Polarity in the glnA Operon: Suppression of the Reg − Phenotype by rho Mutations

Abstract

To determine the ability of mutations in glnA , the gene for glutamine synthetase (GS), to regulate nitrogen assimilatory enzymes, we assayed histidase and GS in 34 glnA (Gln − ) strains. Twenty-five glnA mutants were RegC, synthesizing high levels of histidase regardless of the availability of nitrogen, and nine were Reg − , synthesizing low levels of histidase in medium containing either limiting or excess ammonia. rho mutations were introduced into strains containing glnA point mutations or insertions in glnA, glnL, glnG , or glnF . The Reg − phenotype of strains with glnA point mutations, but not those with glnA or glnF insertions, was altered by the presence of rho , suggesting that glnA (Reg − ) mutations are polar and exert their phenotype by decreasing expression of glnL and glnG . Consistent with this view, no GS protein was detected by two-dimensional gel electrophoresis in glnA (Reg − ) rho + or glnA (Reg − ) rho double mutants, whereas GS protein was detected in cells of 10 of 11 glnA (RegC) strains. Since glnA (Reg − ) rho double mutants synthesize constitutive levels of histidase, GS protein is not necessary for full expression of histidase. Mu d1 insertions in glnL , but not those in glnG , responded to the presence of a rho allele, presumably owing to elevated transcription into glnG from the Mu d1 prophage. Our results suggest that glnA (Reg − ) alleles are polar mutations, and a rho-dependent termination site down-stream is postulated as the basis for the polar phenomenon. The data also indicate that, under some circumstances, a significant portion of glnL and glnG transcription is initiated at the glnA promoter.