4)HAU3 is a temperate Streptomyces phage with cohesive ends and a broad host range that includes Streptomyces hygroscopicus 10-22, a producer of antifungal compounds, but it fails to grow on Streptomyces lividans 66. Two phasmid derivatives were constructed that function as X cosmid vectors in Escherichia coli and as phages in Streptomyces spp.

Streptomycetes are mycelial bactria that produce sporulating aeraial hyphae on solid medai. Bald (bld) mutants fail to form aerial mycelium under at Least some conditions. Bld A encodes the only tRNA species able to read the leucine condon UUA efficeiently, implying the involvement of a TTA-containing gene in initiating aerial growth. One candidate for such a gene was bldH, because the bldH109 mutant of Streptomyces coelicolor resembles bldA mutants in some aspects. In the similar to the Streptomyces griseus A factor-regulated adpAg, was found to complement the blodh109 mutant paritally at both single and multiple copies. The sequence of adpAc form the bldH109 mutant revealed a frameshift. A constructed in frame deletion of adpAc conferred a bald colony phenotype, and the mutant behaved like bldAmutants and bldH109 in its pattern of extracellular signal exchange. Both adpAc and AdpAg contin a TTA codon. A TTA-free version of adpAc was engineered by replacing the TTA leucine condon with a cognate TTG leucine codon. The adpA (TTA-TTG) gene could partially restore aerial mycelium formation to a bldA mutant when it was followed in cis by the gene ornA, as in the natural chromosomal arrangement. This indicated that the UUA condon in adpAc mRNA is the principal target throuth which bldA influences morphological differention. It also implied that translational arrest at the UUA codon in adpAc mRNA caused a polar effect on the downstream ornA, and that the poor translation of both genes contributes extensively to the deficeiency of aerial mycelium formation in bldA mutants. Unlike the situation in S. griseus, adpAc transcrpion does not depend on the host's y-buyrolactone singatlling system, at least in liquid cultures. In addition, sigma factor BldN, which is the homologue of an S. griseus sigma factor AdsA that is absent form adpAg mutants of S. Griseus, Was present in the constructed adpAc, Null Mutant of S. Coelicolor.

The PKS genes for biolsynthesis of the polyether nanchangmycin are organized to encode two sets of proteins (six and seven ORFs, respectivly), but are separated by independent ORFs that encode and eqimerase, epoxidase and epoxide hydrolase, and, notably, and independent ACP. One of the PKS modules lacks a corresponding ACP. We propose that the process of oxidavtive cvclization to form the polyether structrue occurs whten the polyketide chain is still andchored on the independent ACP berore release. 4-O-methyl-L-rhodinose biolsynthesis and its transglycosylation involve four puattive genes, and requlation of nanchang-mycin biosynthesis seems to involve activation as well as repression. In-frame deletion of a KR6 domain generated the anachangmycin aglycone with loss of 4-O -methyl-L-rhodinose and antibacterial activity, in agreement specific biolsynthetic steps.

A cluster encoding genes for the biosyntheresis of meilingmycin, a mzcrolide antibiotic structurally similar to avermectin and milbemycin a 11, was identified among seven uncharactreized polyketide synthase gene clusters isolated from Streptomyces nanchangensis NS 3226 by hybirdization with PCR products using primers derived from the sequences of aevE, aveF and a thioesterase do main of the avermectin biosynthetcin production, confirming that the gene cluster encodes biosynthesis of this important anthelminthic antibiotic compound. A sequenced 8.6-kb fragment had aveC and aveE homologues (meiC and meiE) linked together, as in the avermectin gene cluster, but the arrangement of aveF (meiF) and the thioesterase homolgues differed. The rusults should pave the way to producing novel insecticidal compounds by generating hybrids between the two pathways.

Streptomyces hygroscopicus 10-22 could not be tranformed with any of the commonly used Streptomyces plasmid vectors and was resitant to plaque formation by the Streptomyces phages C31 and R4. Repeated selection reslulted in the isolation of derivatioves of S. hygroscpicus 10-22 that could accept DNA propagated in Streptomyces lividans 66. These new strains, which include three that still produce the original antibiotics, and be used as hosts for gene cloning. Insertion of nonreplicationg vectors by homolgous rctombination and transpositon of Tn 4560 were demonstrated in S. hygroscopcus 10-22