Large decreases in membrane phosphatidylethanolamine and diphosphatidylglycerol upon mutation to duramycin resistance do not change the protonophore resistance of Bacillus subtilis

Duramycin-resistant mutant strains were selected from wild-type Bacillus subtilis (BD99) and its protonophore-resistant mutant derivative, strain AG1A3. Analyses of the membranes of the duramycin-resistant mutants showed that they had little or no phosphatidylethanolamine and diphosphatidylglycerol as determined by chemical detection after thin-layer chromatography. Small amounts of these phospholipids must remain in the mutant strains, however, because during studies of incorporation of exogenous, radioactive fatty acids, label associated with palmitoleic acid was found in chromatographic positions that corresponded to the expected positions of phosphatidylethanolamine and diphosphatidylglycerol. The duramycin-resistant strains both showed elevated levels of phosphatidylglycerol and aminoacyl(lysyl)phosphatidylglycerol. The duramycin-resistant derivative of protonophore-resistant AG1A3 (AG1A3-DR4), but not that of the wild type, also showed a decreased content of neutral relative to polar lipid in the membrane. The composition of neutral lipid in that strain was higher in free fatty acids and lower in 1,2-diacylglycerol than its parent strain. AG1A3-DR4 also contained appreciable levels of lysophosphatidylethanolamine and somewhat elevated diglycosyldiacylglycerol relative to the other strains in the study. The protonophore resistance of AG1A3 was unaltered by mutation to duramycin resistance. Nor was there any change in the efficacy of exogenous palmitoleic acid in diminishing the protonophore resistance of AG1A3-DR4. This phenomenon persists upon dramatic reduction in the content of phosphatidylethanolamine and diphosphatidylglycerol even though those phospholipids are normally the preferred sites of incorporation of the exogenous unsaturated fatty acids that mediate the effect.     

Features of apparent nonchemiosmotic energization of oxidative phosphorylation by alkaliphilic Bacillus firmus OF4.

Oxidative phosphorylation by extremely alkaliphilic Bacillus species violates two major predictions of the chemiosmotic hypothesis: the magnitude of the chemiosmotic driving force, the delta p (electrochemical proton gradient), is too low to account for the phosphorylation potentials observed during growth at pH 10.5 without using a much higher H+/ATP stoichiometry than used during growth at pH 7.5, and artificially imposed diffusion potentials fail to energize ATP synthesis above about pH 9.5 (Guffanti, A. A., and Krulwich, T. A. (1989) Annu. Rev. Microbiol. 43, 435-463). To further examine the latter observation, large valinomycin-mediated potassium diffusion potentials were imposed across starved cells of Bacillus firmus OF4 at various pH values from pH 7.5 to 10.5. As the external pH increased above pH 8, there was a sharp decrease in the rate of ATP synthesis in response to an imposed diffusion potential. The rate of ATP synthesis fell to zero by pH 9.2 and 9.4, respectively, in the presence and absence of a small inwardly directed Na+ gradient. Electrogenic Na+/H+ antiport and Na+/alpha-aminoisobutyric acid symport proceeded at substantial rates throughout. When synthesis was energized by an electron donor, cells under comparable conditions synthesized ATP at rapid rates up to pH 10.5. The proton transfers that occur during respiration-dependent oxidative phosphorylation at pH 10.5 may depend upon specific complexes. Cells grown at pH 7.5, which have one-third the levels of the caa3-type terminal oxidase, and slightly lower levels of certain other respiratory chain complexes than pH 10.5-grown cells, support only low rates of ATP synthesis at pH 10.5, although energy-dependent symport and antiport rates are comparable with those in pH 10.5-grown cells. A model is presented for oxidative phosphorylation by the alkaliphilic Bacillus that involves a nonchemiosmotic direct intramembrane transfer of protons from specific respiratory chain complexes to the F0 sector of the ATPase, whereas remaining respiratory chain complexes extrude protons into the bulk to generate the bulk potential required both for ATP synthesis and other bioenergetic work. A pK-regulated gate or a delocalized proton pathway that fails to work above pH 9.5 are suggested as possible features that account for the loss of efficacy of a bulk-imposed diffusion potential in energizing ATP synthesis above pH 9.4.     

