Small recirculating filters for nitrogen reduction.

Concerned about the negative impacts of nitrogen loading from septic systems on the Chesapeake Bay watershed, Maryland's Anne Arundel County Health Department has pioneered the use of small recirculating sand filters to reduce nitrogen in effluent from residential septic systems. Recirculating sand filters can reduce the total nitrogen in septic-tank effluent by up to 70 percent. Years of experience and the county's participation in the National Onsite Demonstration Project have led to modifications that make the filters more acceptable to homeowners. Use of alternative media, changes in flow patterns, and homeowner education have increased acceptance by homeowners.

Ferrisiderophore reductase activity in Bacillus megaterium.

The release of iron from ferrisiderophores (microbial ferric-chelating iron transport cofactors) by cell-free extracts of Bacillus megaterium was demonstrated. Reductive transfer of iron from ferrisiderophores to the ferrous-chelating agent ferrozine was measured spectrophotometrically. This ferrisiderophore reductase activity (reduced nicotinamide adenine dinucleotide phosphate:ferrisiderophore oxidoreductase) was associated primarily with the cell soluble rather than particulate (membrane) fraction. Ferrisiderophore reductase was inhibited by oxygen and required the addition of a reductant (reduced nicotinamide adenine dinucleotide phosphate was most effective) for maximal activity. The activity was destroyed by both heat and protease treatments and was inhibited by iodoacetamide treatment. Ferrisiderophore reductase activity for several microbial ferrisiderophores was measured; highest activity was displayed for ferrischizokinen, the ferrisiderophore produced by this organism. The Km and Vmax values of the reductase for ferrischizokinen were 2.5 x 10(-4) M and 35.7 nmol/min per mg of the ferrisiderophore reductase reaction. Preliminary fractionation of the cell soluble material by gel filtration chromatography resulted in the demonstration of ferrisiderophore reductase activity in three peaks of different molecular weight. Ferrisiderophore reductase probably mediates entrance of iron into cellular metabolism.

Ferric hydroxamate transport without subsequent iron utilization in Bacillus megaterium.

Iron transport and utilization were examined in Bacillus megaterium Ard1, a mutant that is resistant to the hydroxymate antibiotic A22765 and whose growth is inhibited by the structurally similar hydroxamate Desferal. Rapid, low-level uptake of Desferal-50Fe was observed; such uptake was temperature and energy independent. Gel filtration chromatography of the cytoplasmic fraction of protoplasts labeled with Desferal-55Fe for 30 to 120 s demonstrated only unchanged esferal-55Fe in the cytoplasm. Although B. megaterium Ard1 showed transport of Desferal-59Fe by a process that resembles facilitated diffusion, this organism was unable to transfer iron from this chelate to cellular macromolecules for metabolic use. High-level transport of the ferric hydroxamate schizokinen-59Fe by B. megaterium Ard1 was both temperature and energy dependent. Within 30 s, protoplasts labeled with schizokinen-55Fe contained iron associated with certain macromolecules and in an apparent "pool" of schizokinen-55Fe in the cytoplasmic fraction. Prior transport of Dseferal-55Fe by protoplasts of strain Ard1 did not interfere with subsequent transport and utilization of schizokinen-59Fe. These studies suggest that transport of ferric hydroxamates may occur by a facilitated diffusion-type process; transfer of iron to cellular macromolecules may drive high-level transport of the chelate and may be the step at which energy is required in the iron transport-assimilation process.

Active transport of iron in Bacillus megaterium: role of secondary hydroxamic acids.

