Streptomyces galilaeus ATCC 31133 and ATCC 31671, producers of the anthracyclines aclacinomycin A and 2-hydroxyaklavinone, respectively, formed an anthraquinone, aloesaponarin II, when they were transformed with DNA from Streptomyces coelicolor containing four genetic loci, actI, actIll, actlV, and actVII, encoding early reactions in the actinorhodin biosynthesis pathway. Subcloning experiments indicated that a 2.8-kilobasepair XhoI fragment containing only the actl and actVII loci was necessary for aloesaponarin II for the actll gene, which is the reduction of the keto group at C-9 from the carboxy terminus of the assembled polyketide to the corresponding secondary alcohol. In the presence of the actIll gene, anthraquinones or anthracyclines formed as a result of dehydration and aromatization lack an oxygen function on the carbon on which the keto reductase operated. When S. galiaeus ATCC 31671 was transformed with the DNA carrying the actl, actVII, and actlV loci, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin, the 3-hydroxy analog of aloesaponarin II, and 1-0-methyldesoxyerythrolaccin. The results obtained in these experiments together with earlier data suggest a pathway for the biosynthesis of actinorhodin and related compounds by S. coelicolor.The biosynthesis of the benzoisochromanequinone antibiotic actinorhodin (4), a polyketide antibiotic produced by Streptomyces coelicolor, has been studied intensively in recent years from both the biochemical (7,13,14) and genetic (15,(22)(23)(24)30) viewpoints. Seven classes of blocked mutants have been described and placed in sequential order on the basis of cosynthetic and chemical studies (7,13,30). Studies on the molecular genetics of actinorhodin biosynthesis have provided fundamental knowledge on the structure and organization of antibiotic biosynthesis genes in streptomycetes, including the clustering of antibiotic biosynthesis structural genes (23,24), production of the antibiotic by recombinant strains carrying all of the biosynthesis genes on a plasmid (23), and the potential homology shared by certain structural genes within several different pathways (i.e., the genes encoding polyketide synthases [22]). Actinorhodin

The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pU702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.

The transcriptional organization of the erythromycin biosynthetic gene (ery) cluster of Saccharopolyspora erythraea has been examined by a variety of methods, including S1 nuclease protection assays, Northern blotting, Western blotting, and bioconversion analysis of erythromycin intermediates. The analysis was facilitated by the construction of novel mutants containing a S. erythraea transcriptional terminator within the eryAI, eryAIII, eryBIII, eryBIV, eryBV, eryBVI, eryCIV, and eryCVI genes and additionally by an eryAI ؊10 promoter mutant. All mutant strains demonstrated polar effects on the transcription of downstream ery biosynthetic genes. Our results demonstrate that the ery gene cluster contains four major polycistronic transcriptional units, the largest one extending approximately 35 kb from eryAI to eryG. Two overlapping polycistronic transcripts extending from eryBIV to eryBVII were identified. In addition, seven ery cluster promoter transcription start sites, one each beginning at eryAI, eryBI, eryBIII, eryBVI, and eryK and two beginning at eryBIV, were determined.

A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The crylA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryL4(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryL4(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control. Clavibacter xyli subsp. cynodontis is a fastidious gram-positive coryneform bacterium that naturally inhabits the xylem of Bermuda grass (Cynodon dactylon L.) (6). C. xyli subsp. cynodontis also colonizes the vascular system of corn (Zea mays L.) when artificially inoculated (16). High populations of C. xyli subsp. cynodontis are distributed in the xylem elements following the introduction of inoculum into the tissues of the whorl or stem of corn seedlings or after germination of corn seed infused with the bacterium. C. xyli subsp. cynodontis does not enter developing kernels and is therefore not present in the progeny seed of inoculated plants (5). Additionally, a DNA transformation system and the development of plasmid cloning vectors have been recently reported (13, 19).

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xyleminhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.

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We report the development of an electrotransformation method applicable to all strains of Saccharopolyspora erythraeaexamined to date. Vegetatively grown mycelia were rendered electrocompetent by subjecting mycelial suspensions to ultrasound pulses. The protocol provides an alternative route for the introduction of DNA into filamentous microorganisms otherwise recalcitrant to transformation techniques.

Streptomyces peucetius and Streptomyces strain C5, producers of anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (>99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, p1.][01, and SCP2*, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 106 transformations ,ug of plasmid DNA-' were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage +C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with 4C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.