Genetic and biochemical characterization of the Escherichia coli K-12 fhuB mutation.

The fhuB region of Escherichia coli K-12 was subcloned from pLC4-44 into pP lac to obtain pCPN1. Deletions of this recombinant plasmid were made, and a 1.4-kilobase PstI fragment was further subcloned into the vector plasmid pKK177-2 to obtain pCPN12. The response of tonA and tonB strains and fhuB strains containing the plasmids to 15 hydroxamate siderophores were assayed. Results showed that tonA strains were deficient only in the utilization of ferrichrome-type siderophores, whereas fhuB strains were deficient in the utilization of all hydroxamate-type siderophores. The response of the plasmid-containing fhuB strains to the siderophores showed that the fhuB gene resides on a 1.4-kilobase PstI fragment of DNA. The proteins synthesized by these plasmids were examined in maxicells of strain CSR603. Plasmid pCPN1 expressed five proteins of molecular weights 78,000, 40,000, 30,000, 24,000, and 13,700. By the use of deletions of pCPN1, the approximate order of the genes for these proteins was determined. Plasmid pCPN12 expressed no proteins other than the beta-lactamase proteins in maxicell strain CSR603. However, in maxicell strain BN660, a lon mutant, it expressed a 20,000-molecular-weight protein. Inner membrane vesicles made from tonB and fhuB strains were able to transport [55Fe]ferrichrome and [55Fe]rhodotorulate at rates similar to those obtained in vesicles from tonB+ and fhuB+ strains.     

Effects of pronase on passive ion permeability of the human red blood cell

Summary The effects of pronase fromStreptomyces griseus on sulfate, potassium, sodium, and erythritol permeability of human red blood cells were studied. It was found that the proteolytic enzyme reduces anion permeability, increases cation permeability and has no effect on the nonfacilitated component of the flux of the nonelectrolyte. These findings can be explained on the basis of the fixed charge hypothesis by the assumption that the enzyme exerts its effects by altering the density of positive fixed charges in the membrane.The effects of pronase are qualitatively similar to those of the amino reactive agent, dinitrofluorobenzene (DNFB). Therefore, attempts were made to discover if this similarity is due to alterations of the same membrane sites by the enzyme and the chemical modifier. It was found that the effects of pronase and DNFB were not additive. Hence, the enzyme and the amino reactive agent do not seem to act on two independent and parallel channels. A more detailed analysis of the data suggests that DNFB and pronase affect functionally identical sites.Proteolytic enzymes frequently exhibit some esterase activity. However, the amino-N content of lipid extracts of red cell membranes remained virtually unaltered after exposure of the cells to pronase. This finding indicates that the positive charge of the bulk of the lipid amino groups is not involved in the control of passive ion permeability. The carbohydrate amino groups of the red cell membrane are N-acylated and hence cannot contribute to the positive membrane charge. It seems reasonable to conclude that the effects of pronase on ion permeability are primarily due to alterations of the density of charged protein amino groups in the red cell membrane.     

Chromosomal genes for ColV plasmid-determined iron(III)-aerobactin transport in Escherichia coli.

Four chromosomal genes, tonA (fhuA), fhuB, tonB, and exbB, were required for the transport of iron(III)-aerobactin specified by the plasmids ColV-K311, ColV-K229, ColV-K328, and ColV-K30. These genes also determine the transport system in Escherichia coli for the iron ionophore ferrichrome. Aerobactin and ferrichrome are both iron ligands of the hydroxamate type, but they are of different structure. The ColV plasmids determine an outer membrane protein that serves as a receptor for cloacin. Cloacin-resistant mutants were devoid of iron(III)-aerobactin transport but were unimpaired in ferrichrome transport. We conclude that for iron(III)-aerobactin transport two outer membrane proteins, the TonA and the cloacin receptor protein, have to interact functionally or structurally or both.     

Frequency domain analysis of asymmetry current in squid axon membrane.

The change in capacity of squid axon membrane during hyper- and depolarizations was investigated in the absence of ionic currents after the membrane was treated with pronase. In the presence of the inactivation process (h parameter), failure to observe the gating current in the frequency domain was attributed to the rapid attenuation of the possible capacity change during depolarizations, which is likely to be due to the sodium activation process. Elimination of the h process would therefore enable us to observe the gating current in the frequency domain as the change in the capacitance component of membrane admittance. However, even after the inactivation process was abolished by pronase, the capacity of the axon membrane remained constant when ionic currents were blocked by external tetrodotoxin and internal Cs+ ion. Actually capacity was observed to decrease slightly with depolarization, contrary to the prediction based on the magnitude of gating currents.     

