Use of bacteriophage transposon Mu d1 to determine the orientation for three proC-linked phosphate-starvation-inducible (psi) genes in Escherichia coli K-12

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Abstract
We have previously used the bacteriophage transposon Mu d1 (which encodes the lacZY structural genes but without their promoter) to construct strains that have lacZY fused to phosphate-regulated promoters in Escherichia coli K-12. Among 18 identified phosphate-starvation-inducible (psi) genes, three (the phoA and two new genes: psiF and psiG) are closely linked to the proC region. The gene order (clockwise) is phoA psiF proC psiG phoB phoR. Using these mutants containing Mu d1 insertions, we devised and tested a new method to determine their orientation. In this procedure, mutants with deletions that are selectable by their ability to grow at 42 degrees C are tested for the presence of Mu d1 and of neighboring genes. Some difficulties arose during analysis of suspected deletion-containing strains derived from Mu d1 lysogens (which also contained a Tn5 element) that were caused by Mu d1 and transposon transpositions and other possible genome rearrangements. Nevertheless, we have shown that the phoA and psiF genes are transcribed clockwise and the psiG gene is transcribed counterclockwise towards proC. Because phoA, but not psiF, gene expression requires the phoB+ (positive regulator) gene product, the phoA and psiF genes do not constitute an operon. On the other hand, the psiG:lacZ fusion-bearing strain may have a fusion to the promoter-distal end of the phoB gene. This implies that phoB expression is phosphate regulated. We believe that this method may be useful in general to elucidate the direction of gene transcription.