A phoA structural gene mutation that conditionally affects formation of the enzyme bacterial alkaline phosphatase.
The phoA503 mutant was identified as a mutant that shows a novel phoA regulatory phenotype. The phoA503 allele dramatically reduces the synthesis of bacterial alkaline phosphatase activity during Pi starvation in an otherwise wild-type host and during the logarithmic growth phase in a phoR or phoU background. Near-normal amounts of enzyme activity are found in phoR phoA503 or phoU phoA503 mutants when starved for carbon, nitrogen, or sulfur or during the stationary phase, however. Marker rescue and DNA sequence analysis located the phoA503 mutation to the phoA coding region. It is a C-to-T transition that would cause a substitution of Val for Ala-22 in the mature protein. Transcriptional and translational lacZ fusions to both wild-type and mutant alleles demonstrated that phoA gene expression is unaltered. Also, the mutant protein was secreted and processed as efficiently as the wild type. Furthermore, the subunits appeared to dimerize and to be stable in the periplasm. But, greater than 98% of the dimers were inactive and found exclusively as isozyme 1. An activation of preformed phoA503 dimers occurred during the stationary phase with the concomitant conversion into isozymes 2 and 3. We propose that the phoA503 mutation affects a late stage in the formation of active enzyme. An unknown change when Pi is present during stationary-phase growth leads to formation of active dimers, which is responsible for this new conditional phenotype.
Agrawal
D K
DK
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Wanner
B L
BL
D000469:Alkaline Phosphatase / Q000235:genetics*; D000483:Alleles; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004789:Enzyme Activation; D005798:Genes, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D000469:Alkaline Phosphatase