- Volume 137, Issue 12, 1991
Volume 137, Issue 12, 1991
- Biochemistry
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Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness
More LessA serum-resistant strain of Proteus mirabilis was used to determine whether changes in the composition of surface components could be detected following induction of progressive stages of cell form defectiveness by β-lactam antibiotics. The critical stage was the conversion from filaments to the spheroplast form, which was accompanied by increased susceptibility to the bactericidal action of human serum. Inner and outer membranes of the bacterium, its filament form and its spheroplast form were separated by sucrose density-gradient centrifugation after digestion of peptidoglycan, followed by osmotic lysis of the cells. Outer membranes of the bacterial and the filament forms sedimented at the same density, whilst the outer membrane fraction of the spheroplast form sedimented in a region of lesser density. In addition, the amounts of two major outer-membrane proteins as well as the O-polysaccharide content of the lipopolysaccharide were reduced in the spheroplast form. These results indicate a general disorganization in structure and assembly of components in regard to their interactions with one another in the outer membrane of the spheroplast form.
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A complex chitinolytic system in exponentially growing mycelium of Mucor rouxii: properties and function
More LessEnzymological evidence has been sought for the purported involvement of chitinolysis in vegetative growth of filamentous fungi. A procedure has been developed for the production of fast growing and morphologically homogeneous exponential phase mycelium of the non-septate dimorphic zygomycete Mucor rouxii. A partially purified extract of this material has been subjected to gel-permeation chromatography and the chitinolytic activity of eluate fractions has been assessed using colloidal and nascent chitin and 3,4-dinitrophenyl tetra-N-acetylchitotetraoside [3,4-DNP-(GlcNAc)4] as substrates. Exponentially growing (t d = 1·1 h) mycelium consisting of single short-branched hyphae contains at least seven chitinases. The two particulate ones have not been studied in detail. The soluble chitinases hydrolyse (pseudo)chito-oligomers by random cleavage of internal β-1,4-bonds (and not by processing) and have a minimum chain-length requirement of n = 4. They are clearly distinct from β-N-acetylglucosaminidase (β-GlcNAc’ase) with respect to their chromatographic behaviour, substrate chain-length specificity, inhibition by chitobionolactone oxime (K 1 = 175 μm), and non-inhibition by the specific β-GlcNAc’ase inhibitor N-acetylglucosaminono-1,5-lactone oxime. Their pH optima are similar (6·5–7·0), and all can hydrolyse 3,4-DNP-(GlcNAc)4 as well as nascent chitin. With respect to their charge, response to protease treatment, behaviour upon gel-permeation chromatography and ability to use colloidal chitin as a substrate, the soluble chitinases do, however, represent two distinct groups. Type A chitinases are acidic, display partial latency, show an unusual affinity to dextran gel and act weakly on colloidal chitin. Type B chitinases are basic (or neutral) and non-zymogenic, do not behave anomalously upon gel filtration and can degrade preformed chitin. An hypothesis is presented for the function of the complex chitinolytic system of the fungal hypha in branching and, possibly, also in apical growth.
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Expression of active yeast pyruvate decarboxylase in Escherichia coli
More LessWe have shown by appropriate modification of the translational signals and using the strong T7 RNA polymerase promoter ϕ10, that a cloned Saccharomyces cerevisiae pyruvate decarboxylase gene (pdc1) can be expressed in Escherichia coli. This protein, which migrated as a single band on SDS-polyacrylamide gels, was found to have a subunit molecular mass of approximately 62 kDa, similar to that of the enzyme produced by yeast. Polyclonal antibodies raised against purified yeast pyruvate decarboxylase recognized this bacterially produced protein. We found that this recombinant enzyme is active, indicating that the homotetramer encoded by the pdc1 gene is functional.
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Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity.