Staining of proteoglycans in mouse lung alveoli. II. Characterization of the Cuprolinic Blue-positive, anionic sites

Summary The nature of Cuprolinic Blue-positive anionic filaments in mouse lung alveoli has been characterized. The contrast of filaments in the alveolar basement membrane of type I epithelial cells was lost on treatment with nitrous acid and pronase (without prefixation). In contrast, neither neuraminidase, chondroitinase ABC or AC, norStreptomyces hyaluronidase had any effect. Treatment with pronase (after prefixation) and 2.0m MgCl₂ (after prefixation) also had no effect, indicating that the filaments are heparan sulphate proteoglycans. The filaments in the alveolar basement membrane of type II epithelial cells and in the capillary basement membrane of the endothelial cells were also nitrous acid sensitive, but chondroitinase ABC-insensitive. A model in which the whole alveolus contains a single layer of heparan sulphate-containing proteoglycan monomers is proposed. Furthermore, the collagen fibril associated filaments remained unaffected after treatment with nitrous acid, neuraminidase orStreptomyces hyaluronidase, or after digestion with pronase (after prefixation) and treatment with 2.0m MgCl₂ (after prefixation). These filaments, however, could no longer be detected when digestion with chondroitinase ABC or pronase (without prefixation) was applied; chondroitinase AC treatment clearly affected the filaments, although they still were visible. These results indicate that the filaments are dermatan sulphate-containing proteoglycans. Some functional aspects of the proteoglycans are discussed.     

Molecular characterization of the iron-hydroxamate uptake system in Staphylococcus aureus

To investigate iron uptake, a chromosomal locus containing three consecutive open reading frames, designated fhuC, fhuB, and fhuD, was identified in Staphylococcus aureus. Whereas the fhuC gene encodes an ATP-binding protein, fhuB and fhuD code for ferrichrome permeases and thus resemble an ATP-binding cassette transporter. A fhuB knockout mutant showed impaired uptake of iron bound to the siderophores but not of ferric chloride, suggesting that this operon is specific for siderophore-mediated iron uptake.     

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.     

Proteolytic digestion and labelling studies of the organization of the proteins in the outer membrane of Escherichia coli.

The arrangement of the proteins in the outer membrane of Escherichia coli was examined by treating intact cells and isolated membrane preparations with fluorescamine and with pronase. Intact wild-type cells, or those of a mutant in which the core region of the lipopolysaccharide was absent, were equally resistant to pronase treatment. The protein components of isolated outer membrane preparations varied in their rate of digestion and labelling with fluorescamine. The N-terminal portion of protein B was removed by pronase to yield a fragment (protein Bp) still embedded in the membrane. Protein Bp was not significantly enriched in nonpolar amino acids, suggesting that protein B may not be held in the membrane primarily by hydrophobic interactions. This was confirmed by reconstitution experiments in which protein B could be reassociated with itself, without lipopolysaccharide or phospholipid, in the presence of divalent cation such that pronase digestion of the reassociated material gave protein Bp.     

Surface immunoglobulins (SmIg) in human B-lymphocytes: effect of enzymatic digestion]

In order to determine the number of circulating B lymphocytes which can synthetize their surface membrane immunoglobulins, we have evaluated by immunofluorescence their presence before and after enzymatic exposure to pronase. After pronase treatment the majority of lymphocytes carrying SmIg has been disappeared (from 7.8% to 1.9%; p < .0.0005). After 18 hours incubaton only a small proportion (4.6%) of B lymphocytes risynthetized their SmIg. This observation support the hypothesis that a subset of lymphocytes present in the peripheral blood carries on its membrane only cytophilic SmIg and therefore may interfere in the determination of the number of B lymphocytes.     

Iron-hydroxamate transport into Escherichia coli K12: localization of FhuD in the periplasm and of FhuB in the cytoplasmic membrane

Summary ThefhuB, fhuC andfhuD genes encode proteins which catalyze transport of iron(III)-hydroxamate compounds from the periplasm into the cytoplasm ofEscherichia coli. ThefhuB, C, D genes were cloned downstream of a strong phage T7 promoter and transcribed by T7 RNA polymerase. The overexpressed FhuD protein appeared in two forms of 31 and 28 kDa and was released upon conversion of vegetative cells into spheroplasts, suggesting synthesis of FhuD as a precursor and export into the periplasm. The very hydrophobic FhuB protein was found in the cytoplasmic membrane. These properties, together with the previously found homologies in the FhuC protein to ATP-binding proteins, display the characteristics of a periplasmic binding protein dependent transport system across the cytoplasmic membrane. The molecular weight of FhuB and the sequence offhuC, as previously published by us, was confirmed. FhuB exhibited double the size of most hydrophobic proteins of such systems and showed homology between the amino- and carboxy-terminal halves of the protein, indicating duplication of an original gene and subsequent fusion of the two DNA fragments.     

