Sodium extrusion by bacteria is generally attributed to secondary antiport of Na' for H4 energized by the proton circulation. Streptococcs faecalis is an exception, in that sodium expulsion from intact cells requires the generation ofATP but does not depend on the protonmotive force. Unfortunately, studies with everted membrane vesicles failed to reveal the expected sodium pump; instead, the vesicles contained a conventional secondary Na4/H4 antiporter. We report here that everted membrane vesicles prepared in the presence of protease inhibitors retain an ATP-driven sodium transport system. The evidence includes the findings that (i) accumulation of2-Na4 by these vesicles is resistant to reagents that dissipate the protonmotive force but requires ATP and (ii) the vesicles contain a sodium-stimulated ATPase that is distinct from FIFO ATPase, and whose presence is correlated with sodium transport activity. Sodium movements appear to be electroneutral and are accompanied by movement of H4 in the opposite direction. When membranes are incubated in the absence of protease inhibitors, a secondary Na4/H4 antiport activity emerges, possibly by degradation ofthe sodium pump. We suggest that S. faecalis expels Na4 by means of an ATP-driven primary transport system that mediates exchange ofNa4 for H+. The Na+/ H+ antiporter seen in earlier membrane preparation is an artefact of proteolytic degradation.Virtually all cells expel sodium ions from the cytoplasm by active transport ofone kind or another. In animal cells, this is the task of the Na+-K+ ATPase. In bacteria, and probably also-in lower eukaryotes and in plants, sodium is extruded by exchange for protons. Since all these organisms maintain a substantial electrochemical proton gradient (interior alkaline and negative), antiport of Na+ for H4 can expel Na4 against a substantial gradient and no direct coupling to ATP, or other energy donor, should be required. This hypothesis, first proposed by Mitchell (1) in the context of his chemosmotic theory, has received substantial support from bacterial physiologists (for review, see ref.2), and it is generally accepted that Escherichia coli, Azotobacter, alkalophilic bacilli, and other bacteria extrude sodium by secondary Na4/H+ antiport (2-7). The discovery by MacDonald and his colleagues (8, 9) of a light-driven sodium pump in halobacteria was the first indication ofdiversity in bac-. terial sodium transport mechanisms. However, even in this case, the major pathway of sodium extrusion may be secondary Na+/H4 antiport; the light-energized pump appears to make but a minor contribution to the total Na+ flux (10).From the beginning, Na+ extrusion by Streptococcusfaecalis did not quite conform to the hypothesis of secondary Na+/H+ antiport, in that both net Na+ movement and 'Na4/Na+ exchange were observed only in cells capable of generating ATP (11). In a detailed study with intact cells, we (12) demonstrated that glycolyzing cells could extrude Na4 against a 100-fold concentration gradient in the presence of reagents t...

The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to ∼2.2 Å resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.

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Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 ,g of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform Lphase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.

Clostridium perfringens 11268 CDR (Rif' Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (RiV Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 ,ug/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rif Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.