Abstract
Escherichia coli responds to an increase in medium osmolarity by accumulating K+ and glutamate. At low osmolarity a large fraction of cytoplasmic K+ serves to balance charge on macromolecular anions. That fraction of K+ is here referred to as "bound," as distinguished from "free" K+ that serves to balance charge of small anions. At higher osmolarity where cytoplasmic K+ increases markedly, the bound fraction decreases but the absolute amount of bound K+ expressed per unit of dry weight increases. The increase in bound K+ can be explained largely by the reduction of cytoplasmic putrescine at high osmolarity. At high osmolarity, glutamate is the major cytoplasmic anion, equal to at least 70% of free cytoplasmic K+. A sudden increase in the osmolarity of the medium stimulates glutamate synthesis with a lag of only about a minute; glutamate synthesis is almost totally dependent on K+ uptake. The high rate of flow of nitrogen through the glutamate pool under control conditions of growth at low osmolarity indicates that glutamate accumulation immediately after shift to high osmolarity must be due to inhibition of utilization of glutamate in the synthesis of other nitrogen-containing compounds rather than stimulation of glutamate synthesis. In agreement with this reasoning we find the kinetics of glutamate accumulation to be independent of the specific path of synthesis, whether by glutamate dehydrogenase or by glutamate synthase. Synthesis of glutamate appears to be required to attain normal values of the electrical membrane potential after shift to high osmolarity.
Highlights
Transport system for them, Prop, is synthesized constitutively and is activated by high osmolarity [12,13,14]
We have examined the kinetics of glutamate accumulation from NH: in a strain thatis wild-type for glutamate synthesis (Fig. 2 A )
When ammonium ions were omitted from the medium, transient alkalinization of the cytoplasm wasfollowedby restoration of internal pH (Fig.4) with kinetics similar to those seen in complete medium [34]
Summary
State of Cytoplasmic Solutes-It is generally accepted that a significant fraction of cytoplasmic anions is represented by macromoleculessuch as proteins andnucleic acids. To prevent the salts on the filter from removing bound cations, cells on filters were prewashed at room temperature with a glucose solution that was somewhat hypertonic to the growth medium to remove free ions trapped in the filter; such ions could contaminate the solution used to effect downshock If this prewash was omitted, cells retained less K+after cold distilled water washing than shown ' 0. This result indicates that the extenotf downshock where glutamate is made from ammonia by the activity of by washing with a 0.02 osmolar solution at 0 "C is sufficient glutamine synthetase and glutamate synthase, the product of to allow for equilibration of the cytoplasmic andexternal the gltB and gltD genes, and for a gltB mutant where the sole compartments, aswell as for release of bound K+by exchange path for glutamate synthesis is glutamate dehydrogenase,the with Na+ in the shock solution. Downshock at room temperature orat 37 "C those forK+,ranging from about 25% [33] to 50%(34) of K+. removed less cell K+ [6](data not shown) and increased the The initial impression is of an anion "gap," suggesting the minimum osmolarity of the growth medium required to attain existence of other cytoplasmic anions that make up the difequilibrium
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