The use of dextran as a blocking agent on nitrocellulose membrane in the analysis of sporozoite antigens of Plasmodium vivax

Dextran (molecular weight, 71,200) has been found to block the unbound sites of the nitrocellulose membrane, to which antigens have been electroblotted from acrylamide gel, for use in assaying monoclonal antibodies. The use of polysaccharide as a blocking agent allows the antigens on the nitrocellulose membrane to be digested with pronase and subsequently reacted with monoclonal antibodies. Sporozoite antigens of Plasmodium vivax, after being digested with pronase, completely lost their antigenicity to bind to the sporozoite-specific monoclonal antibodies, thus suggesting that they are proteins or protein conjugates in nature. The method described here for qualitative determination of protein antigens requires as little as 2000 sporozoites for each assay.     

The role of the plasma membrane in the development of Dictyostelium discoideum. II. Developmental and topographic analysis of polypeptide and glycoprotein composition

Previous workers have shown in a variety of ways that cell contact is required for the differentiation of Dictyostelium discoideum. Because interactions between cells are probably mediated by molecules on their plasma membranes, we have characterized the polypeptide composition of the membrane of cells at different stages of development. At least 55 polypeptides are found in the plasma membrane of vegetative cells. The polypeptide composition of the plasma membranes changes considerably during development. Treatment of intact cells with pronase indicated that many of the altered components appear to be located on the external surface of the plasma membrane where they could participate in interactions between cells. Similar digestion of the isolated membranes destroys most of their polypeptides, indicating that the bulk of the proteins of the plasma membrane are not completely embedded in the membrane. Several polypeptides appear to change in sensitivity to pronase during development. There are several changes in glycoprotein composition which occur between log phase and aggregation phase. An almost complete change in glycoprotein species occurs between aggregation and pre-culmination. Unlike the polypeptides, the glycoproteins are very resistant to pronase treatment in intact cells. However, some are pronase sensitive in isolated membranes.     

The role of the plasma membrane in the development of Dictyostelium discoideum. II. Developmental and topographic analysis of polypeptide and glycoprotein composition

Previous workers have shown in a variety of ways that cell contact is required for the differentiation of Dictyostelium discoideum. Because interactions between cells are probably mediated by molecules on their plasma membranes, we have characterized the polypeptide composition of the membrane of cells at different stages of development. At least 55 polypeptides are found in the plasma membrane of vegetative cells. The polypeptide composition of the plasma membranes changes considerably during development. Treatment of intact cells with pronase indicated that many of the altered components appear to be located on the external surface of the plasma membrane where they could participate in interactions between cells. Similar digestion of the isolated membranes destroys most of their polypeptides, indicating that the bulk of the proteins of the plasma membrane are not completely embedded in the membrane. Several polypeptides appear to change in sensitivity to pronase during development. There are several changes in glycoprotein composition which occur between log phase and aggregation phase. An almost complete change in glycoprotein species occurs between aggregation and pre-culmination. Unlike the polypeptides, the glycoproteins are very resistant to pronase treatment in intact cells. However, some are pronase sensitive in isolated membranes.     

Demonstration of sialic acid groups in the glomerular basement membrane of the rat with phosphotungstic acid at low pH

Summary This paper reports an unrecognized aspect of phosphotungstic acid staining at low pH. It provides an on-section staining method in which sialic acid-containing molecules can be demonstrated in the laminae rarae of the rat glomerular basement membrane. The staining in the basement membrane became negative after perfusion with the following cations: protamine sulphate, hexadimethrine, Alcian Blue, Ruthenium Red and Toluidine Blue. Blocking ws not achieved with Alcian Blue at about pH 1. The staining was also abolished after mild methylation and demethylation restored the contrast. This is suggestive of the involvement of carboxyl groups. Prior digestion with pronase, trypsin and neuraminidase rendered the laminae rarae negative, whereas hyaluronidase, chondroitinase ABC and crude heparinase were without effect. This indicates that sialic acid groups are detected by this method and that heparan sulphate does not interfere. The staining of the epithelial plasma membrane, also carrying sialic acid groups, remained positive after neuraminidase treatment. It is presumed that this method can be applied successfully for detecting changes in the sialic acid content of the laminae rarae in rat glomerular basement membranes under normal and pathological conditions.     

Intermediate steps in cellular iron uptake from transferrin. Detection of a cytoplasmic pool of iron, free of transferrin.

