Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100.
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Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100. (+info)
Complete genomic sequence of the lytic bacteriophage phiYeO3-12 of Yersinia enterocolitica serotype O:3.
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phiYeO3-12 is a T3-related lytic bacteriophage of Yersinia enterocolitica serotype O:3. The nucleotide sequence of the 39,600-bp linear double-stranded DNA (dsDNA) genome was determined. The phage genome has direct terminal repeats of 232 bp, a GC content of 50.6%, and 54 putative genes, which are all transcribed from the same DNA strand. Functions were assigned to 30 genes based on the similarity of the predicted products to known proteins. A striking feature of the phiYeO3-12 genome is its extensive similarity to the coliphage T3 and T7 genomes; most of the predicted phiYeO3-12 gene products were >70% identical to those of T3, and the overall organizations of the genomes were similar. In addition to an identical promoter specificity, phiYeO3-12 shares several common features with T3, nonsubjectibility to F exclusion and growth on Shigella sonnei D(2)371-48 (M. Pajunen, S. Kiljunen, and M. Skurnik, J. Bacteriol. 182:5114-5120, 2000). These findings indicate that phiYeO3-12 is a T3-like phage that has adapted to Y. enterocolitica O:3 or vice versa. This is the first dsDNA yersiniophage genome sequence to be reported. (+info)
Characterization of different plaque-forming and defective temperate phages in Agrobacterium.
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Four Agrobacterium tumefaciens temperate phages (PB2A, PB6(omega), PV-1(LV-1) and PS8), were shown to have the same genome size. Moreover hybridization experiments by the heteroduplex method and electron microscopy showed a 100% homology between these four phage genomes. Indications for lysogeny were found by direct means for the Agrobacterum timefaciens strain 396, Agrobacterium radiobacter strain 8149 and Agrobacterium species 0362 and by the electron microscope negative staining technique for the A.tumefaciens strains b6-806,b6-6,b6s3,b2as,cv-1,4452,11156,11158,396, and 925; for A.radiobacter strains tr-1 and 8149, the latter being bi-lysogenic, and for the A. species 0362. These isolated phage particles, most of which appear to be defective, could be grouped into different classes. No particles could be detected in the lysates of A. tumefaciens RV3, A. radiobacter strains 4718 and S1005, and A. species 0363. Further characterization by genome size was carried out for the defective temperate phages PB6-806, P4452,P8149 and P0362. No evidence for homology between PB6-806 and PB6 omega could be found. The defective phages PB6-806 and P4452 showed the same morphology but a different genome size, whereas the two phages P0362 and P8149 had a very different morphology and genome size. (+info)
A one-tube nested polymerase chain reaction for the detection of mycobacterium bovis in spiked milk samples: an evaluation of concentration and lytic techniques.
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The objective of this study was to evaluate the use of a one-tube nested polymerase chain reaction (OTN PCR) with 5 concentration and lytic treatments for the detection of Mycobacterium bovis in experimentally inoculated milk samples (spiked samples). OTN PCR and the following treatments were tested in inoculated samples: 1) centrifugation; 2) C18-carboxypropylbetaine + capture resin 1 + Proteinase K (CB18-CH-PK); 3) centrifugation + capture resin 1 + Proteinase K; 4) centrifugation + capture resin 2 + Proteinase K; and 5) centrifugation + immunomagnetic separation (IMS). The OTN PCR and the 5 treatments were evaluated in 2 different sets of spiked milk samples. One set consisted of 10-fold serial dilutions of a phenol-killed M. bovis in milk to final concentrations ranging from 5 to 50,000 cells/ml of milk. The other set of samples consisted of 2.5 serial dilutions of milk spiked with M. bovis to final concentrations ranging from 20.5 to 5,000 cells/ml of milk. Each treatment was repeated 5 times at each cell concentration. CB18-CH-PK and IMS were significantly more sensitive than other treatments. The lowest detection limit for these techniques was 20-50 cells/ ml of spiked milk. The specificity of OTN PCR in this study was high as demonstrated by the lack of DNA amplification products when M. bovis cells were not present in the samples. [The OTN PCR used in conjunction with CB18-CH-PK or IMS could be effectively used as a diagnostic and/or screening test for the detection of M. bovis in milk from herds with bovine tuberculosis.] (+info)
Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B.
