The promoter region of the pColV-K30-encoded operon specifying biosynthesis and transport of the siderophore aerobactin was subjected to deletion analysis to determine the smallest DNA sequence affording iron regulation of a iucA'-'lacZ gene fusion. A 78-base-pair (bp) region containing the main (P1)
The cloned fur (ferric uptake regulation) gene of Escherichia coli K12 was ligated to an expression vector which was inducible with nalidixic acid. The Fur protein was isolated in a single step by immobilized metal-ion-affinity chromatography over zinc iminodiacetate agarose. The amino acid composition of the isolated protein agreed with that predicted from the gene sequence and indicated post-transcriptional removal of the N-terminal methionine residue. All four cysteines were shown to be present as thiols. Proteolysis with trypsin and chymotrypsin yielded large fragments identifiable on polyacrylamide gel electrophoresis. Various divalent metal ions were found by a nitrocellulose filter binding assay to effect non-specific interaction of the Fur dimer with DNA with a dissociation constant of 7 x 10(-12) M. A much smaller value, 2.5 x 10(-17) M, was measured by gel mobility retardation assay for binding of Fur to a DNA fragment containing the operator sequences of the aerobactin promoter.
Summary. The hydrophobicity-hydrophilicity of various strains of Staphylococcus has been studied by a technique involving partitioning of the cells between aqueous and hydrocarbon phases. S. aureus was typically hydrophobic, and to a greater degree in stationary-than in exponential-phase cultures. Mutants that lacked teichoic acid, protein A or coagulase production were hydrophobic, indicating that none of these factors was responsible for hydrophobicity. The presence of a capsule rendered strains hydrophilic. Thus, determination of hydrophobicity may be a useful additional test for capsulation. However, a non-capsulate S. aureus strain was hydrophilic. Trypsin treatment converted strains from hydrophobic to hydrophilic. Isolated bacterial cell wall preparation, either crude or purified, and peptidoglycan were hydrophilic. These results indicate that the determinant of hydrophobicity is a protein or protein-associated molecule localised at the cell surface of the organism, i.e., a component of either the cell wall, cell membrane, or both. Twenty-five strains of twelve coagulase-negative species were examined and most ( 1 8 ) were hydrophobic, again indicating that protein A is not a major determinant of hydrophobicity in these staphylococci. Four of seven hydrophilic strains were capsulate; three strains of S. sciuri were hydrophilic but non-capsulate.
Paracoccus denitrificans grown in a complex medium was highly susceptible to lysozyme, in contrast to cells grown in a complex medium supplemented with Mg2+ and Ca2+ or in a succinate-salts medium. The complex medium was deficient in divalent cations needed for optimum outer membrane stability. The major change in molecular compositions of outer membranes isolated from cells grown under the different conditions was a higher ratio of ornithine-containing lipid to phospholipid in complex-medium-grown cells (0.63) than in cells grown in complex medium with Mg2' and Ca2+ (0.22) or in succinate-salts medium (0.14). We suggest that the dipolarionic ornithine-containing lipid is less dependent than acidic phospholipids on divalent cations for its incorporation into the outer membrane.Paracoccus denitrificans is a member of the a subdivision of the purple bacteria (28). This important group includes the purple non-sulfur bacteria, rhizobacteria, agrobacteria, rickettsiae, and Nitrobacter spp. This subdivision is composed predominantly of soil bacteria which tend to form intimate associations with eucaryotic cells and includes the endosymbiont ancestor of the mitochondrion (28). The organisms are thus of general biological interest. Although the outer membranes of these bacteria have been studied to some extent (1, 2), the main paradigm of outer membrane structure and function is still medically significant, mainly enteric, bacteria (8,14,17). Studies of the structure and function of the outer membrane of P. denitrificans are thus justified on the basis of the phylogeny of the organism.When P. denitrificans is grown in a complex medium, exponential-phase cells are lysozyme susceptible without further treatment (9,18 Iron-regulated proteins occur in the outer membrane, including high-Mr proteins induced upon iron deprivation (10) and a 23,000-Mr protein produced only in an iron-containing medium (22).In this paper we describe our investigations of outer membranes isolated from cells of the organism grown in complex medium, complex medium with Mg2' and Ca2+, and succinate-salts medium. We wanted to identify the changes in molecular composition associated with divalentcation deficiency and outer membrane stability. The major change under the different growth conditions was in the ratio of ornithine-containing lipid to phospholipid. P. denitrificans ATCC 13543 was grown to stationary phase with shaking at 30°C in complex medium (18), complex medium supplemented with 0.81 mM MgSO4 and 0.36 mM CaCI2, or succinate-salts medium (3). Our previously described procedure (16) for the isolation of cytoplasmic and outer membranes was used, with the following modifications. Cell envelopes were washed twice in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (pH 7.4) without MgCl2, and the sucrose gradients were 10 ml of 2.25 M, 12 ml of 1.44 M, and 10 ml of 0.77 M sucrose in 10 mM HEPES buffer (pH 7.4). Cell envelopes and cytoplasmic and outer membranes were made 1 mM in MgCI2 for recovery by ultracentrifugatio...
Deprivation of Paracoccus denitrificans of iron in sodium molybdate‐containing medium caused a slower rate of growth and lower final cell yield, in contrast to our previous studies in non‐sodium molybdate‐containing medium, where iron deprivation had little effect on growth rate. Five high Mr outer membrane proteins and catechol production were induced in iron‐deprived cultures. The fifth protein, Mr 72 000, was produced later than the others. Growth of iron‐deprived cells in medium containing 20 μM ferric citrate repressed siderophore and iron deprivation‐induced protein production, and led to production of an Mr 23 000 outer membrane protein (half maximum production after 5 h). Synthesis of the Mr 23 000 and high Mr proteins appeared to be mutally exclusive, and to be regulated by the cell's iron status. Cells inoculated into medium containing 20 μM ferric citrate took up 92% of the iron within 1 h, suggesting the occurrence of a nonsiderophore mediated, ‘low affinity’ iron uptake pathway.
Both peritoneal macrophages and cell populations containing multinucleate giant cells have been shown to release preferentially acid phosphatase and beta-glucuronidase. In both instances the relative proportion of the released enzyme is the same and is higher than the release of cytoplasmic LDH indicating that cell death is not the major cause of the released hydrolases. Stimulation with heat aggregated IgG increases both the specific activity/mg of soluble cellular protein as well as the relative proportion of the released enzyme. Populations containing multinucleate giant cells, however, release a higher relative proportion of acid phosphatase than beta-glucuronidase which may indicate that lysosomes with a lower beta-glucuronidase content may be involved. Such a selective release of lysosomal material may aid the extracellular degradation of closely opposed undigestible material in instances of granulomatous inflammation.
Production of a 23,000-Mr major outer membrane protein of Paracoccus denitrificans ATCC 13543 was dependent upon the addition of iron to a succinate-salts medium. The 23,000-Mr protein was not produced in an iron-deficient medium, but production of five outer membrane proteins in the 85,000-to 72,000-Mr range and of catechol were induced. The 23,000-Mr protein was not produced in a complex medium even when ferric citrate was added to the medium. Production of the protein was influenced by the carbon source and was decreased by peptone.
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