Mitomycin resistance in Streptomyces lavendulae includes a novel drug-binding-protein-dependent export system

Sequence analysis of Streptomyces lavendulae NRRL 2564 chromosomal DNA adjacent to the mitomycin resistance locus mrd (encoding a previously described mitomycin-binding protein [P. Sheldon, D. A. Johnson, P. R. August, H.-W. Liu, and D. H. Sherman, J. Bacteriol. 179:1796-1804, 1997]) revealed a putative mitomycin C (MC) transport gene (mct) encoding a hydrophobic polypeptide that has significant amino acid sequence similarity with several actinomycete antibiotic export proteins. Disruption of mct by insertional inactivation resulted in an S. lavendulae mutant strain that was considerably more sensitive to MC. Expression of mct in Escherichia coli conferred a fivefold increase in cellular resistance to MC, led to the synthesis of a membrane-associated protein, and correlated with reduced intracellular accumulation of the drug. Coexpression of mct and mrd in E. coli resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. Taken together, these data provide evidence that MRD and Mct function as components of a novel drug export system specific to the mitomycins.     

Identification of AHBA Biosynthetic Genes Related to Geldanamycin Biosynthesis in Streptomyces hygroscopicus 17997

To clone and study the geldanamycin biosynthetic gene cluster in Streptomyces hygroscopicus 17997, we designed degenerate primers based on the conserved sequence of the ansamycin 3-amino-5-hydroxybenzoic acid (AHBA) synthase gene. A 755-bp polymerase chain reaction product was obtained from S. hygroscopicus 17997 genomic DNA, which showed high similarity to ansamycin AHBA synthase genes. Through screening the cosmid library of S. hygroscopicus 17997, two loci of separated AHBA biosynthetic gene clusters were discovered. Comparisons of sequence homology and gene organization indicated that the two AHBA biosynthetic gene clusters could be divided into a benzenic and a naphthalenic subgroup. Gene disruption demonstrated that the benzenic AHBA gene cluster is involved in the biosynthesis of geldanamycin. However, the naphthalenic AHBA genes in the genome of Streptomyces hygroscopicus 17997 could not complement the deficiency of the benzenic AHBA genes. This is the first report on the AHBA biosynthetic gene cluster in a geldanamycin-producing strain. W. He and L. Wu contributed equally to this work.     

Insights into the biosynthesis of the benzoquinone ansamycins geldanamycin and herbimycin, obtained by gene sequencing and disruption

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.     

Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid, the starter unit of rifamycin biosynthesis in amycolatopsis Mediterranei S699

To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/d-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-d-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.     

Molecular basis of mitomycin C resistance in streptomyces: structure and function of the MRD protein

Mitomycin C (MC) is a potent anticancer agent. Streptomyces lavendulae, which produces MC, protects itself from the lethal effects of the drug by expressing several resistance proteins. One of them (MRD) binds MC and functions as a drug exporter. We report the crystal structure of MRD and its complex with an MC metabolite, 1,2-cis-1-hydroxy-2,7-diaminomitosene, at 1.5 A resolution. The drug is sandwiched by pi-stacking interactions of His-38 and Trp-108. MRD is a dimer. The betaalphabetabetabeta fold of the MRD molecule is reminiscent of methylmalonyl-CoA epimerase, bleomycin resistance proteins, glyoxalase I, and extradiol dioxygenases. The location of the binding site is identical to the ones in evolutionarily related enzymes, suggesting that the protein may have been recruited from a different metabolic pathway.     

Insights into the Biosynthesis of the Benzoquinone Ansamycins Geldanamycin and Herbimycin, Obtained by Gene Sequencing and Disruption

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.     

purU, a source of formate for purT-dependent phosphoribosyl-N-formylglycinamide synthesis.

A gene designated purU has been identified and characterized. purU is adjacent to tyrT at min 27.7 on the Escherichia coli chromosome. The gene codes for a 280-amino-acid protein. The C-terminal segment of PurU from residues 84 to 280 exhibits 27% identity with 5'-phosphoribosylglycinamide (GAR) transformylase, the product of purN. Primer extension mapping and assays of lacZ in a promoter probe vector identified two promoters giving mono- and bi-cistronic purU mRNA. Neither mRNA was regulated by purines. Mutations in either of two pairs of genes are required to block synthesis of 5'-phosphoribosyl-N-formylglycinamide (FGAR) from GAR: purN purT (purT encodes an alternative formate-dependent GAR transformylase) or purN purU. On the basis of the growth of purU, purN, and purU purN mutants, it appears that PurU provides the major source of formate for the purT-dependent synthesis of FGAR.     

