CouO and NovO: C-methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and novobiocin antibiotic biosynthesis

During the biosynthesis of the streptomycete aminocoumarin antibiotics novobiocin and the dimeric coumermycin A(1), the bicyclic coumarin scaffold is C-methylated adjacent to the phenolic oxygen. The SAM-dependent C-methyltransferases NovO and CouO have been heterologously expressed and purified from Escherichia coli and shown to act after the aminocoumarin ring has been constructed by prior action of Nov/CouHIJK. Neither C-methyltransferase works on the tyrosyl-derived S-pantetheinyl intermediates tethered to NovH or on the subsequently released free aminocoumarin. NovL ligates the aminocoumarin to prenylhydroxybenzoate to yield novobiocic acid, which is the substrate for NovO before it is O-glycosylated by NovM. In coumermycin assembly, the corresponding ligase CouL makes the bis-amide by tandem ligation of two aminocoumarins to a dicarboxypyrrole. CouO works on both the mono- and bis-amides for mono- and di-C-methylation adjacent to the phenolic hydroxyl before it is glycosylated by CouM. Thus, the specific timing of C-methylation in the aminocoumarin antibiotic pathways is established.     

Characterization of the aminocoumarin ligase SimL from the simocyclinone pathway and tandem incubation with NovM,P,N from the novobiocin pathway

Simocyclinone D(8) consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A(1), the phenolic hydroxyl group of the aminocoumarin in simocyclinone is not glycosylated with a decorated noviosyl moiety that is the pharmacophore for targeting bacterial DNA gyrase. We have expressed the Streptomyces antibioticus simocyclinone ligase SimL, purified it from Escherichia coli, and established its ATP-dependent amide bond forming activity with a variety of polyenoic acids including retinoic acid and fumagillin. We have then used the last three enzymes from the novobiocin pathway, NovM, NovP, and NovN, to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetraenoate moiety, to evaluate antibacterial activity.     

An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489.

The aminocoumarin antibiotic coumermycin A1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL, encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalysed the formation of both amide bonds of coumermycin A1, i.e. between the central 3-methylpyrrole-2,4-dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer. The activity was strictly dependent on the presence of ATP and Mn2+ or Mg2+. The apparent Km values were determined as 26 micro m for the 3-methylpyrrole-2,4-dicarboxylic acid and 44 micro m for the aminocoumarin moiety, respectively. Several analogues of the pyrrole dicarboxylic acid were accepted as substrates. In contrast, pyridine carboxylic acids were not accepted. 3-Dimethylallyl-4-hydroxybenzoic acid, the acyl component in novobiocin biosynthesis, was well accepted, despite its structural difference from the genuine acyl substrate of CouL.     

Chemoenzymatic formation of novel aminocoumarin antibiotics by the enzymes CouN1 and CouN7

The aminocoumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 are highly potent inhibitors of the bacterial type II topoisomerase DNA gyrase. The key pharmacophore of both clorobiocin and coumermycin A1, the 5-methyl-2-pyrrolylcarbonyl moiety, targets the ATP-binding site of GyrB. The 5-methyl-2-pyrrolylcarbonyl group is transferred by the acyltransferases Clo/CouN7 from the carrier proteins Clo/CouN1 to the 3'-hydroxyl of the l-noviosyl scaffold during the late steps of clorobiocin and coumermycin A1 biosynthesis. We first examined the substrate specificity of the purified thiolation domain protein CouN1 in becoming primed by the phosphopantetheinyltransferase Sfp using a variety of synthetic CoA analogues of the 5-methyl-2-pyrrolylcarbonyl moiety. The acyl-S-CouN1 thioesters were then assayed as donors to the 3'-OH group of descarbamoylnovobiocin by the acyltransferase CouN7, resulting in 21 novel variants with heterocyclic acyl groups installed on the noviosyl moiety of the aminocoumarin scaffold. Scaleup of a 5-methylthiophene derivative yielded a compound with activity against both Gram-negative and Gram-positive bacteria. The minimal inhibitory concentration found for the Gram-positive bacteria was comparable to that of novobiocin.     

Cloning and analysis of the simocyclinone biosynthetic gene cluster of <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> Tü 6040

The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.     