Cloning of the Bacillus firmus OF4 cls gene and characterization of its gene product

The gene that codes for cardiolipin (CL) synthase and an adjacent gene that codes for a MecA homolog in the alkaliphilic bacteria Bacillus firmus OF4 have been cloned and sequenced (GenBank accession number U88888). The cls gene contains 1509 nucleotides, corresponding to a polypeptide of 57.9 kDa. The predicted amino acid sequence has 129 identities and 100 similarities with the Escherichia coli CL synthase. Homologies were also noted with polypeptide sequences from putative cls genes from Bacillus subtilis and Psuedomonas putida. Conserved histidine, tyrosine, and serine residues may be part of the active site and participate in phosphatidyl group transfer. The B. firmus OF4 cls gene product was inserted into plasmid pET3 to form a recombinant plasmid pDG2, which overproduces CL synthase in E. coli. A membrane fraction containing the overproduced enzyme converts phosphatidylglycerol to CL and glycerol. The B. firmus enzyme is stimulated by potassium phosphate, inhibited by CL and phosphatidate, and has a slightly higher pH optimum than the E. coli enzyme.     

Protonophore-resistance and cytochrome expression in mutant strains of the facultative alkaliphile Bacillus firmus OF4.

Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse the protonmotive force (delta mu H+) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa3, b, c and o in strain CC2. Strain CC2 also maintained a higher delta mu H+ than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of delta mu H+ than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o. Cells of stran CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium, neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of delta mu H+. Instead, both resistant alkaliphile strains maintained a higher delta mu H+ and a correspondingly higher delta Gp than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa3-type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk delta mu H+.     

Growth and bioenergetics of alkaliphilic Bacillus firmus OF4 in continuous culture at high pH.

The effect of external pH on growth of alkaliphilic Bacillus firmus OF4 was studied in steady-state, pH-controlled cultures at various pH values. Generation times of 54 and 38 min were observed at external pH values of 7.5 and 10.6, respectively. At more alkaline pH values, generation times increased, reaching 690 min at pH 11.4; this was approximately the upper limit of pH for growth with doubling times below 12 h. Decreasing growth rates above pH 11 correlated with an apparent decrease in the ability to tightly regulate cytoplasmic pH and with the appearance of chains of cells. Whereas the cytoplasmic pH was maintained at pH 8.3 or below up to external pH values of 10.8, there was an increase up to pH 8.9 and 9.6 as the growth pH was increased to 11.2 and 11.4, respectively. Both the transmembrane electrical potential and the phosphorylation potential (delta Gp) generally increased over the total pH range, except for a modest fall-off in the delta Gp at pH 11.4. The capacity for pH homeostasis rather than that for oxidative phosphorylation first appeared to become limiting for growth at the high edge of the pH range. No cytoplasmic or membrane-associated organelles were observed at any growth pH, confirming earlier conclusions that structural sequestration of oxidative phosphorylation was not used to resolve the discordance between the total electrochemical proton gradient (delta p) and the delta Gp as the external pH is raised. Were a strictly bulk chemiosmotic coupling mechanism to account for oxidative phosphorylation over the entire range, the deltaGp/deltap ration (which would equal the H+/ATP ratio) would rise from about 3 at pH 7.5 to 13 at pH 11.2, dropping to 7 at pH 11.4 only because of the rise in cytoplasmic pH relative to other parameters. Moreover, the molar growth yields on malate were higher at pH 10.5 than at pH 7.5, indicating greater rather than lesser efficiency in the use of substrate at the more alkaline pH.     

Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4.

Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains.     

Mutation affecting expression of spectinomycin resistance in Bacillus subtilis.

