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Query: UMLS:C0023241 (Legionella)
 6,990 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A chemically defined liquid medium has been developed for the study of the physiology and antigen production of the Legionnaires disease bacterium. The medium contains basal salts, vitamins, alpha-ketoglutaric acid, pyruvate, 0.05% l-cysteine, 0.05% glutathione, and a mixture of 20 additional amino acids, each of 0.01% final concentration, except serine, which was at 0.1%. The medium in shake culture at 37 degrees C with increased CO2 at pH 6.5, supports the maximum rate of growth, the highest cell yields, and the maximum cell surface antigen as distinguished by specific fluorescein isothiocyanate-conjugated antibody. Studies during the development of this medium showed that CO2, pyruvate, and alpha-ketoglutarate strongly stimulated growth; that cysteine and methionine were required for growth; and that serine, threonine, histidine, tyrosine, and tryptophane were energy sources. Glutathione substituted for cysteine, but cystine did not. The organisms did not use glucose and polysaccharides, as judged by cell yields when these carbohydrates were present or absent. The chelators malate, citrate, and ethylenediaminetetraacetic acid totally inhibited growth. Beta-mercaptoethanol, thioglycolate, dithiothreitol, and Tween 80 (0.05%) inhibited growth strongly or completely. Catalase activity was extremely weak or absent. Morphology varied, depending upon conditions and phases of growth. In general, filamentous forms became chains of cigar-shaped bacilli fragmenting to pairs and becoming coccoidal in the late stationary pha-e of growth. The organism grew at 25, 30, and 37 degrees C. Although they varied in their growth characteristics, 10 isolates were passed for five transfers in the chemically defined broth, giving maximum rates of growth, cell yields, and antigen production.
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PMID:Development of a chemically defined liquid medium for growth of Legionella pneumophila. 3 86

The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.
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PMID:Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein. 131 95

The utilization of amino acids and other compounds as carbon and energy sources by Legionella pneumophila was examined. Based on the stimulation of oxygen consumption in washed-cell suspensions, glutamate, serine, threonine, and tyrosine were the only amino acids which were utilized as energy sources. Other stimulators of oxygen uptake were lactate, pyruvate, acetate, fumarate, and succinate. Citrate was a good stimulator only when the bacteria were grown in the presence of the substrate. Radiolabeling studies showed that [14C]glutamate was rapidly metabolized, with the label distributed evenly in all cell fractions. [14C]pyruvate and [14C]acetate were incorporated into the lipid-containing cell fraction, whereas glucose and glycerol were found in both the lipid- and polysaccharide-containing cell fractions. Radiorespirometry of differentially labeled [14C]glucose indicated that this compound was metabolized primarily by the pentose phosphate and Entner-Doudoroff pathways rather than by the glycolytic pathway.
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PMID:Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources. 613 45

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.
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PMID:Substrate utilization by Legionella cells after cryopreservation in phosphate buffer. 614 14

The amino acids required for growth and as energy sources by 10 strains of Legionella pneumophila were determined by using a chemically defined medium. All strains required arginine, cysteine, isoleucine, leucine, threonine, valine, methionine, and phenylalanine or tyrosine. Most strains (7 of 10) required serine, and two strains had to be supplied proline before growth could be established. All 10 strains used serine and, to a lesser extent, threonine as the sole sources of carbon and energy. The Y serine calculated was 94.9 +/- 8.5 g (dry weight) of cells/mol of serine. Assuming that the value of Y adenosine 5'-triphosphate is 10.5, these results indicate that oxidative catabolism of 1 mol of serine yielded approximately 9 mol of adenosine 5'-triphosphate. This high yield suggests that although serine was the major source of carbon, other amino acids may also be metabolized.
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PMID:Amino acid requirements of Legionella pneumophila. 676 47

Flagella were isolated from virulent Legionella pneumophila serogroups 1, 2, and 3. Antiserum made against purified serogroups 1 flagellin agglutinated live, flagellated serogroups 1, 2, and 3 but not heat-killed or nonflagellated bacteria. A single line of identity was seen in immunodiffusion slides between the flagella isolated from the three serogroups and antibody to flagellin isolated from serogroups 1, 2, and 3. Indirect immunoperoxidase staining showed that antibody to flagellin isolated from serogroup 1 organisms reacted with flagella on serogroup 1, 2, and 3 bacteria. Indirect immunoperoxidase staining was also showed that antibody to flagellin isolated from serogroup 1 L. pneumophila did not react with the serogroup-specific cell surface antigen, thus demonstrating that the flagella- and the serogroup-specific antigen are separate antigens. The amino acid content of the flagella from the three serogroups was essentially the same, with aspartate, glutamate, alanine, and threonine comprising 41% of the total. Thirty-five percent of the amino acids were hydrophobic, and there were not detectable amounts of cysteine, tryptophan, or tyrosine.
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PMID:Immunological and biochemical relationships among flagella isolated from Legionella pneumophila serogroups 1, 2, and 3. 679 82

