kujimycin-b and lankacidins

We extensively analyzed the giant linear plasmid pSLA2-L in Streptomyces rochei 7434AN4, a producer of two structurally unrelated polyketide antibiotics, lankacidin and lankamycin. It was found that amine oxidase LkcE oxidizes an acyclic amine to an imine, which is in turn converted to the 17-membered carbocyclic lankacidin. Heterologous expression and translational fusion experiments indicated the modular-iterative mixed polyketide biosynthesis of lankacidin. Concerning to lankamycin biosynthesis, starter unit biosynthesis and the post-PKS modification pathway were elucidated by feeding and gene inactivation experiments. It was shown that pSLA2-L contains many regulatory genes, which constitute the signaling molecule/receptor system for antibiotic production and morphological differentiation in this strain. Two signaling molecules, SRB1 and SRB2, that induce production of lankacidin and lankamycin were further isolated and their structures were elucidated. Each contains a 2,3-disubstituted butenolide skeleton, and the stereochemistry at C-1' position is crucial for inducing activity.

Topics: Anti-Bacterial Agents; Erythromycin; Macrolides; Multigene Family; Plasmids; Streptomyces

Topics: 4-Butyrolactone; Anti-Bacterial Agents; Bacterial Proteins; Biosynthetic Pathways; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Erythromycin; Genes, Bacterial; Macrolides; Magnetic Resonance Spectroscopy; Molecular Structure; Mutation; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Streptomyces

New signaling molecules that induce lankacidin and lankamycin production in Streptomyces rochei were extracted from the culture filtrate and purified by Sephadex LH20 and silica gel chromatography with the help of bioassay. Chiral HPLC and ESI-MS analyses indicated the presence of two active components--SRB1 and SRB2--and their molecular formulas were established to be C15H24O5 and C16H26O5, respectively. By extensive NMR analysis, SRB1 and SRB2 were determined to be 2-(1'-hydroxy-6'-oxo-8'-methylnonyl)-3-methyl-4-hydroxybut-2-en-1,4-olide and 2-(1'-hydroxy-6'-oxo-8'-methyldecyl)-3-methyl-4-hydroxybut-2-en-1,4-olide, respectively. These structures were finally confirmed by chemical synthesis and the absolute configuration at C-1' was determined to be R in each case. The synthetic 1'R isomers induced production of lankacidin and lankamycin at around 40 nM concentrations. SRB1 and SRB2 are therefore distinct from the well-known 2,3-disubstituted γ-butyrolactone molecules such as A-factor, virginia butanolide, and SCB1 and and belong, like avenolide, recently isolated from Streptomyces avermitilis, to the γ-butenolide family.

Topics: 4-Butyrolactone; Erythromycin; Isomerism; Macrolides; Magnetic Resonance Spectroscopy; Molecular Conformation; Streptomyces

The structures of the large ribosomal subunit of Deinococcus radiodurans (D50S) in complex with the antibiotic lankamycin (3.2 Å) and a double antibiotic complex of lankamycin and lankacidin C (3.45 Å) have been determined, in continuation of previous crystallographic studies on lankacidin-D50S complex. These two drugs have been previously reported to inhibit ribosomal function with mild synergistic effect. Lankamycin, a member of the macrolide family, binds in a similar manner to erythromycin. However, when in complex with lankacidin, lankamycin is located so that it can form interactions with lankacidin in the adjacent ribosomal binding site. When compared to the well-documented synergistic antibiotics, Streptogramins A and B, the pair of lankacidin and lankamycin bind in similar sites, the peptidyl transferase center and nascent peptide exit tunnel, respectively. Herein, we discuss the structural basis for antibiotic synergism and highlight the key factors involved in ribosomal inhibition.

Topics: Anti-Bacterial Agents; Binding Sites; Crystallography; DNA Footprinting; Drug Synergism; Erythromycin; Inhibitory Concentration 50; Macrolides; Models, Molecular; Molecular Structure; Ribosome Subunits, Large; RNA, Ribosomal, 23S; X-Ray Diffraction

Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.

Topics: Anti-Bacterial Agents; Binding Sites; Crystallography, X-Ray; Deinococcus; Drug Discovery; Drug Synergism; Erythromycin; Macrolides; Models, Molecular; Molecular Structure; Ribosome Subunits, Large, Bacterial; Ribosomes

Our previous studies revealed that the srrX and srrA genes carried on the large linear plasmid pSLA2-L constitute a gamma-butyrolactone-receptor system in Streptomyces rochei. Extensive transcriptional analysis has now showed that the Streptomyces antibiotic regulatory protein gene srrY, which is also carried on pSLA2-L, is a target of the receptor/repressor SrrA and plays a central role in lankacidin and lankamycin production. The srrY gene was expressed in a growth-dependent manner, slightly preceding antibiotic production. The expression of srrY was undetectable in the srrX mutant but was restored in the srrX srrA double mutant. In addition, SrrA was bound specifically to the promoter region of srrY, and this binding was prevented by the addition of the S. rochei gamma-butyrolactone fraction, while the W119A mutant receptor SrrA was kept bound even in the presence of S. rochei gamma-butyrolactone. Furthermore, the introduction of an intact srrY gene under the control of a foreign promoter into the srrX or srrA(W119A) mutant restored antibiotic production. All of these results confirmed the signaling pathway from srrX through srrA to srrY, leading to lankacidin and lankamycin production.

