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

The surface properties of Legionella pneumophila were examined by analyzing outer membrane (OM) proteins, lipopolysaccharides (LPS), and cellular fatty acids after growth within Acanthamoeba polyphaga and in vitro under various nutrient-depleted conditions. Intra-amoeba-grown legionellae were found to differ in several respects from cells grown in vitro; most notably, they contained a 15-kDa OM protein and a monounsaturated straight-chain fatty acid (18:1(9)). These compounds were also found in abundant quantities in the host amoeba. Immunoblot analysis of intra-amoeba-grown legionellae with antiacanthamoebic serum revealed that both the bacterial whole cells and Sarkosyl-extracted OMs contained amoebic antigens. The findings suggest that the 15-kDa OM protein is likely to be of amoebic origin and associates with the OM of the bacterium. It is proposed that disruption of amoebic membranes, as a result of intra-amoebic infection, may liberate macromolecules, including a 15-kDa polypeptide, a major constituent of the amoebic membrane, which adhere to the surface of the legionellae. Growth under specific nutrient depletions also had a significant effect on the surface composition of L. pneumophila. Cells grown under phosphate depletion were markedly sensitive to protease K digestion and contained lower levels of LPS, as observed by silver staining of the digests on polyacrylamide gels. Intra-amoeba-grown cells contained more bands than the in vitro-grown organisms, reflecting further differences in the nature of the LPS. The whole-cell fatty acids of the phosphate-depleted cells were appreciably different from those of cells grown under other nutritional conditions. We found no evidence for expression of iron-regulated OM proteins under iron depletion.
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PMID:Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila. 833 82

The lipopolysaccharide (LPS, endotoxin) of Legionella pneumophila serogroup 1 (Philadelphia 1) exhibits peculiar chemical features which may account for its importance as a bacterial virulence factor. The O-chain of this LPS constitutes a homopolymer of an unusual sugar, 5-acetamidino-7-acetamido-8-O-acetyl-3,5,7, 9-tetradeoxy-D-glycero-L-galacto-nonulosonic acid (legionaminic acid) of which about 10-75 residues are present. Due to the lack of free hydroxyl groups, this homopolymer renders the cell surface highly hydrophobic and, therefore, supports adherence to the membrane of target cells including alveolar macrophages. Investigations of the serological specificity of the serogroup 1 LPS revealed a monoclonal antibody (mAb 3/1) which recognizes an epitope located in the environment of the 8-O-acetyl group of legionaminic acid. According to epidemiological studies, this determinant appears to be associated with L. pneumophila virulence. The outer core oligosaccharide of L. pneumophila LPS exhibits also hydrophobic properties due to the presence of N- and O-acetyl groups as well as 6-deoxy sugars. The inner core expresses both similarities and differences as compared to enterobacterial core oligosaccharides in containing Kdo but lacking heptose and phosphate groups. Lipid A possesses some unique structural features since its backbone consists of a bisphosphorylated beta-GlcpN3N-(1-->6)-GlcpN3N disaccharide with only amide-linked acyl groups having 14-22 carbon atoms. Long chain fatty acids [28:0(27-oxo) and 27:0-dioic] possessing the double length as enterobacterial acyl groups, may be responsible for the low endotoxicity of L. pneumophila lipid A.
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PMID:The lipopolysaccharide of Legionella pneumophila serogroup 1 (strain Philadelphia 1): chemical structure and biological significance. 852 18

The bacterium Legionella pneumophila is the responsible agent for Legionnaires' disease and has recently been shown to harbor a gene encoding a kinase that confers resistance to the aminoglycoside antibiotic spectinomycin (Suter, T. M., Viswanathan, V. K., and Cianciotto, N. P. (1997) Antimicrob. Agents Chemother. 41, 1385-1388). We report the overproduction, purification, and characterization of this spectinomycin kinase from an expressing system in Escherichia coli. The purified protein shows stringent substrate specificity for spectinomycin with Km = 21.5 microM and kcat = 24.2 s-1 and does not bind other aminoglycosides including kanamycin, amikacin, neomycin, butirosin, streptomycin, or apramycin. Purification of spectinomycin phosphate followed by characterization by mass spectrometry and 1H, 13C, and 31P NMR established the site of phosphorylation to be at the hydroxyl group at position 9. Thus this enzyme is designated APH(9)-Ia (where APH is aminoglycoside kinase). The enzyme was inactivated by the electrophilic ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine, consistent with a nucleophilic residue such as Lys lining the nucleotide binding pocket. Site-directed mutagenesis of Lys-52 and Asp-212 to Ala confirmed that these residues were important for catalysis, with Lys-52 playing a potential role in ATP binding and Asp-212 in phosphoryl transfer. Thio and solvent isotope effect experiments in the presence of either Mg2+ or Mn2+ were consistent with a kinetic mechanism in which phosphate transfer does not contribute significantly to the rate-limiting step. These results establish that APH(9)-Ia is a highly specific antibiotic resistance kinase and provides the requisite mechanistic information for future structural studies.
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PMID:Spectinomycin kinase from Legionella pneumophila. Characterization of substrate specificity and identification of catalytically important residues. 961 79