PCR screening reveals considerable unexploited biosynthetic potential of ansamycins and a mysterious family of AHBA‐containing natural products in actinomycetes

Aims

Ansamycins are a family of macrolactams that are synthesized by type I polyketide synthase (PKS) using 3‐amino‐5‐hydroxybenzoic acid (AHBA) as the starter unit. Most members of the family have strong antimicrobial, antifungal, anticancer and/or antiviral activities. We aimed to discover new ansamycins and/or other AHBA‐containing natural products from actinobacteria.

Methods and Results

Through PCR screening of AHBA synthase gene, we identified 26 AHBA synthase gene–positive strains from 206 plant‐associated actinomycetes (five positives) and 688 marine‐derived actinomycetes (21 positives), representing a positive ratio of 2·4–3·1%. Twenty‐five ansamycins, including eight new compounds, were isolated from six AHBA synthase gene–positive strains through TLC‐guided fractionations followed by repeated column chromatography. To gain information about those potential ansamycin gene clusters whose products were unknown, seven strains with phylogenetically divergent AHBA synthase genes were subjected to fosmid library construction. Of the seven gene clusters we obtained, three show characteristics for typical ansamycin gene clusters, and other four, from Micromonospora spp., appear to lack the amide synthase gene, which is unusual for ansamycin biosynthesis. The gene composition of these four gene clusters suggests that they are involved in the biosynthesis of a new family of hybrid PK‐NRP compounds containing AHBA substructure.

Conclusions

PCR screening of AHBA synthase is an efficient approach to discover novel ansamycins and other AHBA‐containing natural products.

Significance and Impact of the Study

This work demonstrates that the AHBA‐based screening method is a useful approach for discovering novel ansamycins and other AHBA‐containing natural products from new microbial resources.     

Cloning and functional analysis of the naphthomycin biosynthetic gene cluster in Streptomyces sp. CS

Naphthomycins (NATs) are 29-membered naphthalenic ansamacrolactam antibiotics with antimicrobial and antineoplastic activities. Their biosynthesis starts from 3-amino-5-hydroxy-benzoic acid (AHBA). By PCR amplification with primers for AHBA synthase and amino-dehydroquinate (aDHQ) synthase, a genomic region containing orthologs of these genes was identified in Streptomyces sp. CS. It was confirmed to be involved in naphthomycin biosynthesis by deletion of a large DNA fragment, resulting in abolishment of naphthomycin production. A 106 kb region was sequenced, and 32 complete ORFs were identified, including five polyketide synthase genes, eight genes for AHBA synthesis, and putative genes for modification, regulation, transport or resistance. Targeted inactivation and complementation experiments proved that the halogenase gene nat1 is responsible for the chlorination of C-30 of NATs. The nat1 mutant could also be complemented with asm12, the halogenase gene of ansamitocin biosynthesis. Likewise, an asm12 mutant could be complemented with nat1, suggesting a similar catalytic mechanism for both halogenases. A putative hydroxylase gene, nat2, was also inactivated, whereupon the biosynthesis of NATs was completely abolished with a tetraketide desacetyl-SY4b accumulated, indicating the participation of nat2 in the formation of the naphthalene ring. The information presented here expands our understanding of the biosynthesis of naphthalenic ansamycins, and may pave the way for engineering ansamacrolactams with improved pharmaceutical properties.     

Novel family of cholesterol esterases produced by actinomycetes bacteria

Although cholesterol esterase (CHE; EC 3.1.1.13) is widespread in nature, CHEs from Streptomyces lavendulae and Streptomyces sp. X9 are the only known CHEs produced by actinomycetes. We purified CHEs from S. avermitilis JCM5070, and S. griseus IFO13350 and identified four new CHEs from actinomycetes. The enzymic properties of the CHEs from Streptomyces sp. X9, S. avermitilis, and S. griseus including substrate specificity, sensitivity to inhibitors and optimal conditions for catalysis were similar. We identified genes for the CHEs from Streptomyces sp. X9 and S. avermitilis and the encoded predicted sequences comprised 217 and 214 amino acid residues, respectively, with 64% similarity. The CHEs from Streptomyces sp. X9 and S. avermitilis were also 54 and 57% similar, respectively, to S. lavendulae CHE, indicating that these CHEs are orthologs. Phylogenetic analysis showed that they are distantly related to the conventional lipase/esterase type CHEs from mammals, yeasts and other bacteria. The actinomycetes CHEs did not have the Gly-Xaa-Ser-Xaa-Gly sequence that is conserved in the lipase/esterase family. A database search showed that orthologs of this type of CHE were restricted to actinomycetes. These findings imply that the actinomycetes CHEs constitute a novel family of cholesterol esterases.     

