Characterization of the 5' flanking region of the Escherichia coli ppa gene encoding inorganic pyrophosphatase: mutations in the ribosome-binding site decrease the level of ppa mRNA.

We have previously cloned and sequenced the ppa gene, encoding inorganic pyrophosphatase (PPase), of Escherichia coli K12 [Lahti, R., Pitkäranta, T., Valve, E., Ilta, I., Kukko-Kalske, E. & Heinonen, J. (1988) Journal of Bacteriology 170, 5901-5907]. In this work mutations were constructed in the 5' flanking region of E. coli ppa and the effect on expression was determined. The minimum length of the fully active ppa5' flanking region was shown to be 117 bp. Further deletion decreased the activity, and upon deletion to nucleotide -37 the promoter activity was totally lost. A clear point of inflection was observed in the inactivation upon deletion over the nucleotide -50. This is consistent with the fact that by binding to promoters RNA polymerase holoenzyme generally covers the -50 to +20 region in E. coli genes. When the -35 sequence of ppa, AAGACA, was mutated to AAAACA, ppa expression, as measured by PPase production, decreased to 20% of the wild-type, whereas by the change of the -10 sequence, TATAAT, to TTTAAT or TATAAA, the ppa gene was totally inactivated. Furthermore, when the ribosome-binding site (RBS) sequence, AGGAAA, was altered to AAGAAA, PPase production decreased to 19% of the wild-type. Surprisingly, when the RBS sequence was mutated to the consensus RBS sequence, AGGAGG, the intracellular levels of both ppa mRNA and PPase decreased drastically. The implications of these results are discussed.

Intracellular PPi concentration is not directly dependent on amount of inorganic pyrophosphatase in Escherichia coli K-12 cells.

No correlation was observed between the level of inorganic pyrophosphatase (PPase) and the intracellular concentration of PPi in Escherichia coli cells. In exponentially growing cells the intracellular PPi concentration was in every case 1.5 nmol/mg (dry weight) or about 0.5 mM, even though the amount of PPase was varied from 15 to 2,600% of the control amount by mutation or by using a multicopy plasmid with an inserted gene (ppa) encoding PPase. The PPi concentration could, however, be increased or decreased from the control level under some stressful conditions.

Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene.