Induction of cadBA in an Escherichia coli lysine auxotroph transformed with a cad-gfp transcriptional fusion
- 128 Downloads
CadBA functions as a part of overall Escherichia coli response to low extracellular pH. A gfpmut3 structural gene transcriptionally fused to the cadBA promoter (Pcad) was used as a reporter to monitor changes in intracellular lysine as a potential factor influencing cadBA induction. Different patterns of cadBA induction were observed in two E. coli strains with different lysine biosynthetic capabilities. In E. coli ZK126 (pJBA25-Pcad), a lysine prototroph, maximum levels of induction were detected 3 h after the transfer of bacterial cells under inducing conditions (pH 5.8; 3.4 μM extracellular lysine). The induction subsequently decreased until hour 7 after which no further change in expression was observed. However, in the lysine depleted strain E. coli ATCC 23812 (pJBA25-Pcad) which is an auxotroph for lysine, no decrease in cadBA expression was observed over time under the same induction conditions. Although no time dependent statistical differences in intracellular lysine were observed, bacterial cells depleted for no longer than 4 h (1.38 ± 0.25 μmol lysine/g cell dry weight) exhibited more rapid induction of cadBA (after 3 h) and a lower maximum level of induction compared to cells with relatively lower intracellular lysine (approximately 1.08 μmol/g cell dry weight). For the latter, the detectable level of induction was delayed for 1 h but the maximum level of induction response was higher.
KeywordsEscherichia coli Lysine auxotroph cad operon Green fluorescent protein Transcriptional fusion Intracellular lysine
This research was supported by Hatch grant H8311 administered by the Texas Agricultural Experiment Station and Texas Advanced Technology Program, grant #: 000517-0220-2001.The authors would like to express their thanks to Dr. Deborah Siegele (Texas A&M University, College Station, TX) and Julia Sonka (University of Arkansas, Fayetteville, AR) for assistance in the preparation of this manuscript.
- Gale EF (1946) The bacterial amino acid decarboxylases. Adv Enzymol 6:1–32Google Scholar
- Ingraham JL, Marr AG (1996) Effect of temperature, pressure, pH, and osmotic stress on growth. In: Neidhardt FC, Curtiss RIII, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella: Cellular and molecular biology, vol 2, 2nd edn. ASM Press, Washington, D.C, pp 1570–1578Google Scholar
- Li X, Erickson AM, Ricke SC (1999) Comparison of minimal media and inoculum concentration to decrease the lysine growth assay response time of an Escherichia coli lysine auxotroph mutant. J Rapid Methods Autom Microbiol 7:279–290Google Scholar
- Sambrook J, Frish EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.YGoogle Scholar
- Slonczewski JL, Foster JW (1996) pH-regulated genes and survival at extreme pH. In: Neidhardt FC, Curtiss RIII, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella: Cellular and molecular biology, vol 1, 2nd edn. ASM Press, Washington, D.C, pp 1539–1549Google Scholar