Salmonella typhimurium pgtB mutants conferring constitutive expression of phosphoglycerate transporter pgtP independent of pgtC.
PgtC is one of the three components of the atypical "two-component" pgt regulatory system. To investigate whether functional PgtC required for the induction of pgtP expression could be bypassed in the signal transduction process, we sought, and succeeded in isolating, intergenic suppressors arising in the low-copy mini-F plasmid, pSJ11, bearing the entire pgt system except for a 168-bp deletion near the end of the pgtC gene. By transport assays, these suppressors were found to confer constitutive pgtP expression. Intriguingly, all five mutations reside near the 5' end of the pgtB gene, at codons 19 and 21. One mutation alters Arg-19 to Gln, two alter Ala-21 to Thr, one alters Ala-21 to Val, and one alters Ala-21 to Ile. Appropriate strains in which the pgtP promoter was fused to lacZ and which bore the pgtB mutations with and without mutations in pgtC and pgtA genes were constructed, and the epistatic relationships of the wild-type pgtC allele, a mutant pgtA allele, and an essentially total deletion of pgtC to the constitutive pgtB mutations were determined. In the mutant strains bearing the Ala-21 --> Ile and Ala-21 --> Val substitutions, the level of constitutive pgtP-lacZ reporter expression was not affected by the presence of the wild-type pgtC allele, nor was it affected by the total absence of PgtC in the case of the Ala-21 --> Val alteration examined; however, in the mutant strains bearing the Ala-21 --> Thr and the Arg-19 --> Gln substitutions, the extent of constitutive pgtP-lacZ reporter expression was markedly enhanced by the presence of wild-type pgtC allele and, in the case of the Arg-19 -->Gln change examined, by the total absence of PgtC as well. These results indicate that PgtC contains no domain necessary for the kinase activity; that PgtB can be activated in the absence of PgtC mutational alterations of the protein itself; and that PgtB and PgtC interact in the signaling process, with PgtC functioning to activate and modulate the kinase activity of Pgtb. In all strains, the replacement of the wild type pgtA allele with a mutant pgtA allele completely abolished expression of the pgtP-lacZ reporter, indicating that functional pgtA is essential for the constitutivity. His-457 of PgtB, a potential site of autophosphorylation, is also required for the constitutivity because its change to Val drastically reduced pgtP-lacZ reporter expression. The structural basis for the activation of the altered PgtB is discussed in terms of putative structure of PgtB in the membrane.
Niu
S
S
Boston Biomedical Research Institute, Massachusetts 02114, USA.
Jiang
S Q
SQ
Hong
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator; D017930:Genes, Reporter / Q000235:genetics; D005988:Glyceric Acids / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D027361:Organic Anion Transporters; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012486:Salmonella typhimurium / Q000235:genetics* / Q000254:growth & development; D015398:Signal Transduction / Q000235:genetics; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D005988:Glyceric Acids; D027361:Organic Anion Transporters; C056780:PgtP protein, Salmonella typhimurium; D006639:Histidine; C005156:3-phosphoglycerate