TY - JOUR
T1 - The sodium/proton antiporter is part of the pH homeostasis mechanism in Escherichia coli
AU - Zilberstein, D.
AU - Agmon, V.
AU - Schuldiner, S.
AU - Padan, E.
PY - 1982
Y1 - 1982
N2 - The E. coli chromosome has been shown to bear at 89.5 min a locus designated phs, which determines the Na+/H+ antiporter activity. The mutant -DZ3 has previously been shown to be simultaneously impaired in Na+ extrusion capacity and in growth on the Na+-co-transported substrates, melibiose and glutamate. This mutant when mated with the wild type yielded wild type-like recombinants that appeared at a map distance of 1.5 min from metB recombinants. Furthermore, genotypes containing repressed operon of glutamate which cannot grow on this substrate still bear the normal phs locus and served as donors for trannsduction of DZ3 to yield wild type-like transductants. The mutant DZ3 also been shown to be impaired in growth at alkaline pH. This fact allowed us to investigate the role of the Na+/H+ antiporter in pH homeostasis in E. coli. A pH controlled growth system and rapid filtration technique were used to compare the wild type and the mutant DZ3 with respect to both internal pH and growth during transfers of logarithmically growing cells to different external pHs. Following the shift in external pH of the wild type, a transient state was initiated by reduction of ΔpH (pH gradient) across the membrane. Subsequently, at a specific time course pH homeostasis was re-established. Whereas the capacity of the pH homeostasis mechanism was found to be a function of both the span of the external pH shift as well as the rate at which the change occurs, inhibition of protein synthesis did not affect this process. After stepwise transfer of growing wild type cells from pH 7.2 to 8.3 to 8.6, and then to 8.8, or from 7.2 to 6.4, the ΔpH was initially zero at each step and growth ceased. Subsequently, within 6 min at the most, the ΔpH was built up to a magnitude that yielded an internal pH of 7.6-7.8 and thereafter growth was resumed at the initial rate. However, if the shift was made abruptly from pH 7.2 to 8.6, the lag was longer and the buildup of the ΔpH was slower. The shift between pH 7 to 8.8 appeared to be the limit of the pH homeostasis capacity since the wild type grew normally when allowed to adapt by step transfers over this range and failed to restore both normal internal pH and optimal growth if the transition was made in one step. Since the mutant DZ3 behaved like the wild type after transfer from pH 7.2 to 6.4, but exhibited progressive failure to control internal pH and to grow at the alkaline shifts, we conclude that the Na+/H+ antiporter is an absolute requirement for pH adaptability at alkaline pH. It is suggested that the collaborative functioning of this antiporter with the primary proton pumps extruding protons is the basis of the pH homeostasis mechanism at alkaline pH. In all cases, both in the wild type and the mutant, recovery of pH homeostasis always preceded initiation of growth, indicating a tight coupling between the two processes.
AB - The E. coli chromosome has been shown to bear at 89.5 min a locus designated phs, which determines the Na+/H+ antiporter activity. The mutant -DZ3 has previously been shown to be simultaneously impaired in Na+ extrusion capacity and in growth on the Na+-co-transported substrates, melibiose and glutamate. This mutant when mated with the wild type yielded wild type-like recombinants that appeared at a map distance of 1.5 min from metB recombinants. Furthermore, genotypes containing repressed operon of glutamate which cannot grow on this substrate still bear the normal phs locus and served as donors for trannsduction of DZ3 to yield wild type-like transductants. The mutant DZ3 also been shown to be impaired in growth at alkaline pH. This fact allowed us to investigate the role of the Na+/H+ antiporter in pH homeostasis in E. coli. A pH controlled growth system and rapid filtration technique were used to compare the wild type and the mutant DZ3 with respect to both internal pH and growth during transfers of logarithmically growing cells to different external pHs. Following the shift in external pH of the wild type, a transient state was initiated by reduction of ΔpH (pH gradient) across the membrane. Subsequently, at a specific time course pH homeostasis was re-established. Whereas the capacity of the pH homeostasis mechanism was found to be a function of both the span of the external pH shift as well as the rate at which the change occurs, inhibition of protein synthesis did not affect this process. After stepwise transfer of growing wild type cells from pH 7.2 to 8.3 to 8.6, and then to 8.8, or from 7.2 to 6.4, the ΔpH was initially zero at each step and growth ceased. Subsequently, within 6 min at the most, the ΔpH was built up to a magnitude that yielded an internal pH of 7.6-7.8 and thereafter growth was resumed at the initial rate. However, if the shift was made abruptly from pH 7.2 to 8.6, the lag was longer and the buildup of the ΔpH was slower. The shift between pH 7 to 8.8 appeared to be the limit of the pH homeostasis capacity since the wild type grew normally when allowed to adapt by step transfers over this range and failed to restore both normal internal pH and optimal growth if the transition was made in one step. Since the mutant DZ3 behaved like the wild type after transfer from pH 7.2 to 6.4, but exhibited progressive failure to control internal pH and to grow at the alkaline shifts, we conclude that the Na+/H+ antiporter is an absolute requirement for pH adaptability at alkaline pH. It is suggested that the collaborative functioning of this antiporter with the primary proton pumps extruding protons is the basis of the pH homeostasis mechanism at alkaline pH. In all cases, both in the wild type and the mutant, recovery of pH homeostasis always preceded initiation of growth, indicating a tight coupling between the two processes.
UR - http://www.scopus.com/inward/record.url?scp=0020329209&partnerID=8YFLogxK
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C2 - 6277945
AN - SCOPUS:0020329209
SN - 0021-9258
VL - 257
SP - 3687
EP - 3691
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 7
ER -