Initial characterization of novobiocic acid noviosyl transferase activity of NovM in biosynthesis of the antibiotic novobiocin

The aminocoumarin class of antibiotics, exemplified by novobiocin, is composed of tripartite l-noviosylaminocoumarin prenylbenzoate natural products. The decorated noviosyl sugar component interacts with the target bacterial enzyme DNA gyrase. We have subcloned the putative 40 kDa l-noviosyl transferase from Streptomyces spheroides into Escherichia coli, expressed it in soluble form, and purified it to homogeneity as a C-terminal His(8) fusion protein. The aglycone novobiocic acid, obtained from selective degradation of novobiocin, and TDP-l-noviose, obtained by an 11-step chemical synthesis from l-rhamnose, were shown to be robust substrates for NovM to produce the desmethyldescarbamoyl novobiocin intermediate with a k(cat) of >300 min(-1). NovM displays activity with variant coumarin aglycones, suggesting it may be a promiscuous catalyst for noviosylation of a range of planar scaffolds. Conversely, NovM shows no activity with and is inhibited by TDP-l-rhamnose (K(i) = 83.5 +/- 5.5 microM), the sugar donor that most closely structurally resembles the natural substrate TDP-l-noviose. The NovM reaction products generated during the course of this work will serve as substrates for subsequent analysis of the NovP and NovN tailoring enzymes that impart the noviose decorations required for DNA gyrase binding and antibiotic activity.     

Novobiocin biosynthesis: inactivation of the putative regulatory gene<Emphasis Type="Italic"> novE</Emphasis> and heterologous expression of genes involved in aminocoumarin ring formation

The left ends of the biosynthetic gene clusters of novobiocin ( nov), clorobiocin ( clo) and coumermycin A(1) ( cou) from Streptomyces spheroides (syn. S. caeruleus) NCIMB 11891, S. roseochromogenes var. oscitans DS 12.976 and S. rishiriensis DSM 40489 were cloned and sequenced. Sequence comparison suggested that novE, cloE and couE, respectively, represent the borders of these three clusters. Inactivation of novE proved that novE does not have an essential catalytic role in novobiocin biosynthesis, but is likely to have a regulatory function. The gene products of novF and cloF show sequence similarity to prephenate dehydrogenase and may produce 4-hydroxyphenylpyruvate (4HPP) as a precursor of the substituted benzoate moiety of novobiocin and clorobiocin. Coumermycin A(1) does not contain this benzoate moiety, and correspondingly the coumermycin cluster was found not to contain a functional novF homologue. The coumermycin biosynthetic gene cluster apparently evolved from an ancestral cluster similar to those of novobiocin and clorobiocin, and parts of the ancestral novF homologue have been deleted in this process. No homologue to novC was identified in the gene clusters of clorobiocin and coumermycin, questioning the postulated involvement of novC in aminocoumarin biosynthesis. Heterologous expression of novDEFGHIJK in Streptomyces lividans resulted in the formation of 2,4-dihydroxy-alpha-oxy-phenylacetic acid, suggesting that at least one of the proteins encoded by these genes may participate in a hydroxylation reaction.     

Mass spectrometric pathway monitoring of secondary metabolites: systematic analysis of culture extracts of Streptomyces species

Streptomyces spheroides, Streptomyces rishiriensis, and Streptomyces roseochromogenes are producers of the aminocoumarin-type antibiotics novobiocin, coumermycin A(1), and clorobiocin, respectively, all of which are bacterial gyrase inhibitors. In an attempt to develop a general analytical method for pathway monitoring of secondary metabolites from culture extracts of these strains, we used superior mass spectrometric methods. The aim was to develop and apply a technique for the rapid analysis of Streptomyces culture extracts with respect to those substances, thereby providing a method for screening extracts of genetically modified strains for new pharmaceutically active antibiotics with improved pharmacological effects. The combination of full scan mass spectrometry (MS), parent ion scan MS, product ion scan MS, and in-source collision-induced fragmentation prior to product ion scans (pseudo-MS(3) scan), using characteristic fragmentation of the central aminocoumarin unit, was employed for the detection and structural interpretation of expected and new intermediates. We were able to show the applicability of this methodology to the three culture extracts, where the main intermediates could be found, and to demonstrate its use for interpretation of secondary metabolite biosynthesis. Some new compounds were discovered, including bis-carbamoylated novobiocin, hydroxylated clorobiocin, and several structurally and not yet fully elucidated coumermycin derivatives or precursors.     

Genetic engineering of antibiotic biosynthesis for the generation of new aminocoumarins

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A₁ are inhibitors of gyrase and highly effective antibacterial agents. Their biosynthetic gene clusters have been cloned from the respective Streptomyces producer strains, and the function of nearly all genes contained therein has been elucidated by genetic and biochemical methods. Efficient methods have been developed for the genetic manipulation and the heterologous expression of the clusters, and more than 100 new derivatives of these antibiotics have been generated by metabolic engineering, mutasynthesis and chemoenzymatic synthesis, providing a model for the power of genetic and genomic methods for the generation of new bioactive compounds.     

