We report the isolation and genetic characterization of novel TnlOdTc and TnlOOOdKn insertion mutations in and near the lip locus of the Escherichia coli chromosome. The TnlOdTc and TnlOOOdKn mutations define two genes, lpA and lipB, involved in lipoic acid biosynthesis. Two representative alleles (lip-2 and lip-9) from the previously reported genetic class of lipoic acid auxotrophic mutants (A. A. Herbert and J. R. Guest, J. Gen. Microbiol. 53:363-381, 1968) were assigned to the lipA complementation group. We have cloned the E. coli lip locus and developed a recombinant plasmid-based genetic system for fine-structure physical-genetic mapping of mutations in this region of the E. coli chromosome. We also report that a recombinant plasmid containing a 5.2-kbp PvuH restriction fragment from the E. coli lip locus produced three proteins of approximately 8, 12, and 36 kDa by using either a maxiceli or in vitro transcription translation expression system. The 36-kDa protein was identified as the gene product encoded by the lipA locus. Finally, we have identified a previously unreported lipoylated protein that functions in the glycine cleavage system of E. coli.

A physical map of the chromosome of the erythromycin-producing actinomycete Saccharopolyspora erythraea NRRL 2338 has been constructed using the restriction enzymes Asel and Dral. The map was constructed by a variety of methods including linking clone analysis, cross-hybridizations using labelled macrorestriction fragments, gene probing, two-dimensional PFGE and restriction enzyme site generation. Analysis of the individual macrorestriction patterns of the 17 Asel-, 6 Drab and 22 AsellDral-digested fragments indicated a chromosome size of about 8 Mb. Linking clones for five contiguous Asel fragments were obtained, covering 32% of the chromosome. The linkage of an additional eight Asel fragments was aided by the finding that the rRNA operons of S. erythraea contain an Asel site within the 165 ( r a ) gene. Generation of 5. erythraea strains that contain additional DraI sites within selected Asel fragments, followed by PFGE analysis and Southern hybridization to determine specific linkages, facilitated the completion of the Asel map. The entire DraI map was constructed by gene probing and cross-hybridizations. PFGE analysis of agarose-embedded DNA prepared in either the presence or absence of proteinase K suggested that the S. erythraea NRRL 2338 chromosome is linear. A total of 15 genes or gene clusters were mapped to specific Asel and Dral fragments, including the erythromycin-biosynthetic gene cluster and the rRNA operons.

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.

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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.