Characterization of an Escherichia coli mdoB mutant strain unable to transfer sn-1-phosphoglycerol to membrane-derived oligosaccharides

A procedure for the isolation of mutants affected in components containing glycerol derived from phospholipids yielded two mutant strains that contain membrane-derived oligosaccharides (MDO) devoid of glycerol (Rotering, H., Fiedler, W., Rollinger, W., and Braun, V. (1984) FEMS Microbiol. Lett. 22, 61-68). MDO are found in the periplasmic space of Escherichia coli and other Gram-negative bacteria, and they may comprise up to 7% of the cells dry weight. The biosynthesis of MDO is osmoregulated (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095) and linked to the metabolism of phospholipids (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368-1372). This leads to substitution of MDO with sn-1-phosphoglycerol and phosphoethanolamine (Kennedy, E. P., Rumley, M. K., Schulman, P., and van Golde, L. M. G. (1976) J. Biol. Chem. 251, 4208-4213). MDO also contain succinate in O-ester linkage. We now report that one mutant strain lacks phosphoglycerol transferase I activity and thus is unable to transfer sn-1-phosphoglycerol residues from phosphatidylglycerol to MDO. The mdoB gene affected in this mutant has been located at 99.2 min on the E. coli chromosome. The ethanolamine content of MDO isolated from the mutant strain is elevated, whereas the number of succinate residues is not affected. The only phenotype of mdoB mutants we found is a dramatic reduction of the diglyceride content observed in dgk mdoB double mutants when the beta-glucoside arbutin is present in the growth medium.     

Regulation of the synthesis of membrane-derived oligosaccharides in Escherichia coli. Assay of phosphoglycerol transferase I in vivo

Membrane-derived oligosaccharides are periplasmic constituents of Escherchia coli and other Gram-negative bacteria. Oligosaccharides in this family may be variously substituted with O-succinyl ester residues, and with sn-1-phosphoglycerol and phosphoethanolamine residues derived from membrane phospholipids. Membrane-derived oligosaccharides appear to be important in osmoregulation, because their synthesis is under strict control (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1092-1095). Maximum rate of synthesis is at very low osmolarity of the medium. Phosphoglycerol residues are transferred from phosphatidylglycerol to membrane-derived oligosaccharides, or to certain beta-glucoside acceptors, in a reaction catalyzed by phosphoglycerol transferase I, an enzyme of the inner membrane (Jackson, B. J., and Kennedy, E.P. (1983) J. Biol. Chem. 258, 2394-2398). We now report that this enzyme catalyzes the transfer of phosphoglycerol residues to arbutin (p-hydroxyphenyl-beta-D-glucoside) added to the medium with Km similar to that observed with the cell-free enzyme. The active site of the enzyme must therefore be on the periplasmic face of the inner membrane. We assayed phosphoglycerol transferase I in vivo and found that it is present and completely active even in cells growing in medium of very high osmolarity, in which the synthesis of membrane-derived oligosaccharides is severely reduced. We conclude that osmotic regulation must occur at the stage of the synthesis of oligosaccharide chains. A study of the kinetics of transfer of phosphoglycerol residues to membrane-derived oligosaccharides in vivo revealed that synthesis of the polyglucose chains must stop abruptly upon transfer of cells from medium of low to high osmolarity, inconsistent with a model postulating simple dilution of some rate-limiting enzyme during growth at the higher osmolarity.     

Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity.

Phosphoglycerol transferase I, an enzyme of the inner, cytoplasmic membrane of Escherichia coli, catalyzes the in vitro transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to the model substrate arbutin (p-hydroxyphenyl-beta-D-glucoside). The products are a phosphoglycerol diester derivative of membrane-derived oligosaccharides or arbutin, respectively, and sn-1,2-diglyceride (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983). Because this enzyme has its active site on the outer aspect of the inner membrane, it also catalyzes the transfer of phosphoglycerol residues to arbutin added to the medium (J.-P. Bohin and E. P. Kennedy, J. Biol. Chem. 259:8388-8393, 1984). When strains bearing the dgk mutation, which are defective in the enzyme diglyceride kinase, are grown in medium containing arbutin, they accumulate large amounts of sn-1,2-diglyceride, a product of the phosphoglycerol transferase I reaction. Growth is inhibited under these conditions. A further mutation in such a dgk strain, leading to the loss of phosphoglycerol transferase I activity, should result in the phenotype of arbutin resistance. We have exploited this fact to obtain strains with such mutations, designated mdoB, that map near min 99. Such mutants lack detectable phosphoglycerol transferase I activity, cannot transfer phosphoglycerol residues to arbutin in vivo, and synthesize membrane-derived oligosaccharides devoid of phosphoglycerol residues. These findings offer strong genetic support for the function of phosphoglycerol transferase I in membrane-derived oligosaccharide biosynthesis.     

