichia coli and P. aeruginosa PBP3s, respectively. The downstream sequence of pbpC encoded convergently transcribed homologs of the E. coli soxR gene and the Mycobacterium bovis adh gene. The pbpC gene product was expressed from the T7 promoter in E. coli and was exported to the cytoplasmic membrane of E. coli cells and could bind [3H] penicillin. By using a broad-host-range vector, pUCP27, the pbpC gene was expressed in P. aeruginosa PAO4089. [3H]penicillin-binding competition assays indicated that the pbpC gene product had lower affinities for several PBP3-targeted beta-lactam antibiotics than P. aeruginosa PBP3 did, and overexpression of the pbpC gene product had no effect on the susceptibility to the PBP3-targeted antibiotics tested. By gene replacement, a PBP3x-defective interposon mutant (strain HC132) was obtained and confirmed by Southern blot analysis. Inactivation of PBP3x caused no changes in the cell morphology or growth rate of exponentially growing cells, suggesting that pbpC was not required for cell viability under normal laboratory growth conditions. However, the upstream sequence of pbpC contained a potential sigma(s) recognition site, and pbpC gene expression appeared to be growth rate regulated. [3H]penicillin-binding assays indicated that PBP3 was mainly produced during exponential growth whereas PBP3x was produced in the stationary phase of growth.