Ser331 appears constitutively phosphorylated under all conditions examined

yaB-lac fusion strains. Heavy suspensions of double Mu, A lysogens were incubated on miniimal agar plates containing lactose as sole carbon source and Debio 1347 price different sources of sulfur. The plates were incubated at 420C for 12 h and then at 370C until colonies appeared. RESULTS Isolation ofMu-induced cysB auxotrophs. Since application of the Casadaban method to construct lac fusions requires the isolation of Mu-induced auxotrophs, strain EC1201 was mutagenized with Mu c as described in Materials and Methods. After ampicillin counterselection at 300C, the mutagenized cultures were scored for cysteine auxotrophs and temperature sensitivity. Temperature-sensitive cysteine auxotrophs were then examined for the sites of their presumed Mu insertions by nutritional tests and biochemical assays of cysteine enzymes. Those Cys- isolates that did not grow on O-acetyl-Lserine or sulfite, and that had low to undetectable levels of sulfite reductase and O-acetylserine sulflhydrylase when grown on L-djenkolate, were presumed to be cysB. The presence of phage Mu insertions in the cysB gene was finally confirmed by a two-step procedure: first, Cys- mutants of EC1201 strain were transduced to Cys+ prototrophy using phage P1 grown on strain Cu152, and transductants were scored for Trp+ phenotype. The cysB locus is closely linked by P1 PubMed ID: transduction with the trp operon, and the presence of Trp+ colonies among Cys+ recombinants indicated that cysteine auxotrophy had resulted from a mutation in the cysB gene. In the second step, Cys+ transductants were checked for thermoresistance; among five strains only those of EC1206 gave Cys+ transductants that could grow at 420C, and this strain was chosen for further experiments. The thernosensitivity of Cys+ transductants of other mutants probably resulted from the presence of one or more additional copies of Mu c in the chromosome. Isolation of cysB-lac fusion strains. The lac structural genes were first transposed to a site near the cysB promoter by lysogenizing strain EC1206 with the AMu hybrid phages Xpl and Xpl23. Since the X-Mu hybrid phages carry no A attachment site and the host chromosome is deleted for the lac operon, lysogenization in such a situation should occur preferentially via recombination between the homologous regions of Mu DNA. Because of the lack of the lac promoter, lac structural genes ordinarily are not expressed on these phage, but they can be if fused to a promoter on the host chromosome. Heat induction of the Mu c prophage was used to generate deletions at the insertion site of Mu, which occasionally result in a fusion between lac genes and a nearby promoter. Since the direction of transcription of the cysB gene was unknown, it was necessary to insert the lac genes in both orientations. This was achieved by using two different lambda phages carrying either the c or the S end of phage Mu. Because the lac genes are oriented in the same direction on both phages, lyosogenization through pieces from opposite ends of phage Mu integrates the lac genes in opposite orientations. Only the lysogen having the lac genes inserted in the same direction of transcription as the cysB gene was expected to give the desired fusions after heat induction of the prophage. Owing to a lack of information regarding a relationship between cysB gene expression and growth on varying sulfur sources, fusions of the VOL. 147, 1981 REGULATION OF THE cysB GENE 747 strains were further characterized. Fine-structure mapping of X pha