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Both IgG and IgA Abs have been implicated in host defense against bacterial infections, although their relative contributions remain unclear. We generated a unique panel of human chimeric Abs of all human IgG and IgA subclasses with identical V genes against porin A, a major subcapsular protein Ag of Neisseria meningitidis and a vaccine candidate. Chimeric Abs were produced in baby hamster kidney cells, and IgA-producing clones were cotransfected with human J chain and/or human secretory component. Although IgG (isotypes IgG1-3) mediated efficient complement-dependent lysis, IgA was unable to. However, IgA proved equally active to IgG in stimulating polymorphonuclear leukocyte respiratory burst. Remarkably, although porin-specific monomeric, dimeric, and polymeric IgA triggered efficient phagocytosis, secretory IgA did not. These studies reveal unique and nonoverlapping roles for IgG and IgA Abs in defense against meningococcal infections. (+info)
ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guerin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes.
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We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guerin (BCG)-infected human macrophages. ATP increases the cytosolic Ca(2+) concentration in macrophages by mobilizing intracellular Ca(2+) via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca(2+) via P2X purinoceptors. The relative contribution of these receptors and Ca(2+) sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca(2+) in UTP-desensitized macrophages (in Ca(2+)-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca(2+) store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca(2+)-mobilizing P2Y purinoceptor in addition to the P2Y(2) subtype is expressed on human macrophages. ATP and the Ca(2+) ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca(2+)-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethyl ester (an intracellular Ca(2+) chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca(2+) mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage. (+info)
Functional activity of anti-Neisserial surface protein A monoclonal antibodies against strains of Neisseria meningitidis serogroup B.
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Neisserial surface protein A (NspA) is currently being investigated with humans as a candidate vaccine for the prevention of meningococcal disease. Although NspA is highly conserved, the ability of anti-NspA antibodies to bind to or elicit complement-mediated bactericidal activity against diverse Neisseria meningitidis serogroup B strains is controversial. To evaluate strain differences in NspA surface accessibility and susceptibility to bactericidal activity, we prepared murine immunoglobulin G2a anti-NspA monoclonal antibodies (MAbs) and evaluated their functional activity against 10 genetically diverse N. meningitidis serogroup B strains. By colony Western blot, all 10 strains expressed NspA as detected by one or more MAbs. By flow cytometry, two MAbs were found to bind to the bacterial surface of 6 of the 10 strains. In addition, two strains showed variable NspA surface accessibility for the MAbs despite being uniformly positive for NspA expression by colony Western blotting. Only 4 of the 10 strains were susceptible to anti-NspA complement-mediated bacteriolysis. Passively administered MAb protected infant rats from developing bacteremia after challenge with N. meningitidis serogroup B strain 8047 (surface binding positive, susceptible to anti-NspA bacteriolysis), was poorly protective against strain BZ232 (surface binding variable, resistant to bacteriolysis), and did not protect against strain M986 (surface binding negative, resistant to bacteriolysis). Finally, NspA does not appear to be critical for causing bacteremia, as an NspA knockout from strain 8047 was highly virulent in infant rats. Taken together, these findings suggest that an NspA-based vaccine will need to incorporate additional antigens to elicit broad protection against N. meningitidis serogroup B. (+info)
Taxonomic position and seasonal variations in marine neritic environment of some gram-negative antibiotic-producing bacteria.
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Six marine bacteria which synthesize macromolecular antibiotics were isolated from neritic waters on the French Mediterranean coast, and their frequency recorded over two successive years. They appeared in relatively large numbers during the period August to December, and can be identified as marine pseudomonads; however, the low guaninecytosine ratio of their DNA, lack of catalase and specific self-inhibition are not compatible with the characteristics of the genus Pseudomonas. Two produced violacein, usually synthesized by bacteria belonging to the genus Chromobacterium. Their taxonomic position is discussed. (+info)