~~ ~~Theprs gene, encoding phosphoribosylpyrophosphate synthetase, is preceded by a leader, which is 302 bp long in Escherichia coli and 417 bp in Salmonella typhimurium. A potential open reading frame (ORF) extends across the prs promoter and into the leader. The region between the prs coding region and an upstream gene (hemA) in E. coli and S. typhimurium was cloned, sequenced and shown to encode two ORFs of unknown function. ORF 1 encodes a 23 kDa protein and ORF 2 a 31 kDa protein, as observed by denaturing PAGE of extracts of cells bearing plasmids encoding the Oms. Both ORFs are transcribed in the same direction as theprs gene with ORF 2 extending into the prs leader. Northern blot analysis showed that the prs message in E. coli was on 1.3 and 2.7 kb transcripts. The shorter transcript encoded theprs gene only, while the longer transcript also encoded the two ORFs. Thus, the prs gene is transcribed from two promoters, the first promoter (PI) originating upstream of ORF 1, and expressing the prs gene in a tricistronic operon and a second promoter (PJ, located within the ORF 2 coding frame, which transcribes the prs gene only. The transcripts encoding prs only were 20 times as abundant as the tricistronic transcripts under all conditions examined. This was the case whether cells containing plasmid-encoded or only chromosomally encoded copies of the hernA-prs region were probed for these transcripts. Derepression of the prs gene upon pyrimidine starvation was shown to be due to an increase in the amount of message originating from the promoter P,.

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.

An Escherichia coli strain which is temperature-sensitive for growth due to a mutation @IS-2) causing a defective phosphoribosyl diphosphate (PRPP) synthase has been characterized. The temperature-sensitive mutation was mapped to a 276 bp Hindlll-BssHII DNA fragment located within the open reading frame specifying the PRPP synthase polypeptide. Cloning and sequencing of the mutant allele revealed two mutations. One, a G + A transition, located in the ninth codon, was responsible for the temperatureconditional phenotype and resulted in a serine residue at this position. The wild-type codon at this position specified a glycine residue that is conserved among PRPP synthases across a broad phylogenetic range. Cells harbouring the glycine-to-serine alteration specified by a plasmid contained approximately 50% of the PRPP synthase activity of cells harbouring a plasmid-borne wildtype allele, both grown at 25 OC. The mutant enzyme had nearly normal heat stability, as long as it was synthesized at 25 OC. In contrast, there was hardly any PRPP synthase activity or anti-PRPP synthase antibody cross-reactive material present in cells harbouring the glycine to serine alteration following temperature shift to 42 O C . The other mutation was a C + T transition located 39 bp upstream of the G + A mutation, i.e. outside the coding sequence and close to the Shine-Dalgarno sequence. Cells harbouring only the C + T mutation in a plasmid contained approximately three times as much PRPP synthase activity as a strain harbouring a plasmid-borne wild-type prs allele. In cells harbouring both mutations, the C + T mutation appeared to compensate for the G + A mutation by increasing the amount of a partially defective enzyme at the permissive temperature.