Kinetics of radioactive iron transport were examined in three strains of Bacillus megaterium. In strain ATCC 19213, which secretes the ferric-chelating secondary hydroxamic acid schizokinen, (59)Fe(3+) uptake from (59)FeCl(3) or the ferric hydroxamate Desferal-(59)Fe(3+) was rapid and reached saturation within 3 min. In strain SK11, which does not secrete schizokinen, transport from (59)FeCl(3) was markedly reduced; the two ferric hydroxamates Desferal-(59)Fe(3+) or schizokinen-(59)Fe(3+) increased both total (59)Fe(3+) uptake and the (59)Fe(3+) appearing in a cellular trichloroacetic acid-insoluble fraction, although 10 min was required to reach saturation. Certain characteristics of transport from both ferric hydroxamates and FeCl(3) suggest that iron uptake was an active process. The growth-inhibitory effect of aluminum on strain SK11 was probably due to the formation of nonutilizable iron-aluminum complexes which blocked uptake from (59)FeCl(3). Desferal or schizokinen prevented this blockage. A strain (ARD-1) resistant to the ferric hydroxamate antibiotic A22765 was isolated from strain SK11. Strain ARD-1 failed to grow with Desferal-Fe(3+) as an iron source, and it was unable to incorporate (59)Fe(3+) from this source. Growth and iron uptake in strain ARD-1 were similar to strain SK11 with schizokinen-Fe(3+) or the iron salt as sources. It is suggested that the ferric hydroxamates, or the iron they chelate, may be transported by a special system which might be selective for certain ferric hydroxamates. Strain ARD-1 may be unable to recognize both the antibiotic A22765 and the structurally similar chelate Desferal-Fe(3+), while retaining its capacity to utilize schizokinen-Fe(3+).

Inhibition of iron uptake and deoxyribonucleic acid synthesis by Desferal in a mutant strain of Bacillus subtilis.

In the Bacillus subtilis mutant 1D-4, the hydroxamate Desferal inhibited growth, iron uptake, and deoxyribonucleic acid synthesis but did not quantitatively affect synthesis of ribonucleic acid and protein.

Diversity of siderophore genes encoding biosynthesis of 2,3-dihydroxybenzoic acid in Aeromonas spp.

Most species of the genus Aeromonas produce the siderophore amonabactin, although two species produce enterobactin, the siderophore of many enteric bacteria. Both siderophores contain 2,3-dihydroxybenzoic acid (2,3-DHB). Siderophore genes (designated aebC, -E, -B and -A, for aeromonad enterobactin biosynthesis) that complemented mutations in the enterobactin genes of the Escherichia coli 2,3-DHB operon, entCEBA(P15), were cloned from an enterobactin-producing isolate of the Aeromonas spp. Mapping of the aeromonad genes suggested a gene order of aebCEBA, identical to that of the E. coli 2,3-DHB operon. Gene probes for the aeromonad aebCE genes and for amoA (the entC-equivalent gene previously cloned from an amonabactin-producing Aeromonas spp.) did not cross-hybridize. Gene probes for the E. coli 2,3-DHB genes entCEBA did not hybridize with Aeromonas spp. DNA. Therefore, in the genus Aeromonas, 2,3-DHB synthesis is encoded by two distinct gene groups; one (amo) is present in the amonabactin-producers, while the other (aeb) occurs in the enterobactin-producers. Each of these systems differs from (but is functionally related to) the E. coli 2,3-DHB operon. These genes may have diverged from an ancestral group of 2,3-DHB genes.

Novel heme-binding component in the serum of the channel catfish (Ictalurus punctatus).

The serum of the channel catfish (Ictalurus punctatus) was examined for heme- and hemoglobin-binding proteins. Electrophoretic mobility retardation assays failed to detect a hemoglobin-binding material similar to mammalian haptoglobin; however, a heme-binding component (not previously described) was identified in catfish serum. The heme-binding component was purified by gel filtration chromatography; electrophoretic analyses suggested it to be composed of two polypeptide subunits of molecular masses about 115 and 98 kDa. This composition is inconsistent with hemopexin, the known heme-binding serum protein of mammals. Although it was not fully saturated with heme, the catfish component contained detectable heme in normal sera. When complexed by the binding material, heme was used as an iron source by isolates of the bacterial Gram-negative genus Aeromonas; the capacity of other bacteria to use the complex was not tested. The physiological function of the catfish heme-binding serum protein is presently not clear.

Evaluation of iron-chelating agents in cultured heart muscle cells. Identification of a potential drug for chelation therapy.

Primary cultures of neonatal rat cardiac muscle cells incorporated radioiron from both [55Fe]transferrin and 59FeCl3 (added simultaneously). To evaluate the effect of iron chelators on such uptake, deferri chelators were added 6 hr after addition of the radioiron sources. The microbial chelator agrobactin was significantly more effective than the drug defoxamine in reduction of 55Fe uptake from [55Fe]transferrin; both chelators halted 59Fe3+ uptake. Agrobactin may have potential in chelation therpay for iron-overload disease. Certain other microbial chelators lowered radioiron uptake from either [55Fe]transferrin of 59FeCl3. These chelators should be useful inhibitors for studies of animal cell iron uptake and intracellular iron flow.

Iron-chelating hydroxamic acid (schizokinen) active in initiation of cell division in Bacillus megaterium.

Bacillus megaterium ATCC 19213 secretes a cell division-initiating "schizokinen" (SK) which accumulates during its culture cycle to a concentration inversely proportional to the iron added to a sucrose-mineral salts medium. Secreted SK was purified from culture filtrates as a red Fe (III) chelate, and a fraction with similar biological properties was obtained from whole cells. Infrared spectra of SK, and analyses of unhydrolyzed and acid-hydrolyzed preparations indicated it to be a secondary hydroxamate; visible absorption maxima of the ferric complex showed pH dependency typical of ferric monohydroxamates. Schizokinen preparations from cultures grown at "normal" and at low Fe concentrations were similar biologically and in certain of their chemical properties, but their R(F) values and infrared spectra suggested nonidentity. Significant lag reduction of B. megaterium was effected by 0.2 mmug of SK per ml; the Fe (III)-SK chelate and "iron-free" SK were equally effective. A 50-mmug amount produced half-maximal growth response of the siderochrome auxotroph, Arthrobacter JG-9. Schizokinen also overcame ferrimycin A inhibition of three Bacillus species. These properties relate the B. megaterium schizokinen to the trihydroxamate siderochromes, although SK appears to be a monohydroxamate.

Enhancement of copper toxicity by siderophores in Bacillus megaterium.

Chelation of copper by siderophores enhanced the toxicity of copper for Bacillus megaterium. Although this antibacterial activity appeared to be rapidly bactericidal, it could be partly reversed by addition of deferrisiderophore , or of ferrisiderophore at high concentration, immediately after exposure of cells to the cupric-siderophore complex.

Repression of phenolic acid-synthesizing enzymes and its relation to iron uptake in Bacillus subtilis.

Excretion of the metal-chelating phenolic acid, 2,3-dihydroxybenzoate, by a tryptophan-requiring strain (M-13) of Bacillus subtilis was inversely proportional to the iron added to the medium. Addition of iron as the ferric chelates of two secondary hydroxamates (ferri-schizokinen and Desferal) markedly reduced excretion. Synthesis of 2,3-dihydroxybenzoate from chorismate by extracts of B. subtilis M-13, grown in low-iron medium, was unaltered by additions of FeSO(4), FeCl(3), ferrischizokinen, 2,3-dihydroxybenzoate, the 2,3-dihydroxybenzoate-iron complex, or by extracts of cells grown in high-iron medium (which contained no demonstrable 2,3-dihydroxybenzoate-synthesizing activity) to the extracts of "low-iron cells." Iron control seemed to involve repression of synthesis of the enzymes in the 2,3-dihydroxybenzoate pathway. Both ferri-schizokinen and 2,3-dihydroxybenzoate plus iron enhanced considerably the otherwise minimal repressive effects of iron at low concentrations. Ferri-schizokinen delayed derepression of the pathway in B. subtilis M-13, and reduced its rate of synthesis after derepression. Addition of FeSO(4) to derepressed cells of B. subtilis M-13 halted synthesis of the enzymes after a lag period. The effect of the ferric hydroxamates was related to the capacity of B. subtilis M-13 to incorporate (59)Fe(3+) from Desferal-(59)Fe(3+). Cellular accumulation of (59)Fe(3+) from Desferal-(59)Fe(3+) after 20 min was nearly double that incorporated from (59)FeCl(3).

Iron requirements and aluminum sensitivity of an hydroxamic acid-requiring strain of Bacillus megaterium.

Bacillus megaterium strain ATCC 19213 secretes a ferric-chelating secondary hydroxamic acid, whereas a mutant (strain SK11) derived from it cannot produce a hydroxamate. Strain SK11 could be cultivated in a sucrose-mineral salts medium (treated with Chelex 100 to reduce trace metals) in the absence of added hydroxamate, if the inoculum was high. The lowest iron supplements necessary for maximal growth of both strains were equivalent (0.01 to 0.04 mug of iron per ml). Addition of either aluminum (0.5 mug/ml) or chromium (0.1 mug/ml) to the medium prevented full growth of strain SK11 at the minimal iron concentration, although elevated iron (1 mug/ml) reversed this inhibition. The iron-free secondary hydroxamate, Desferal, also abolished aluminum and chromium inhibition of strain SK11, producing maximal population densities at the low iron concentration. Growth of the hydroxamate-producing strain 19213 was not altered significantly by the aluminum or chromium levels which inhibited strain SK11. However, strain 19213 responded to these metals by increasing its secretion of a secondary hydroxamate. It was concluded that aluminum and chromium interfered with iron incorporation, either directly or by formation of nonutilizable aggregates with iron. The secondary hydroxamates may have overcome this interference by solubilization of iron for delivery to a single uptake process, or the ferric-hydroxamate chelate may enter the cell by an alternate route.

Acquisition of iron from host sources by mesophilic Aeromonas species.

The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.

Iron acquisition and virulence in the motile aeromonads: siderophore-dependent and -independent systems.

During an infection, a microbial pathogen must acquire all of its iron from the host. Aeromonas isolated producing the siderophore amonabactin obtain iron either from host Fe-transferrin (siderophore dependent) or from host heme-containing molecules (siderophore independent). Isolates producing the siderophore enterobactin do not utilize Fe-transferrin in serum and probably rely exclusively on host heme iron.

Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.

Physiological control of amonabactin biosynthesis in Aeromonas hydrophila.

Amonabactin is a siderophore from Aeromonas hydrophila which is produced in two biologically active forms composed of the phenolate 2,3-dihydroxybenzoic acid (DHB), lysine, glycine, and either trytophan (amonabactin T) or phenylalanine (amonabactin P). Amonabactin biosynthetic mutants (generated by chemical mutagenesis) that either produced no amonabactin or overproduced the siderophore were isolated and identified on chrome azurol S siderophore detection agar. Amonabactin-negative mutants were of two categories. One type produced no phenolates and used exogenous DHB to synthesize amonabactin (both forms) while the other type excreted DHB but not amonabactin. This suggests an amonabactin biosynthetic pathway composed of two segments, one producing DHB and the other assembling amonabactin from DHB and the amino acids. Overproduction mutants used amonabactin poorly or not at all, indicating that they contained lesions in amonabactin utilization. Adding the analog D-tryptophan to wild-type A. hydrophila cultures reduced synthesis of both amonabactin T and amonabactin P and lengthened the lag phase in iron restricted medium. The tryptophan and phenylalanine forms of amonabactin may be synthesized by a single assembly pathway that contains a novel enzyme (sensitive to D-tryptophan) which inserts either tryptophan or phenylalanine into amonabactin.

Ferrous iron transport in Streptococcus mutans.

Radioiron uptake from 59FeCl3 by Streptococcus mutans OMZ176 was increased by anaerobiosis, sodium ascorbate, and phenazine methosulfate (PMS), although there was a 10-min lag before PMS stimulation was evident. The reductant ascorbate may have provided ferrous iron. The PMS was reduced by the cells, and the reduced PMS then may have generated ferrous iron for transport; reduced PMS also may have depleted dissolved oxygen. We conclude that S. mutans transports only ferrous iron, utilizing reductants furnished by glucose metabolism to reduce iron prior to its uptake.