The microbial diversity of a deteriorated biological phosphorus removal reactor was investigated by methods not requiring direct cultivation. The reactor was fed with media containing acetate and high levels of phosphate (P/C weight ratio, 8:100) but failed to completely remove phosphate in the effluent and showed very limited biological phosphorus removal activity. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA was used to investigate the bacterial diversity. Up to 11 DGGE bands representing at least 11 different sequence types were observed; DNA from the 6 most dominant of these bands was further isolated and sequenced. Comparative phylogenetic analysis of the partial 16S rRNA sequences suggested that one sequence type was affiliated with the alpha subclass of the Proteobacteria, one was associated with the Legionella group of the gamma subclass of the Proteobacteria, and the remaining four formed a novel group of the gamma subclass of the Proteobacteria with no close relationship to any previously described species. The novel group represented approximately 75% of the PCR-amplified DNA, based on the DGGE band intensities. Two oligonucleotide rRNA probes for this novel group were designed and used in a whole-cell hybridization analysis to investigate the abundance of this novel group in situ. The bacteria were coccoid and 3 to 4 microm in diameter and represented approximately 35% of the total population, suggesting a relatively close agreement with the results obtained by the PCR-based DGGE method. Further, based on electron microscopy and standard staining microscopic analysis, this novel group was able to accumulate granule inclusions, possibly consisting of polyhydroxyalkanoate, inside the cells.
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PMID:Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor. 1004 91

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.
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PMID:Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila. 1072 74

Most studies focusing on detecting microorganisms in air by polymerase chain reaction (PCR) have used a liquid impinger to sample bioaerosols, mainly because a liquid sample is easy to be processed by PCR analysis. Nevertheless, the use of multiple-hole impactors for the analysis of bioaerosols by PCR has not been reported despite its great utility in culture analysis. In this study we have modified the impaction onto an agar surface sampling method to impaction onto a liquid medium using the MAS-100 air sampler (Merck) (single-stage multiple-hole impactor). To evaluate the recovery of airborne microorganisms of both sampling methods, a suspension containing Escherichia coli was artificially aerosolized and bioaerosols were collected onto Tergitol-7 agar and phosphate-buffered saline (PBS) with the MAS-100. A linear regression analysis of the results showed a strong positive correlation between both sampling methods (r = 0.99, slope 0.99, and y intercept 0.07). Afterwards, the method of impingement into a liquid medium was used to study airborne Legionella pneumophila by PCR. A total of 64 samples were taken at a wastewater treatment plant, a chemical plant, and an office building and analyzed by culture and PCR. Results showed that three samples were positive both by PCR and plate culture, and that nine samples negative by plate culture were positive by PCR, proving that L. pneumophila was present in bioaerosols from these three different environments. The results demonstrate the utility of this single-stage multiple-hole impactor for sampling bioaerosols, both by culture and by PCR.
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PMID:Detection of Legionella pneumophila in bioaerosols by polymerase chain reaction. 1135 74

