Pgad Introduction

Pgad operon in Lactococcus lactis2

Pgad is chloride-sensitive promoter which was first discovered in Lactococcus lactis1, which is a gram-positive bacterium which can live in acidic environment. Pgad operon (Fig. 1) provides hydrochloric acid feedback mechanism to adjust intracellular metabolism, in order to survive in acidic environment2. In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of Pgad2. The gene before Pgad, named gadR, which is constitutively expressed under the control of PgadR, is a positive regulator of Pgad coupled genes while intracellular chloride is level elevated2. When intracellular pH decreases, the expression of gadB and gadC is enhanced due to the action of gadR and confers glutamate-dependent acid resistance in L. lactis2.

J. Sanders et al. tried to develop chloride-sensitive expression cassette using Pgad operon3. They constructed the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes PgadR, gadR, Pgad and the starting codon ATG, and replaced downstream report genes3. They manage transforming the cassette to E. coli and varying the expression of report genes under different sodium chloride concentrations3. In our project, we try to build light-coupled chloride expression switch based on this design.

References

• Sanders, J.W. et al. Identifcation of a sodium chloride-regulated promoter in Lactococcus lactis by single-copy chromosomal fusion with a reporter gene. Mol Gen Genet 257, 681-685(1998)
• Sanders, J.W. et al. A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation. Molecular microbiology 27, 299-310(1998).
• Sanders, J.W., Venema, G. & Kok, J. A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis. Applied and environmental microbiology 63, 4877(1997).