A mutation that affects the expression of spectinomycin resistance in a spectinomycin-resistant (spcA), conditionally asporogenic strain of Bacillus subtilis has been designated srm (spectinomycin resistance modifier). This mutation resulted in altered colony morphology and increased growth rate and sporulation efficiency in the presence of spectinomycin.     

Evidence for multiple terminal oxidases, including cytochrome d, in facultatively alkaliphilic Bacillus firmus OF4.

The terminal oxidase content of Bacillus firmus OF4, a facultative alkaliphile that grows well over the pH range of 7.5 to 10.5, was studied by difference spectroscopy. Evidence was found for three terminal oxidases under different growth conditions. The growth pH and the stage of growth profoundly affected the expression of one of the oxidases, cytochrome d. The other two oxidases, cytochrome caa3 and cytochrome o, were expressed under all growth conditions tested, although the levels of both, especially cytochrome caa3, were higher at more alkaline pH (P.G. Quirk, A.A. Guffanti, R.J. Plass, S. Clejan, and T.A. Krulwich, Biochim. Biophys. Acta, in press). These latter oxidases were identified in everted membrane vesicles by reduced-versus-oxidized difference spectra (absorption maximum at 600 nm for cytochrome caa3) and CO-reduced-versus-reduced difference spectra (absorption maxima at 574 and 414 nm for cytochrome o). All three terminal oxidases were solubilized from everted membranes and partially purified. The difference spectra of the solubilized, partially purified cytochrome caa3 and cytochrome o complexes were consistent with these assignments. Cytochrome d, which has not been identified in a Bacillus species before, was tentatively assigned on the basis of its absorption maxima at 622 and 630 nm in reduced-versus-oxidized and CO-reduced-versus-reduced difference spectra, respectively, resembling the maxima exhibited by the complex found in Escherichia coli. The B. firmus OF4 cytochrome d was reducible by NADH but not by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine in everted membrane vesicles. Cytochrome d was expressed under two conditions: in cells growing exponentially at pH 7.5 (but not at pH 10.5) and in cells stationary phase at either pH 7.5 or 10.5. Protein immunoblots with antibodies against subunit I of the E. coli cytochrome d complex reacted only with membrane vesicles that contained spectrally identifiable cytochrome d. Additional evidence that this B. firmus OF4 cytochrome is related to the E. coli complex was obtained with a solubilized, partially purified fraction of cytochrome d that also reacted with antibodies against the subunits of the E. coli cytochrome d.     

Transfer of Tn925 and plasmids between Bacillus subtilis and alkaliphilic Bacillus firmus OF4 during Tn925-mediated conjugation.

Conjugative transposon Tn925 was transferred to alkaliphilic Bacillus firmus OF4 during mating experiments, as monitored by the acquisition of tetracycline resistance at pH 7.5 and confirmed by Southern analysis of chromosomal DNA from transconjugants. Tetracycline resistance could not be demonstrated at pH 10.5, but transconjugants retained resistance upon growth at pH 7.5 after having grown for several generations at pH 10. When the Bacillus subtilis donor strain contained plasmids, either pUB110 or pTV1, in addition to Tn925, transfer of the plasmid to the alkaliphile occurred during conjugation, either together with or independently of the transfer of the transposon. The plasmids were stable in B. firmus OF4, expressing their resistance markers for kanamycin or chloramphenicol at pH 7.5 after growth of the transformants at high pH. Transconjugant B. firmus OF4, which carried Tn925, could serve as the donor in mating experiments with B. subtilis lacking the transposon. These studies establish a basis for initiation of genetic studies in this alkaliphilic Bacillus species, including the introduction of cloned genes and the use of transposon-mediated insertional mutagenesis.     

An essential role for a membrane lipid in cytokinesis. Regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine

Phosphatidylethanolamine (PE) is a major membrane phospholipid that is mainly localized in the inner leaflet of the plasma membrane. We previously demonstrated that PE was exposed on the cell surface of the cleavage furrow during cytokinesis. Immobilization of cell surface PE by a PE-binding peptide inhibited disassembly of the contractile ring components, including myosin II and radixin, resulting in formation of a long cytoplasmic bridge between the daughter cells. This blockade of contractile ring disassembly was reversed by removal of the surface-bound peptide, suggesting that the PE exposure plays a crucial role in cytokinesis. To further examine the role of PE in cytokinesis, we established a mutant cell line with a specific decrease in the cellular PE level. On the culture condition in which the cell surface PE level was significantly reduced, the mutant ceased cell growth in cytokinesis, and the contractile ring remained in the cleavage furrow. Addition of PE or ethanolamine, a precursor of PE synthesis, restored the cell surface PE on the cleavage furrow and normal cytokinesis. These findings provide the first evidence that PE is required for completion of cytokinesis in mammalian cells, and suggest that redistribution of PE on the cleavage furrow may contribute to regulation of contractile ring disassembly.     

Mutation of an inosine-producing strain of Bacillus subtilis to DL-methionine sulfoxide resistance for guanosine production.

An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide. Among the mutants derived, guanosine producers were observed frequently. The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar. Inosine production decreased concomitantly. When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine. In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain. It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine.     

Mutation of an inosine-producing strain of Bacillus subtilis to DL-methionine sulfoxide resistance for guanosine production.

An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide. Among the mutants derived, guanosine producers were observed frequently. The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar. Inosine production decreased concomitantly. When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine. In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain. It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine.     

Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lipid A mutants.

Polymyxin-resistant pmrA mutants of Salmonella typhimurium differed from their parents in that they were resistant to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-lysozyme, tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-deoxycholate, and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-bacitracin. Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate released about 50% less lipopolysaccharide from the pmrA strains than from the parental strains when the bacteria were grown in L-broth containing 2 mM Ca2+. Protamine, polylysine, octapeptin, benzalkonium chloride, cold NaCl, cold MgCl2, or cold tris(hydroxymethyl)aminomethane hydrochloride (pH 7.2) caused no leakage or markedly less leakage of periplasmic beta-lactamase from a pmrA mutant than from its parent strain. pmrA mutants were more resistant than their parent strains to protamine and polylysine but not to octapeptin or benzalkonium chloride, as measured by the ability of these agents to kill the bacteria or to sensitize them to deoxycholate-induced lysis. The pmrA strains did not differ from their parent strains in sensitivity to several antibiotics, in porin function (as measured by cephaloridine diffusion across the outer membrane), or in outer membrane-associated phospholipase A activity.     

The cadC gene product of alkaliphilic Bacillus firmus OF4 partially restores Na+ resistance to an Escherichia coli strain lacking an Na+/H+ antiporter (NhaA).

A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes that are apparent homologs of the cadA and cadC genes of cadmium resistance-conferring plasmid pI258 of Staphylococcus aureus. E. coli NM81 transformed with pJB22 had enhanced membrane Na+/H+ antiporter activity that was cold labile and that decreased very rapidly following isolation of everted vesicles. Subclones of pJB22 containing cadC as the only intact gene showed identical complementation patterns in vivo and in vitro. The cadC gene product of S. aureus has been proposed to act as an accessory protein for the Cd2+ efflux ATPase (CadA) (K. P. Yoon and S. Silver, J. Bacteriol. 173:7636-7642, 1991); perhaps the alkaliphile CadC also binds Na+ and enhances antiporter activity by delivering a substrate to an integral membrane antiporter. A 6.0-kb fragment overlapping the pJB22 insert was isolated to complete the sequence of the cadA homolog. A partial sequence of a region approximately 2 kb downstream of the cadA locus shares sequence similarity with plasmids from several gram-positive bacteria. These results suggest that the region of alkaliphile DNA containing the cadCA locus is present on a transposon that could reside on a heretofore-undetected endogenous plasmid.     

Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a delta cyo delta cyd strain complemented by genes from Bacillus firmus OF4.

Escherichia coli GK100, with deletions in the operons encoding its two terminal oxidases, cytochrome bo and ctyochrome bd, was complemented for growth on succinate by a recombinant plasmid (pMS100) containing a 3.4-kb region of DNA from alkaliphilic Bacillus firmus OF4. The complementing DNA was predicted to encode five proteins, but neither sequence analysis nor complementation experiments with subclones provided insight into the basis for the complementation. Cytochrome difference spectra of everted membrane vesicles from the transformed strain had characteristics of a cytochrome bd spectrum but with features different from those observed for alkaliphile membranes. To determine the bacterial source and identity of the structural genes for the cytochrome bd in the transformed mutant, the complex was extracted and partially purified. On sodium dodecyl sulfate-polyacrylamide gels, two polypeptides were resolved from the preparation, 43 (subunit I) and 27 (subunit II) kDa. An internal peptide from subunit I was sequenced, and it yielded the same primary sequence as is found in positions 496 to 510 of E. coli appC. Consistent with the microsequencing results pMS100 failed to complement a triple mutant of E. coli carrying a deletion in appB as well as in the cyo and cyd loci. The deduced sequence of AppBC had been predicted to be very similar to the sequence of CydAB (J. Dassa et al., Mol. Gen. Genet. 229:341-352, 1991) but this is the first demonstration that the former is indeed a cytochrome bd terminal oxidase. The enzyme catalyzed oxygen uptake coupled to quinol or N,N,N',N'-tetramethyl-p-phenylenediamine oxidation, and the activity was sensitive to cyanide. No cross-reactivity to subunit-specific polyclonal antibodies directed against the two individual subunits of cyd-encoded cytochrome bd was detected. Since this is the second cytochrome bd discovered in E. coli, it is proposed that the two complexes be designated cytochrome bd-I (cydAB-encoded enzyme) and cytochrome bd-II (appBC-encoded enzyme). In addition, cbdAB is suggested as a more appropriate gene designation for cytochrome bd than either appBC or cyxAB.     

ATP-dependent cadmium transport by the cadA cadmium resistance determinant in everted membrane vesicles of Bacillus subtilis.

Resistance to cadmium conferred by the staphylococcal plasmid pI258 occurs by means of energy-dependent efflux, resulting in decreased intracellular accumulation of cadmium. Recent sequence information suggested that efflux is mediated by a P-type ATPase. The cadA gene was previously expressed in Bacillus subtilis, conferring resistance to cadmium. Everted membrane vesicles were prepared from B. subtilis cells harboring either a plasmid containing the cadA system or the vector plasmid alone. 109Cd2+ transport into the everted membranes was measured in the presence of various energy sources. Cadmium transport was detected only in the presence of ATP as an energy source. The production of an electrochemical proton gradient (delta mu H+) by using NADH or phenazine methosulfate plus ascorbate was not able to drive transport. Reagents which dissipate delta pH abolished calcium transport due to the Ca2+/H+ antiporter but only partially inhibited cadmium transport. Inhibition of transport by the antibiotic bafilomycin A1 occurred at concentrations comparable to those which inhibit P-type ATPases. A band corresponding to the cadA gene product was identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antibodies to the protein were prepared.     

Inactive form of edeine in the edeine-producing Bacillus brevis Vm 4 cells.

1. Exogenous edeine inhibits the synthesis of DNA and protein, but not that of RNA, in extracts of edeine-producing Bacillus brevis Vm 4 cells. This is analogous to the effect of edeine on extracts obtained from edeine-sensitive cells. 2. Producer cells, in contrast to sensitive ones, are not permeable to exogenous edeine. DNA synthesis in producer cells rendered permeable by toluene treatment becomes sensitive to edeine. 3. No free edeine could be detected in post-log producer cells during maximal synthesis of edeine. Nascent edeine exists in the cell in a biologically inactive form, bound to a fast-sedimenting fraction. Edeine B, identical to the antibiotic present in the medium, is released from this fraction by mild treatment with alkali.