The uptake of transferrin-bound iron by receptor-mediated endocytosis has been the subject of extensive experimental investigation. However, the path followed by iron (Fe) after release from transferrin (Tf) remains obscure. Once Fe is released from Tf within the endosome, it must be transported across the endosomal membrane into the cell. The present investigation describes the presence of a cytoplasmic Tf-free Fe pool which is detectable only when cells are detached from their culture dishes at low temperature, after initial incorporation of diferric transferrin at 37 degrees C. This cellular iron pool was greatly reduced if incubation temperatures were maintained at 37 degrees C or if cells were treated with pronase. Human melanoma cells (SK-MEL-28) in culture were prelabeled by incubation with human 125I-59Fe-transferrin for 2 h, washed, and reincubated at 4 degrees C or 37 degrees C in balanced salt solution in the presence or absence of pronase. The cells were then mechanically detached from the plates and separated into "internalized" and supernatant fractions by centrifugation. Approximately 90% of cellular 59Fe and 20% of 125I-Tf remained internalized when this reincubation procedure was carried out in balanced salt solution at 37 degrees C. However, at 4 degrees C, cellular internalized iron was reduced to approximately 50% of the initial value. The release of this component of cellular 59Fe (approximately 40% of total cell 59Fe) at 4 degrees C was completely inhibited in the presence of pronase and other general proteinases at 4 degrees C and at 37 degrees C, without affecting internalized transferrin levels. Similar results were obtained in fibroblasts and hepatoma cells, indicating that this phenomenon is not unique to melanoma cells. The characterization of this Tf-free cellular Fe pool which is detectable at low temperature may yield valuable insights into the metabolic fate of iron following its transport across the membrane of the endocytotic vesicle.     

Electrical properties of squid axon membrane. II. Effect of partial degradation by phospholipase A and pronase on electrical characteristics.

Passive electrical characteristics of perfused squid axon membrane are investigated. In a previous publication, we reported that the capacitance of intact squid axon membrane is partly frequency dependent. We extended the same measurement to perfused axons. We found that the electrical characteristics of perfused axon membrane are essentially the same as those of intact axons. In this work, we investigated the effects of phospholipase A and pronase on the membrane capacitance. Phospholipase A is known to block the sodium activation and pronase to eliminate the sodium inactivation. Phospholipase A is found to increase the frequency dependent as well as the frequency independent capacitances. Our tentative conclusion is that this enzyme perturbs the lipid structure and decreases its thickness. Pronase is found to increase the frequency dependent capacitance slightly while the capacitance of the lipid layer remains unaltered. Although voltage clamp data indicate that the pronase disrupts the excitatory mechanism extensively, this enzyme has relatively little effect on the overall membrane capacitance.     

Electrical properties of squid axon membrane. II. Effect of partial degradation by phospholipase A and pronase on electrical characteristics

Passive electrical characteristics of perfused squid axon membrane are investigated. In a previous publication, we reported that the capacitance of intact squid axon membrane is partly frequency dependent. We extended the same measurement to perfused axons. We found that the electrical characteristics of perfused axon membrane are essentially the same as those of intact axons. In this work, we investigated the effects of phospholipase A and pronase on the membrane capacitance. Phospholipase A is known to block the sodium activation and pronase to eliminate the sodium inactivation. Phospholipase A is found to increase the frequency dependent as well as the frequency independent capacitances. Our tentative conclusion is that this enzyme perturbs the lipid structure and decreases its thickness. Pronase is found to increase the frequency dependent capacitance slightly while the capacitance of the lipid layer remains unaltered. Although voltage clamp data indicate that the pronase disrupts the excitatory mechanism extensively, this enzyme has relatively little effect on the overall membrane capacitance.     

Ferric citrate transport in Escherichia coli requires outer membrane receptor protein fecA.

Mutants of Escherichia coli K-12 AB2847 and of E. coli K-12 AN92 were isolated which were unable to grow on ferric citrate as the sole iron source. Of 22 mutants, 6 lacked an outer membrane protein, designated FecA protein, which was expressed by growing cells in the presence of 1 mM citrate. Outer membranes showed an enhanced binding of radioactive iron, supplied as a citrate complex, depending on the amount of FecA protein. The FecA protein was the most resistant of the proteins involved in ferric irion iron translocation across the outer membrane (FhuA = TonA, FepA, Cir, or 83K proteins) to the action of pronase P. It is also shown that previously isolated fec mutants (G. C. Woodrow et al., J. Bacteriol. 133:1524-1526, 1978) which are cotransducible with argF all lack the FecA protein. They were termed fecA to distinguish them from the other ferric citrate transport mutants, now designated fecB, which mapped in the same gene region at 7 min but were not cotransducible with ArgF. E. coli W83-24 and Salmonella typhimurium, which are devoid of a citrate-dependent iron transport system, lacked the FecA protein. It is proposed that the FecA protein participates in the transport of ferric citrate.