More LessA family of peptide antimycotics, termed pseudomycins, has been isolated from liquid cultures of Pseudomonas syringae, a plant-associated bacterium. These compounds were purified using Amberlite XAD-2 and reverse-phase liquid chromatography. Pseudomycin A, the predominant peptide in a family of four, showed selective phytotoxicity, and had impressive activity against the human pathogen Candida albicans. Amino acid, mass spectroscopic, and comparative electrophoretic and chromatographic analyses revealed that the pseudomycins are different from previously described antimycotics from P. syringae, including syringomycin, syringotoxin and syringostatins. Pseudomycins A–C contain hydroxyaspartic acid, aspartic acid, serine, arginine, lysine and diaminobutyric acid. The molecular masses of pseudomycins A–C, as determined by plasma desorption mass spectrometry, are 1224,1208 and 1252 Da, respectively. Pseudomycin D, on the other hand, has a molecular mass of 2401 Da and is more complex than pseudomycins A–C.
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Effect of chloromethane on veratryl alcohol and lignin peroxidase production by the fungus Phanerochaete chrysosporium
More LessBiosynthesis of veratryl alcohol, a secondary metabolite considered to be an important component of the lignin-degrading system in the fungus Phanerochaete chrysosporium Burds INA-12, was initiated up to 36 h earlier in fungal cultures supplemented with 0·6 and 1·25 mm-CH3Cl compared with unsupplemented mycelia. Peak concentrations of the idiolyte were also about 70% higher in the presence of 0·6 mm-CH3Cl, although peak levels elicited by 0·2, 0·4 and 1·25mm-CH3Cl were lower than in unsupplemented cultures. Advanced initiation of veratryl alcohol biosynthesis in the presence of CH3C1 was reflected in the earlier appearance of lignin peroxidase (LiP) activity. This effect was most noticeable in cultures supplemented with 0·4 and 0·6 mm-CH3Cl, where LiP was evident up to 24 h before detection in unsupplemented cultures. However, peak levels of LiP produced by CH3Cl-augmented mycelia were always less – below 40% in the case of 1·25 mm-CH3Cl – than those recorded in cultures without supplementation. These effects may be explained by restricted metabolic availability of CH3CI as methyl donor for veratryl alcohol biosynthesis in the early stages of fungal growth.
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Origin of oxidized cellulose degradation products and mechanism of their promotion of cellobiohydrolase I biosynthesis in Trichoderma reesei
More LessCellobiono-1,5-lactone (CBL) induces cellulase, particularly cellobiohydrolase I (CBH I) formation in Trichoderma reesei. When used as a sole source of carbon, it promoted comparably poor growth, because (a) the δ-gluconolactone arising by the action of β-glucosidase is not metabolized by the fungus, and (b) it is a low-V max substrate of the T. reesei β-glucosidase. Induction of higher amounts of CBH I were observed when CBL was supplied as carbon source together with cellobiose than when supplied alone. Maximal CBH I levels formed under these conditions were comparable to those when T. reesei was grown on cellobiose plus β-gluconolactone, an inhibitor of β-glucosidase. These data suggest an indirect effect of CBL on CBH I induction, probably by inhibiting cellobiose hydrolysis. The origin of CBL during growth of T. reesei on cellulose was also investigated: aldonic acids and aldonolactones were released from cellulose by T. reesei culture fluids. Artificially oxidized celluloses gave rise to the appearance of higher concentrations of oxidized products but the addition of cellobiose did not, suggesting that their appearance is not due to a cellobiose-oxidizing enzyme. No accumulation of oxidized cello-oligosaccharides occurred when the action of both cellobiohydrolases (CBH I and II) was simultaneously blocked by monoclonal antibodies. These data suggest that cellulose already contains oxidized chain termini, and that these aldonolactones and aldonic acids are released by the action of the two cellobiohydrolases and not by a ‘cellulose-oxidizing’ enzyme.
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- Development And Structure
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Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions
Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (d-GlcNAc), N-acetylgalactosamine (d-GalNAc), galactose (d-Gal), mannose-like residues (d-Man) or l-fucose (l-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the d-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or d-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (d-Gal-specific), although the reactivity of other d-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for d-Man-like residues); however, they were unreactive with the l-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.
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- Ecology
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Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides
More LessOligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5′-end or enzymically at the 3′-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody–enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gramnegative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.