Topics: 4-Butyrolactone; Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; DNA Fingerprinting; Electrophoretic Mobility Shift Assay; Erythromycin; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Macrolides; Mutagenesis; Mutation; Plasmids; Polymerase Chain Reaction; Protein Binding; Streptomyces

An afsA homologue (srrX) and three gamma-butyrolactone receptor gene homologues (srrA, srrB and srrC) are coded on the giant linear plasmid pSLA2-L in Streptomyces rochei 7434AN4, a producer of two polyketide antibiotics, lankacidin and lankamycin. Construction of gene disruptants and their phenotypic study revealed that srrX and srrA make a gamma-butyrolactone receptor system in this strain. Addition of a gamma-butyrolactone fraction to an srrX-deficient mutant restored the production of lankacidin and lankamycin, indicating that the SrrX protein is not necessary for this event. In addition to a positive effect on antibiotic production, srrX showed a negative effect on morphological differentiation. The receptor gene srrA reversed both effects of srrX, while the second receptor gene homologue srrC had only a positive function in spore formation. Furthermore, disruption of the third homologue srrB greatly increased the production of lankacidin and lankamycin. Electron microscopic analysis showed that aerial mycelium formation stopped at a different stage in the srrA and srrC mutants. Overall, these results indicated that srrX, srrA, srrB and srrC constitute a complex regulatory system for antibiotic production and morphological differentiation in S. rochei.

Topics: 4-Butyrolactone; Amino Acid Sequence; Antibiosis; Bacterial Proteins; Erythromycin; Gene Deletion; Gene Expression Regulation, Bacterial; Macrolides; Microscopy, Electron, Scanning; Molecular Sequence Data; Morphogenesis; Phylogeny; Plasmids; Receptors, GABA-A; Sequence Alignment; Streptomyces

The complete nucleotide sequence of the large linear plasmid pSLA2-L in Streptomyces rochei strain 7434AN4 has been determined. pSLA2-L was found to be 210 614 bp long with a GC content of 72.8% and carries 143 open reading frames. It is especially noteworthy that three-quarters of the pSLA2-L DNA is occupied by secondary metabolism-related genes, namely two type I polyketide synthase (PKS) gene clusters for lankacidin and lankamycin, a mithramycin synthase-like type II PKS gene cluster, a carotenoid biosynthetic gene cluster and many regulatory genes. In particular, the lankacidin PKS is unique, because it may be a mixture of modular- and iterative-type PKSs and carries a fusion protein of non-ribosomal peptide synthetase and PKS. It is also interesting that all the homologues of the afsA, arpA, adpA and strR genes in the A-factor regulatory cascade in Streptomyces griseus were found on pSLA2-L, and disruption of the afsA homologue caused non-production of both lankacidin and lankamycin. These results, together with the finding of three possible replication origins at 50-63 kb from the right end, suggest that the present form of pSLA2-L might have been generated by a series of insertions of the biosynthetic gene clusters into the left side of the original plasmid.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Bacterial Proteins; Erythromycin; Gene Deletion; Gene Expression Regulation, Bacterial; Helix-Turn-Helix Motifs; Macrolides; Molecular Sequence Data; Multigene Family; Plasmids; Plicamycin; Streptomyces

The 200kb linear plasmid pSLA2-L was suggested to be involved in the production of two macrolide antibiotics, lankamycin (Lm) and lankacidin (Lc), in Streptomyces rochei 7434AN4. Hybridization experiments with the polyketide synthase (PKS) genes for erythromycin and actinorhodin identified two eryAI-homologous regions and an actI-homologous region on pSLA2-L. The nucleotide sequence of a 3.6kb SacI fragment carrying one of the eryAI-homologs revealed that it codes for part of a large protein with four domains for ketoreductase, acyl carrier protein, ketosynthase, and acyltransferase. Gene disruption confirmed that the two eryAI-homologs are parts of a large type-I PKS gene cluster for Lm. A 4.8kb DNA carrying the actI-homologous region contains four open reading frames (ORF1-ORF4) as well as an additional ORF, i.e. ORF5, which might code for a thioesterase. Deletion of the ORF2-ORF4 region showed that it is not involved in the synthesis of Lm or Lc. Thus, it was confirmed that pSLA2-L contains two PKS gene clusters for Lm and an unknown type-II polyketide.

Topics: Amino Acid Sequence; Anthraquinones; Anti-Bacterial Agents; DNA, Bacterial; Erythromycin; Genes, Bacterial; Macrolides; Molecular Sequence Data; Multienzyme Complexes; Multigene Family; Mutagenesis; Plasmids; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Streptomyces

Gel-filtration analysis of a mixture of cyclodextrin (CyD) and lankacidin C showed that beta-CyD had strong, gamma-CyD weak and alpha-CyD no affinity for lankacidin C. Lankacidin C production activity, which was assayed by measuring the incorporation of L-[methyl-14C-]methionine into the lankacidin molecule, was the greatest with cells grown in the presence of beta-CyD, less with gamma-CyD and the least with alpha-CyD. Lankamycin and T-2636M, which are by-products in lankacidin C fermentation, were not included by beta-CyD and their production was not stimulated by beta-CyD. It was apparent that the stimulatory effect of CyD was closely related to the formation of an inclusion complex between CyD and the antibiotic. Lankacidin C biosynthesis was repressed by preincubating cells with lankacidin C, while the repressive effect of lankacidin C was abrogated by the inclusion by beta-CyD. Thus, abrogation of feed-back repression seems to be a main mechanism of the effect of CyD. However, alpha-CyD, which had no affinity for lankacidin C, stimulated the production to the least extent and exhibited a complementary effect on the stimulation by beta-CyD or gamma-CyD, alpha-CyD also caused a change in cell morphology and cell-surface hydrophobicity. It was assumed that the modification of the cell surface is a secondary mechanism of the effect of CyD.

Topics: Anti-Bacterial Agents; Cell Membrane; Chromatography, Gel; Cyclodextrins; Dextrins; Erythromycin; Feedback; Fermentation; Macrolides; Starch; Streptomyces