An adenovirus early region 1A protein is required for maximal viral DNA replication in growth-arrested human cells.

Two closely related adenovirus early region 1A proteins are expressed in transformed cells. The smaller of these, which is 243 amino acids in length, is required for the transformation of primary rat cells and for the transformation of immortalized rat cells to anchorage-independent growth. This protein is not required for productive infection of exponentially growing HeLa cells but is required for maximal replication in growth (G0)-arrested human lung fibroblasts (WI-38 cells). To determine the function of this protein in viral replication in these G0-arrested cells, we compared viral early mRNA, early protein, and late protein synthesis after infection with wild type or a mutant which does not express the protein. No differences were found. However, viral DNA synthesis by the mutant was delayed and decreased to 20 to 30% that of wild type in these cells. Viral DNA synthesis was much less defective in growing WI-38 cells, and in the transformed human HeLa cell line it occurred at wild-type levels. Furthermore, the mutant which can express only the 243-amino-acid early region 1A protein induced cellular DNA synthesis in G0-arrested rat cells to the same level as wild-type virus. A mutant which can express only the 289-amino-acid early region 1A protein induced less cellular DNA synthesis in G0-arrested rat cells. We propose that the early region 1A 243-amino-acid protein alters the physiology of arrested permissive cells to allow maximal viral DNA replication. In nonpermissive rodent cells, the 243-amino-acid protein drives G0-arrested cells into S phase. This activity is probably important for the immortalization of primary cells.     

Crystal structure of 3-amino-5-hydroxybenzoic acid (AHBA) synthase.

The biosynthesis of ansamycin antibiotics, including rifamycin B, involves the synthesis of an aromatic precursor, 3-amino-5-hydroxybenzoic acid (AHBA), which serves as starter for the assembly of the antibiotics' polyketide backbone. The terminal enzyme of AHBA formation, AHBA synthase, is a dimeric, pyridoxal 5'-phosphate (PLP) dependent enzyme with pronounced sequence homology to a number of PLP enzymes involved in the biosynthesis of antibiotic sugar moieties. The structure of AHBA synthase from Amycolatopsis mediterranei has been determined to 2.0 A resolution, with bound cofactor, PLP, and in a complex with PLP and an inhibitor (gabaculine). The overall fold of AHBA synthase is similar to that of the aspartate aminotransferase family of PLP-dependent enzymes, with a large domain containing a seven-stranded beta-sheet surrounded by alpha-helices and a smaller domain consisting of a four-stranded antiparallel beta-sheet and four alpha-helices. The uninhibited form of the enzyme shows the cofactor covalently linked to Lys188 in an internal aldimine linkage. On binding the inhibitor, gabaculine, the internal aldimine linkage is broken, and a covalent bond is observed between the cofactor and inhibitor. The active site is composed of residues from two subunits of AHBA synthase, indicating that AHBA synthase is active as a dimer.     

Self-protection mechanism in D-cycloserine-producing Streptomyces lavendulae. Gene cloning, characterization, and kinetics of its alanine racemase and D-alanyl-D-alanine ligase, which are target enzymes of D-cycloserine

An antibiotic, D-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) and d-alanyl-d-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designated alrS and ddlS, respectively, from DCS-producing Streptomyces lavendulae ATCC25233. Each gene product was purified to homogeneity and characterized. Escherichia coli, transformed with a pET vector carrying alrS or ddlS, displays higher resistance to DCS than the same host carrying the E. coli ALR- or DDL-encoded gene inserted into the pET vector. Although the S. lavendulae DDL was competitively inhibited by DCS, the K(i) value (920 microM) was obviously higher (40 approximately 100-fold) than those for E. coli DdlA (9 microM) or DdlB (27 microM). The high K(i) value of the S. lavendulae DDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for the S. lavendulae ALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of the E. coli ALR. We conclude that ALR from DCS-producing S. lavendulae is also one of the self-resistance determinants.     

Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.