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pH gradients through colonies of Bacillus cereus and the surrounding agar
More LesspH-sensitive microelectrodes, constructed with a tip diameter of about 4 μm, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 μm into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1·45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.
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- Genetics And Molecular Biology
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An ISI-like element is responsible for high-level synthesis of extended-spectrum β-lactamase TEM-6 in Enterobacteriaceae
More LessResistance of Escherichia coli strain HB251 to the newer β-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum β-lactamase TEM-6. The corresponding structural gene, blaT-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that blaT-6 differed by two nucleotide substitutions from blaT-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from blaT-6 resulted in the creation of hybrid promoter P6 in which the –10 region was that of TEM-1 promoter P3 whereas the – 35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the IS1-like element in clinical isolates of Enterobacteriaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.
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Molecular cloning and genetic characterization of the rfb region from Yersinia pseudotuberculosis serogroup IIA, which determines the formation of the 3,6-dideoxyhexose abequose
More LessThe rfb region of Yersinia pseudotuberculosis serogroup IIA has been cloned and expression of O antigen in Escherichia coli K12 was demonstrated. Transposon mutagenesis analysis confined the DNA region required for O antigen expression to a 19·3 kb fragment, and the O antigen expressed was visualized by SDS-PAGE and silver staining. Southern hybridization analysis demonstrated significant levels of similarity between the Yersinia rfb region and the 3,6-dideoxyhexose pathway genes rfbF and rfbG, previously isolated from Salmonella enterica LT2, but no similarity to the abequose synthase gene rfbJ of the same strain or the paratose synthase gene rfbS isolated from S. enterica Ty2. The evolutionary relationship between the abequose biosynthetic genes of the two species of Salmonella and Yersinia is discussed.
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Transformation and allelic replacement in Francisella spp.
More LessWe describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella. Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-Tn10-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.
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Unique DNA plasmid pRS64 associated with chromosomal DNAs of the plant pathogenic fungus Rhizoctonia solani
More LessUnique DNA sequences homologous to the linear DNA plasmid pRS64 were investigated in chromosomal DNAs of isolates belonging to anastomosis group 4 (AG-4) of the plant pathogenic fungus Rhizoctonia solani. Chromosome-sized DNAs of isolates RI-64 and 1271 of AG-4 were separated into six bands by orthogonal-field-alternation gel electrophoresis and hybridized to a cloned segment of pRS64. A small chromosome-sized DNA band of approximately 1·1 Mb carried the sequences homologous to pRS64 DNA. Sequences homologous to pRS64 were also maintained within the chromosomal DNA of isolate 1271 of AG-4 which does not possess the plasmid. The plasmid showed no homology to the mitochondrial DNA of isolate 1271. The possibility that the linear plasmid pRS64 may act as a transposable genetic element is discussed.
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Duplication of both xyl catabolic operons on TOL plasmid pWW15
More LessPseudomonas fluorescens MT15 is the host of the large (250 kbp) TOL plasmid pWW15. We have shown by a combination of hybridization, molecular cloning and enzyme assay that pWW15 carries two distinct regions which share homology with the upper pathway operons (xylCMABN) of other TOL plasmids and two distinct regions which are homologous to the meta pathway operons (xylXYZLTEGFJQKIH) of other TOL plasmids. Both the areas of homology to the upper pathway operons appear to carry all of the structural genes for the three catabolic enzymes of the operon. One of the regions of meta pathway operon homology encodes a complete functional pathway, but the second is incomplete and appears to carry only the genes from xylF downstream.
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- Immunology
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Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro
More LessMultiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK–like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E : T) ratio- and time-dependent. Maximal levels of anti-C albicans activity (approximately 60%) were observed after 4 h and at E : T ≥ 300:1. Similar patterns of anti-C albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA–LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA–LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA–LAK cells (E : T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA–LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.