Installation of the pyrrolyl-2-carboxyl pharmacophore by CouN1 and CouN7 in the late biosynthetic steps of the aminocoumarin antibiotics clorobiocin and coumermycin A1

The 5-methyl-2-pyrrolylcarbonyl moiety of the aminocoumarin antibiotics clorobiocin and coumermycin A1 is the key pharmacophore for targeting the ATP-binding site of GyrB for inhibition of the bacterial type-II topoisomerase DNA gyrase. During the late stage of clorobiocin and coumermycin A1 biosynthesis, the pyrrolyl-2-carboxyl group is transferred from the peptidyl carrier proteins Clo/CouN1 to the 3'-hydroxyl of the 4-methoxy-L-noviosyl scaffold by the action of the acyltransferases Clo/CouN7. CouN1 and CouN7 have now been heterologously expressed and purified from Escherichia coli. The apo form of CouN1 is converted to the acyl-holo form by loading with pyrrolyl-2-carboxyl-S-pantetheinyl moieties from synthetic pyrrolyl- and 5-methylpyrrolyl-CoAs by the action of the phosphopantetheinyl transferase Sfp. CouN7 acts as an acyltransferase, moving the pyrrole acyl moieties from CouN1 to the noviose sugar of descarbamoylnovobiocin. When the 5-methylpyrrolyl-2-carboxyl-thioester of CouN1 is the cosubstrate, the in vitro product differs from clorobiocin only in a CH3 for Cl group change on the coumarin ring. Double transfer of this acyl moiety by CouN7 to the penultimate intermediate in coumermycin A1 assembly completes that antibiotic biosynthetic pathway.     

Genetic analysis of the biosynthesis of the pyrrole and carbamoyl moieties of coumermycin A1 and novobiocin

The aminocoumarin antibiotic coumermycin A(1) contains a central and two terminal pyrrole moieties. The coumermycin gene cluster in Streptomyces rishiriensis contains three genes (couN3, couN4 and couN5) that show sequence similarity to genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens and of undecylprodiginine in S. coelicolor. The gene couN3, which codes for a putative L-prolyl-S-PCP dehydrogenase, and the gene couN4, which encodes a putative L-prolyl-AMP ligase, were disrupted using in-frame deletion and insertional inactivation, respectively. HPLC analysis of culture extracts showed that formation of the two terminal pyrrole moieties was abolished in the couN3 (-) und couN4 (-) mutants. The mutants accumulated coumermycin D, which contains only the central pyrrole moiety. This result not only confirmed the involvement of couN3 and couN4 in the biosynthesis of the terminal pyrrole-2-carboxylic acid moieties of coumermycin A(1), but also indicated, for the first time, that the central 3-methylpyrrole-2,4-dicarboxylic acid unit of the coumermycins is formed by a biosynthetic pathway that differs from that used to assemble the terminal pyrrole moieties. novN, a putative carbamoyl transferase gene from the gene cluster for novobiocin biosynthesis in S. spheroides was expressed in the couN3 (-) mutant. This led to the formation of bis-carbamoylated coumermycin D, a novel compound of the coumermycin series.     

DNA gyrase activities from Rhodobacter capsulatus: analysis of target(s) of coumarins and cloning of the gyrB locus

Bacterial DNA gyrase is composed of two subunits, gyrase A and B, and is responsible for negatively supercoiling DNA in an ATP-dependent manner. The coumarin antibiotics novobiocin and coumermycin are known inhibitors of bacterial DNA gyrase in vivo and in vitro. We have cloned, mapped, and partially sequenced Rhodobacter capsulatus gyrB which encodes the gyrase B subunit that is presumably involved in binding to coumarins. DNA gyrase activities from crude extracts of R. capsulatus were detected and it was shown that the R. capsulatus activity is (1) inhibited by novobiocin and coumermycin, (2) ATP-dependent and, (3) present in highly aerated and anaerobically grown cells. We previously observed that when R. capsulatus coumermycin-resistant strains are continuously recultured on media containing coumermycin they sometimes acquired mutations in hel genes (i.e., cytochromes c biogenesis mutations). We discuss the possibility that coumarins may inhibit cytochromes c biogenesis as a second target in R. capsulatus via hel (i.e., a putative ATP-dependent heme exporter).     

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors,clorobiocin

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N-terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3′ position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24-kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5′-adenylyl-β,γ-imidodiphosphate, and with information on the bound conformation of novobiocin in the p24-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented. © 1997 Wiley-Liss Inc.     

Overexpression,purification and characterization of SimL,an amide synthetase involved in simocyclinone biosynthesis

Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tü 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.     

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.     

S-Adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces

The production of antibiotics in different Streptomyces strains has been reported to be stimulated by the external addition of S-adenosylmethionine (SAM) and by overexpression of the SAM synthetase gene metK. We investigated the influence of SAM addition, and of the expression of SAM biosynthetic genes, on the production of the aminocoumarin antibiotic novobiocin in the heterologous producer strain Streptomyces coelicolor M512 (nov-BG1). External addition of SAM did not influence novobiocin accumulation. However, overexpression of a SAM synthase gene stimulated novobiocin formation, concomitant with an increase of the intracellular SAM concentration. Streptomyces genomes contain orthologs of all genes required for the SAM cycle known from mammals. In contrast, most other bacteria use a different cycle for SAM regeneration. Three secondary metabolic gene clusters, coding for the biosynthesis of structurally very different antibiotics in different Streptomyces strains, were found to contain an operon comprising all five putative genes of the SAM cycle. We cloned one of these operons into an expression plasmid, under control of a strong constitutive promoter. However, transformation of the heterologous novobiocin producer strain with this plasmid did not stimulate novobiocin production, but rather showed a detrimental effect on cell viability in the stationary phase and strongly reduced novobiocin accumulation.     

Characterization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis

The aminocoumarin antibiotics clorobiocin and coumermycin A(1) target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-carboxy ester moiety in the ATP-binding site of the enzyme. The pyrrolyl pharmacophore is derived by a four electron oxidation of a prolyl unit while tethered in phosphopantetheinyl thioester linkage to a peptidyl carrier protein (PCP) subunit. l-Proline is selected and activated as l-prolyl-AMP by adenylation domain enzymes (CloN4 and CouN4) and then installed as the thioester on the holo form of the PCP proteins CloN5 and CouN5. Enzymatic oxidation of the prolyl-S-PCP by the flavoprotein dehydrogenase CloN3 can be followed by rapid quench and subsequent electrospray ionization-Fourier transform mass spectrometry analysis of the acyl-S-protein substrate/product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product.     

Role of MbtH-like proteins in the adenylation of tyrosine during aminocoumarin and vancomycin biosynthesis

MbtH-like proteins consist of ～70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes.     

A Forward Chemical Screen Identifies Antibiotic Adjuvants in Escherichia coli

Multi-drug-resistant infections caused by Gram-negative pathogens are rapidly increasing, highlighting the need for new chemotherapies. Unlike Gram-positive bacteria, where many different chemical classes of antibiotics show efficacy, Gram-negatives are intrinsically insensitive to many antimicrobials including the macrolides, rifamycins, and aminocoumarins, despite intracellular targets that are susceptible to these drugs. The basis for this insensitivity is the presence of the impermeant outer membrane of Gram-negative bacteria in addition to the expression of pumps and porins that reduce intracellular concentrations of many molecules. Compounds that sensitize Gram-negative cells to "Gram-positive antibiotics", antibiotic adjuvants, offer an orthogonal approach to addressing the crisis of multi-drug-resistant Gram-negative pathogens. We performed a forward chemical genetic screen of 30,000 small molecules designed to identify such antibiotic adjuvants of the aminocoumarin antibiotic novobiocin in Escherichia coli. Four compounds from this screen were shown to be synergistic with novobiocin including inhibitors of the bacterial cytoskeleton protein MreB, cell wall biosynthesis enzymes, and DNA synthesis. All of these molecules were associated with altered cell shape and small molecule permeability, suggesting a unifying mechanism for these antibiotic adjuvants. The potential exists to expand this approach as a means to develop novel combination therapies for the treatment of infections caused by Gram-negative pathogens.     

In Vitro Activity of Coumermycin A1

The in vitro activity of coumermycin A(1) was compared with that of novobiocin, ampicillin, and minocycline. Coumermycin was found to be the most active antibiotic of the four against Staphylococcus aureus. It was about 50 times more active than novobiocin or minocycline against the strains tested. Coumermycin also showed good activity against group A streptococci and pneumococci, moderate activity against Escherichia coli, indole-positive Proteus species, and Pseudomonas aeruginosa, and poor activity against Klebsiella-Enterobacter and enterococci. Against P. mirabilis, however, coumermycin activity was almost equal to that of ampicillin. The new antibiotic was further found to be greatly reduced in activity in the presence of plasma, but its minimal inhibitory concentration was not greatly affected by inoculum size. Coumermycin was found to be bacteriostatic in its action, and resistance to it developed slowly. Also, cross-resistance was present with novobiocin but absent with ampicillin or minocycline.