Phosphate-independent controls of bacterial alkaline phosphatase synthesis in Escherichia coli K-12

Mary Regina Wilmes-Riesenberg, Purdue University

Abstract

The synthesis of bacterial alkaline phosphatase (Bap) is normally induced several hundredfold when phosphate is growth limiting. This control requires both products of the phoBR operon. PhoB is a transcriptional activator, and PhoR is a sensory protein that detects environmental phosphate levels. Earlier studies showed that in phoR mutants phoA transcription is no longer controlled by phosphate. Instead, its expression is regulated by the creABCD (formerly called phoM) operon. phoR mutants with a wild-type creABCD operon show induced synthesis of Bap on glucose medium, and an alternating pattern of Bap synthesis, termed Bap clonal variation, on TYE agar. Point mutations in the creC gene confer a Bap$\sp-$ phenotype. However, deletion mutations can confer either a Bap$\sp-$ or clonal variation phenotype, depending on whether neighboring genes are deleted and the strain background. Because strains with certain creABCD deletions are Bap variable, this suggested that gene products other than CreC may be involved in regulation of Bap synthesis. To determine which additional genes in the creABCD region are involved, this region was mutagenized with the transposon Tn5, and these insertions were used to construct new deletions. The results showed that (i) strains with non-polar insertions in the creA, creB, and creD genes show normal regulation of Bap synthesis; (ii) a strain with a deletion removing the creA and creB genes shows a Bap induced phenotype; (iii) strains with simple mutations in the creC gene are Bap$\sp-$; (iv) strains with insertions in the neighboring arcA gene are Bap induced; and (v) strains with deletions that remove both the creC and arcA genes are Bap$\sp-$ on glucose and show the Bap clonal variation phenotype on TYE agar. Models are presented to explain how the products of the creABCD operon and the arcA gene regulate Bap synthesis. Several lines of evidence indicate that one or more additional controls regulate bap synthesis in a PhoR-and CreC-independent manner. As described above, arcA mutations restore Bap synthesis in phoR $\Delta$ (creABCD) mutants. We found that an ompR mutation also restored Bap synthesis in phoR $\Delta$ (creABCD) mutants. Furthermore, phoR $\Delta$ (creABCD) mutants synthesize Bap when plated on TYEG agar. In order to study Pi-independent control of Bap synthesis, 74 transposon-induced mutants were isolated with altered Bap phenotypes. Thirty-two Bap$\sp-$ mutants were shown to have insertions in the phoA or phoB genes. The remaining 42 mutants were placed into one of 19 mutant classes based on their Bap phenotypes or linkage data. Many insertions resulted in the induction of a phoR- and CreC-independent control of Bap synthesis, whereas some mutants eliminated the phoR- and CreC-independent Bap synthesis on TYEG agar. Representative insertions from several mutant classes were cloned and the DNA sequence of their fusion junctions were determined. Based on sequence and/or linkage data, the sites of the insertions for 11 mutants classes were identified. Interestingly, 4 mutants that conferred a Bap induced phenotype had insertions in the ackA gene, for acetate kinase. Based on subsequent experiments with these mutants, we conclude that one phoR- and CreC-independent control of Bap synthesis responds to acetyl phosphate or a closely related metabolite. Several other mutations that alter Bap synthesis in a phoR $\Delta$(creABCD) mutant may affect Bap synthesis indirectly by altering the level of acetyl phosphate.

Degree

Ph.D.

Advisors

Wanner, Purdue University.

Subject Area

Microbiology|Genetics|Molecular biology

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