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- Pathogenicity And Medical Microbiology
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Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene
Lecithin-dependent haemolysin (LDH) of Vibrio parahemolyticus was purified from Escherichia coli C600 transformed with a plasmid (pHL591) ligated with a 1·5 kb DNA fragment of V. parahaemolyticus. The final preparation comprised two LDH proteins with different molecular masses which were immunologically crossreactive and had the same enzymic activity. The LDH was a phospholipase hydrolysing both fatty acid esters of phospholipid, i.e. it hydrolysed phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) and then LPC to glycerophosphorylcholine (GPC). From this point of view, LDH should be classified as a phospholipase B. Phospholipase B, however, does not usually show haemolytic activity, because the intermediate (LPC), which is the actual haemolytic agent, is immediately hydrolysed to the final product (GPC). On the other hand, LPC formed by LDH action was comparatively stable, because the rates of the two reactions catalysed by LDH, PC to LPC and LPC to GPC, are almost the same. This is the reason that LDH shows haemolytic activity. Therefore, LDH of V. parahaemolyticus is an atypical phospholipase to be designated as phospholipase A2/lysophospholipase.
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Influence of capsular neuraminic acid on properties of streptococci of serological group B
More LessNeuraminic acid is thought to be a critical virulence factor of group B streptococci. The present study was designed to further characterize a previously described type III group B streptococcus and its transposon-mutagenized asialo capsular mutant. The wild-type group B streptococcus grew as short chains with a uniform turbidity and had diffuse colonies in soft agar media. In contrast, the asialo mutant grew in fluid media as a granular sediment, formed significantly longer chains and had compact colonies in soft agar. These differences, possibly related to the surface charge of the bacteria, could also be demonstrated in salt aggregation tests and hexadecane adherence studies. The wild-type group B streptococcus showed hydrophilic, and the asialo mutant hydrophobic surface properties. Removal of neuraminic acid from the wild-type strain changed the surface properties from hydrophilic to hydrophobic. A similar masking effect of capsular neuraminic acid could be observed in adherence and phagocytosis experiments. In contrast to the wild-type strain, the asialo mutant adhered significantly more to buccal epithelial cells and was phagocytosed more by polymorphonuclear leucocytes. These altered properties might possibly be of importance for group B streptococcal pathogenicity.
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Distinctions in DNA and protein profiles among clinical isolates of Mycoplasma pneumoniae
More LessClinical isolates of Mycoplasma pneumoniae previously shown to exhibit significant sequence divergency in a major 170 kDa adhesin, designated P1, were further characterized using restriction enzyme fingerprinting of genomic DNA and two-dimensional gel electrophoresis of total proteins. Numerous differences in DNA restriction patterns and protein profiles were found, possibly reflecting various degrees of virulence and antigenic potential.
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Isolation and identification of a putative porcine transferrin receptor from Actinobacillus pleuropneumoniae biotype 1
More LessEach of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidinagarose, and the second on Sepharose-coupled porcine transferrin, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides (~ 64 kDa and 99 kDa) from total membranes of Actinobacillus pleuropneumoniae grown under iron-restricted conditions. Both polypeptides were iron-repressible and were identified as potential receptor candidates as they were not isolated when biotinylated human transferrin was used instead of biotinylated porcine transferrin. The 64 kDa polypeptide was the more easily removed from Sepharose-coupled porcine transferrin and only the 99 kDa polypeptide appeared to be an outer-membrane protein. While these results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pleuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role, other interpretations are also possible.
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Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions
More LessMonoclonal antibody (mAb) probes were used to investigate the expression of lipopolysaccharide (LPS) on four Escherichia coli strains, grown under a variety of conditions in batch culture which mimicked some of the in vivo environmental conditions of an infected host. Techniques of silver staining, immunoblotting, whole cell ELISA and flow cytometry were all used to monitor the expression of LPS on the bacteria and the binding of the anti-LPS mAbs. Growth in heat-inactivated sheep serum and magnesium-depleted conditions demonstrated increased expression of LPS core and subsequent increased binding of anti-core mAbs. Magnesium-depleted conditions also resulted in decreased production of O-polysaccharide material. Iron-depleted bacteria showed only minor changes in LPS expression, although increased binding of anti-core mAbs was observed. Nitrogen-deficient/high-carbon conditions, chosen to promote capsule production, resulted in increased expression of O-polysaccharide and decreased binding of anti-core mAbs.
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