A chemically defined medium containing 18 amino acids, inorganic salts, rhamnose, choline, and ferric pyrophosphate has been developed. The final concentrations of salts and amino acids were modeled after yeast extract. This medium supported the growth of four serogroups of Legionella pneumophila. Growth in shake cultures at 37 degrees C produced a lag time of approximately 5 h and a generation time of 4 h with a maximum growth yield of 10 9 colony-forming units per ml. A soluble brown pigment was observed in the stationary phase of growth. The optimal pH was 6.3. Rhamnose and choline were stimulatory; arginine, serine, threonine, cysteine, valine, and methionine were essential. Supplemental iron was not required to attain maximum growth, but iron deprivation caused an extended lag phase.
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PMID:Chemically defined medium for Legionella pneumophila growth. 746 8

After uptake and intracellular multiplication of Legionella pneumophila in MRC-5 lung fibroblasts, important cytoskeletal filament structures, like actin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton. Dysregulation of the cellular phosphorylation and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication of L. pneumophila. We therefore studied expression of phosphoproteins during intracellular growth of L. pneumophila. By using an anti-tyrosine phosphoprotein antibody we showed that proteins phosphorylated on tyrosine residues accumulated progressively during late infection exclusively around or in phagosomes filled with bacteria. In contrast, expression of serine/threonine phosphoproteins did not change. To discern the origin of phosphorylated proteins, the host cells were treated with cycloheximide, an inhibitor of eukaryotic protein synthesis. The newly synthesized proteins were labeled metabolically with [(35)S]methionine-cysteine and immunoprecipitated with a phosphotyrosine-specific antibody. Sodium dodecyl sulfate gel electrophoresis gave evidence for synthesis of at least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa) of Legionella origin that were phosphorylated on tyrosine residues 24 h after infection. Treatment of infected host cells with genistein, a tyrosine kinase inhibitor, revealed that tyrosine protein phosphorylation was not important for bacterial uptake but contributed to intracellular growth of L. pneumophila. Bacterial tyrosine phosphoproteins and the observed intracellular structural changes may be important to understanding the process involved in intracellular growth of L. pneumophila.
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PMID:Legionella pneumophila invasion of MRC-5 cells induces tyrosine protein phosphorylation. 1045 91

Differentiation in response to environmental cues is integral to the success of many intracellular pathogens. By characterizing a Legionella pneumophila mutant defective for differentiation in broth and replication in macrophages, we identified a subfamily of major facilitator superfamily transporters, here named Pht (phagosomal transporter), that also is conserved in two other vacuolar pathogens, Coxiella burnetii and Francisella tularensis. Biolog phenotype microarray analysis indicated that PhtA transports threonine, an essential amino acid. Either excess threonine or threonine peptides bypass phtA function. In minimal medium, phtA mutants do not replicate; in rich broth, the bacteria prematurely differentiate to the transmissive phase, as judged by the kinetics of flaA-gfp expression, heat resistance, and sodium sensitivity. PhtA is dispensable for transmissive L. pneumophila to establish and persist within a replication vacuole but is essential for their differentiation to the replicative phase, based on phenotypic and RT-PCR analysis. Accordingly, we propose that the Pht transporter family equips transmissive L. pneumophila, C. burnetii, and F. tularensis to assess their phagosomal nutrient supply before committing to reenter the cell cycle.
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PMID:The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages. 1599 35

Proteins containing FIC (filamentation induced by cyclic adenosine monophosphate) domains are found in both prokaryotic and eukaryotic organisms, but their function has remained elusive. Recent studies indicate that bacterial FIC domain-containing proteins disrupt host cell processes after being delivered into eukaryotic host cells: The Vibrio parahaemolyticus VopS protein interferes with Rho guanine triphosphatase (GTPase) function, and the Legionella pneumophila AnkX protein disrupts the microtubule-dependent transport of vesicles. Analysis of the VopS protein revealed that the FIC domain covalently modifies Rac by transferring adenosine 5'-monophosphate (AMP) to a threonine residue in the switch 1 region of the protein. Thus, FIC domain-mediated AMPylation is involved in the posttranslational regulation of protein function, and this activity has been subverted by microbial pathogens to modulate cellular functions during infection.
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PMID:Bacterial FIC Proteins AMP Up Infection. 1929 28


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