Cooling tower water has frequently been cited as a source of infection in outbreaks of Legionnaires' disease. However, there have been few reports on the presence of legionellae in aerosols from cooling towers. This paper describes our use of an impinger or a six-stage microbial impactor for detecting legionellae in air around a cooling tower contaminated with L. pneumophila (1.2+/-0.3x10(5) CFU/100 ml). Phosphate-buffered saline, Page's saline, 2% yeast extract solution and buffered yeast extract (BYE) broth were tested to evaluate their collection efficiency. These solutions were compared in laboratory experiments using an aerosol of L. pneumophila serogroup (SG) 1. Because BYE broth was the most efficient and storable collecting fluid among them, it was used for outdoor air sampling. In the outdoor air sampling, aerosolized L. pneumophila SG 6 was detected in the air around the cooling tower by the impinger (0.09 CFU/l. air). No legionellae were detected by the impactor with Legionella-selective agar plates (WYOalpha) because the plates were overgrown with fungi. Repetitive element PCR (rep-PCR) and arbitrarily primed PCR (AP-PCR) were employed to assess the epidemiological relationship among Legionella isolates from the air sample and the cooling tower water samples. L. pneumophila SG 6 isolated from the aerosols produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 6 strains from the cooling tower water, suggesting that the bacterium was aerosolized from the cooling tower.
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PMID:Sampling and detection of Legionella pneumophila aerosols generated from an industrial cooling tower. 1151 91

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the methylation pathway or the CDP-choline pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of rather diverse bacteria and based on genomic data, we estimate that more than 10% of all bacteria possess PC. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. A number of symbiotic (Rhizobium leguminosarum, Mesorhizobium loti) and pathogenic (Agrobacterium tumefaciens, Brucella melitensis, Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) bacteria seem to possess the PC synthase pathway and we suggest that the respective eukaryotic host functions as the provider of choline for this pathway. Pathogens entering their hosts through epithelia (Streptococcus pneumoniae, Haemophilus influenzae) require phosphocholine substitutions on their cell surface components that are biosynthetically also derived from choline supplied by the host. However, the incorporation of choline in these latter cases proceeds via choline phosphate and CDP-choline as intermediates. The occurrence of two intermediates in prokaryotes usually found as intermediates in the eukaryotic CDP-choline pathway for PC biosynthesis raises the question whether some bacteria might form PC via a CDP-choline pathway.
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PMID:Biosynthesis of phosphatidylcholine in bacteria. 1254 54

The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4' position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain omega-1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0). The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus, Bartonella henselae, and Legionella pneumophila. In this paper, a mutant of R. leguminosarum was created by placing a kanamycin resistance cassette within acpXL, the gene which encodes the acyl carrier protein for 27OHC28:0. The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0. A small amount of the mutant lipid A may contain an added palmitic acid residue. The mutant is sensitive to changes in osmolarity and an increase in acidity, growth conditions that likely occur in the nodule microenvironment. In spite of the probably hostile microenvironment of the nodule, the acpXL mutant is still able to form nitrogen-fixing root nodules even though the appearance and development of nodules are delayed. Therefore, it is possible that the acpXL mutant has a host-inducible mechanism which enables it to adapt to these physiological changes.
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PMID:A Rhizobium leguminosarum AcpXL mutant produces lipopolysaccharide lacking 27-hydroxyoctacosanoic acid. 1261 48

Lipid A of Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, displays several significant structural differences when compared with Escherichia coli. An especially striking feature of R. leguminosarum lipid A is that it lacks both the 1- and 4'-phosphate groups. Distinct lipid A phosphatases that attack either the 1 or the 4' positions have previously been identified in extracts of R. leguminosarum and Rhizobium etli but not Sinorhizobium meliloti or E. coli. Here we describe the identification of a hybrid cosmid (pMJK-1) containing a 25-kb R. leguminosarum 3841 DNA insert that directs the overexpression of the lipid A 1-phosphatase. Transfer of pMJK-1 into S. meliloti 1021 results in heterologous expression of 1-phosphatase activity, which is normally absent in extracts of strain 1021, and confers resistance to polymyxin. Sequencing of a 7-kb DNA fragment derived from the insert of pMJK-1 revealed the presence of a lipid phosphatase ortholog (designated LpxE). Expression of lpxE in E. coli behind the T7lac promoter results in the appearance of robust 1-phosphatase activity, which is normally absent in E. coli membranes. Matrix-assisted laser-desorption/time of flight and radiochemical analysis of the product generated in vitro from the model substrate lipid IVA confirms the selective removal of the 1-phosphate group. These findings show that lpxE is the structural gene for the 1-phosphatase. The availability of lpxE may facilitate the re-engineering of lipid A structures in diverse Gram-negative bacteria and allow assessment of the role of the 1-phosphatase in R. leguminosarum symbiosis with plants. Possible orthologs of LpxE are present in some intracellular human pathogens, including Francisella tularensis, Brucella melitensis, and Legionella pneumophila.
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PMID:Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase. 1286 41


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