Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon.
The phosphate regulon is negatively regulated by the PstSCAB transporter and PhoU protein by a mechanism that may involve protein-protein interaction(s) between them and the Pi sensor protein, PhoR. In order to study such presumed interaction(s), mutants with defined deletions of the pstSCAB-phoU operon were made. This was done by construction of M13 recombinant phage carrying these mutations and by recombination of them onto the chromosome by using a rep host (which cannot replicate M13) for allele replacement. These mutants were used to show that delta (pstSCAB-phoU) and delta (pstB-phoU) mutations abolished Pi uptake by the PstSCAB transporter, as expected, and that delta phoU mutations had no effect on uptake. Unexpectedly, delta phoU mutations had a severe growth defect, and this growth defect was (largely) alleviated by a compensatory mutation in the pstSCAB genes or in the phoBR operon, whose gene products positively regulate expression of the pstSCAB-phoU operon. Because delta phoU mutants that synthesize a functional PstSCAB transporter constitutively grew extremely poorly, the PhoU protein must have a new role, in addition to its role as a negative regulator. A role for the PhoU protein in intracellular Pi metabolism is proposed. Further, our results contradict those of M. Muda, N. N. Rao, and A. Torriani (J. Bacteriol. 174:8057-8064, 1992), who reported that the PhoU protein was required for Pi uptake.
Steed
P M
PM
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Wanner
B L
BL
D000483:Alleles; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005798:Genes, Bacterial; D005838:Genotype; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009876:Operon; D010710:Phosphates / Q000378:metabolism*; D010957:Plasmids; D018085:Regulon; D015183:Restriction Mapping; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010710:Phosphates; D014157:Transcription Factors; C050770:phoU protein, E coli