Differential protein acetylation induced by novel histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.

Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives.

The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds.

Erythromycin production in Saccharopolyspora erythraea does not require a functional propionyl-CoA carboxylase.

Using an oligonucleotide corresponding to the consensus sequence for the biotin-binding motif, two unlinked genetic loci, bpl1 and bpl2, were cloned from the erythromycin producer Saccharopolyspora erythraea and the nucleotide sequences of a c. 4 kb segment from each determined. The two loci share a virtually identical segment of 1746 nucleotides, coinciding with most of the genes designated bcpA1 and bcpA2 present in bpl1 and bpl2, respectively. The deduced sequences of these genes are highly similar to that of the alpha-chain of mammalian propionyl-CoA carboxylase. Upstream of bcpA2 lies pccB, the gene encoding the beta-chain of this enzyme. Mutant strains carrying frameshift mutations in bcpA1 and pccB were constructed, but we failed to isolate insertional mutants in bcpA2. Propionyl-CoA carboxylase activity was undetectable in the pccB mutant, but was unaffected in the bcpA1-defective strain. These results indicate that pccB encodes the beta-chain of propionyl-CoA carboxylases, and suggest that the alpha-chain of this enzyme, which is likely to be encoded by bcpA2, is shared with some other essential biotin-dependent enzyme. The pccB mutation had no impact on erythromycin production in complex medium.

Characterization of the macromolecular synthesis (MMS) operon from Listeria monocytogenes.

The macromolecular synthesis (MMS) operon consists of three genes: rpsU, which encodes the S21 ribosomal protein in Bacillus subtilis (Bs), rpsU is replaced by orfP23 which encodes a protein of unknown function), dnaG, encoding the DNA primase involved in the initiation of chromosome replication, and rpoD, which encodes the principal sigma subunit of RNA polymerase. The operon was cloned in three segments from Listeria monocytogenes (Lm), initially using a probe designed from a highly conserved region of RpoD. Analysis of the nucleotide sequence revealed three genes: orfP17 (whose product, P17, is homologous to Bs P23), dnaG and rpoD. The Lm DnaG resembles the primase from Escherichia coli through the first two-thirds of the sequence. C-terminal similarity was observed between DnaG from Lm and Bs. Lm RpoD is similar to Bs SigA, shares identical DNA-binding domains with SigA, and is a member of the sigma 43 subgroup of the sigma 70 family.

IS1136, an insertion element in the erythromycin gene cluster of Saccharopolyspora erythraea.

The Saccharopolyspora erythraea eryAI and eryAII genes, which, together with eryAIII, are responsible for the formation of the macrolactone portion of the antibiotic erythromycin, are separated by a 1.46-kb segment, designated IS1136, with the characteristics of an insertion sequence. It contains an open reading frame of 425 codons similar to that of the Anabaena IS891 and is present in four nonidentical copies in the Sac. erythraea genome. Inverted repeats were found near the ends of IS1136, and in the copy in eryA, one of the ends was found to overlap the 5' end of eryAII. Hybridization analysis suggests that IS1136 is confined to Saccharopolyspora species containing eryA-homologous DNA.

Identification of a Saccharopolyspora erythraea gene required for the final hydroxylation step in erythromycin biosynthesis.

In analyzing the region of the Saccharopolyspora erythraea chromosome responsible for the biosynthesis of the macrolide antibiotic erythromycin, we identified a gene, designated eryK, located about 50 kb downstream of the erythromycin resistance gene, ermE. eryK encodes a 44-kDa protein which, on the basis of comparative analysis, belongs to the P450 monooxygenase family. An S. erythraea strain disrupted in eryK no longer produced erythromycin A but accumulated the B and D forms of the antibiotic, indicating that eryK is responsible for the C-12 hydroxylation of the macrolactone ring, one of the last steps in erythromycin biosynthesis.

Biosynthesis of the erythromycin macrolactone and a rational approach for producing hybrid macrolides.

The three eryA genes involved in the formation of the polyketide portion of the macrolide antibiotic erythromycin in Saccharopolyspora erythraea, appear to be organized in a single transcriptional unit on the basis of the results of gene disruption experiments. An insertion sequence-like element of lower G + C content separates eryAI from eryAII. The organization of the enzymatic domains present in the eryA-encoded multifunctional polypeptides, determined by computer-assisted analysis, is presented. This has enabled the determination of a putative dehydratase domain. A rational approach for producing novel macrolides by introducing selected changes in polyketide synthase genes is outlined. The isolation of a lactone intermediate resulting from an early synthesis step in macrolactone formation is also presented.

Modular organization of genes required for complex polyketide biosynthesis.

In Saccharopolyspora erythraea, the genes that govern synthesis of the polyketide portion of the macrolide antibiotic erythromycin are organized in six repeated units that encode fatty acid synthase (FAS)-like activities. Each repeated unit is designated a module, and two modules are contained in a single open reading frame. A model for the synthesis of this complex polyketide is proposed, where each module encodes a functional synthase unit and each synthase unit participates specifically in one of the six FAS-like elongation steps required for formation of the polyketide. In addition, genetic organization and biochemical order of events appear to be colinear. Evidence for the model is provided by construction of a selected mutant and by isolation of a polyketide of predicted structure.

Cloning of genes involved in erythromycin biosynthesis from Saccharopolyspora erythraea using a novel actinomycete-Escherichia coli cosmid.

Two plasmids were constructed that replicate in Saccharopolyspora (Sac.) erythraea, Escherichia coli and Streptomyces (S.) lividans, and used for the cloning of a locus involved in the synthesis of the macrolide antibiotic erythromycin (Er). Plasmid pAL7002 contains the thiostrepton-resistance gene (tsr), a replicon-containing fragment from pJVI and pUC9. Plasmid pNJI contains the lambda cos site but is otherwise similar to pAL7002. A library of total DNA from Sac. erythraea was constructed in pNJI and probed in colony hybridizations with a DNA fragment containing ermE, the Sac. erythraea ErR-encoding gene. Plasmids obtained were subsequently introduced into EryA mutants of Sac. erythraea blocked in synthesis of Er (Ery-) and transformants were screened for restoration of Er production (Ery+). Several plasmids were found to convert two mutants to Ery+, but a third EryA strain could not be restored to Ery+ by any of the plasmids employed. A 5-kb segment, designated eryAI, responsible for restoring the Ery+ phenotype in the EryA strains, was identified and mapped in the segment 12 to 17 kb downstream from ermE. Gene disruption experiments indicated that the 5-kb length of eryAI is fully internal to an eryAI-containing transcript. In Southern blots it was shown that one of the EryA strains carried a small deletion in eryAI and that, in at least some of the transformants restored to Ery+, the deletion had been replaced by the wild-type eryAI allele.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta.

Bacillus thuringiensis subsp. kurstaki HD-73 produces a crystal protein which is lethal to many lepidopteran larvae. The gene encoding this crystal protein has been isolated from a 75-kb plasmid and engineered into a recombinant Escherichia coli plasmid for analysis. The complete nucleotide sequences of the coding region and 387-bp 5' and 376-bp 3' to the coding region have been determined. The 3537-bp of the coding region specify a protein of Mr 133 330. The full-length gene and several 3' -truncated derivatives of the gene were examined in both E. coli and in an E. coli minicell-expression system to determine if the carboxy end of the protein is essential for toxicity. The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product.

Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings.

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.