Identification of the diacylglycerol kinase structural gene of Rhizobium meliloti 1021.

The cyclic beta-1,2-glucans of Rhizobium may function during legume nodulation. These molecules may become highly substituted with phosphoglycerol moieties from the head group of phosphatidylglycerol; diglyceride is a by-product of this reaction (K. J. Miller, R. S. Gore, and A. J. Benesi, J. Bacteriol. 170:4569-4575, 1988). We recently reported that R. meliloti 1021 produces a diacylglycerol kinase (EC 2.7.1.107) activity that shares several properties with the diacylglycerol kinase enzyme of Escherichia coli (W. P. Hunt, R. S. Gore, K. J. Miller, Appl. Environ. Microbiol. 57:3645-3647, 1991). A primary function of this rhizobial enzyme is to recycle diglyceride generated during cyclic beta-1,2-glucan biosynthesis. In the present study, we report the cloning and initial characterization of a single-copy gene from R. meliloti 1021 that encodes a diacylglycerol kinase homolog; this homolog can complement a diacylglycerol kinase deficient strain of E. coli. The sequence of the rhizobial diacylglycerol kinase gene was predicted to encode a protein of 137 amino acids; this protein shares 32% identity with the E. coli enzyme. Analysis of hydropathy and the potential to form specific secondary structures indicated a common overall structure for the two enzymes. Because diglyceride metabolism and cyclic beta-1,2-glucan biosynthesis are metabolically linked, future studies with diacylglycerol kinase mutants of R. meliloti 1021 should further elucidate the roles of the cyclic beta-1,2-glucans in the Rhizobium-legume symbiosis.     

Osmotic regulation of biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

The osmotic regulation of the biosynthesis of membrane-derived oligosaccharides (MDO) in strains UB1005 and DC2 of Escherichia coli K-12 was examined; this regulation was previously reported by Clark (J. Bacteriol. 161:1049-1053, 1985) to be different from that observed by Kennedy for other strains of E. coli (Proc. Natl. Acad. Sci. USA 79:1092-1095, 1982). Osmotic regulation of the synthesis of MDO in UB1005 and DC2 is in fact indistinguishable from that previously reported for other strains of E. coli, with maximum production of MDO occurring in the medium of lowest osmolarity. The report of Clark to the contrary was apparently based on the inadequate methods for the measurement of MDO employed in that study. MDO are localized in the periplasm of wild-type E. coli cells. However, strain DC2, selected for hypersensitivity to a range of antibiotics, released most of its MDO into the medium, apparently as a result of greater outer membrane permeability.     

Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti.

Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids. The periplasmic cyclic glucans of Rhizobium spp. are also involved in specific rhizobium-plant interaction. These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli. E. coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function. We have now isolated the constitutive ACP of R. meliloti and determined its primary structure. We have also examined its function, together with those of ACPs from E. coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E. coli ACP acylase enzyme. All four ACPs act as acceptors of acyl residues, but only the E. coli ACP functions in the transglucosylase system.     

Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues

In a previous study (Miller, K.J., Kennedy, E.P. and Reinhold, V.N. (1986) Science 231, 48-51) it was reported that the biosynthesis of periplasmic cyclic beta-1,2-glucans by Agrobacterium tumefaciens is strictly osmoregulated in a pattern closely similar to that found for the membrane-derived oligosaccharides of Escherichia coli (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. USA 79, 1092-1095). In addition to the well-characterized neutral cyclic glucan, the periplasmic glucans were found to contain an anionic component not previously reported. Biosynthesis of the anionic component is osmotically regulated in a manner indistinguishable from that of the neutral cyclic beta-1,2-glucan. We now find that the anionic component consists of cyclic beta-1,2-glucans substituted with one or more sn-1-phosphoglycerol residues. The presence of sn-1-phosphoglycerol residues represents an additional, striking similarity to the membrane-derived oligosaccharides of E. coli.     

The biosynthesis of membrane-derived oligosaccharides. A membrane-bound phosphoglycerol transferase

Membrane-derived oligosaccharides, found in the Escherichia coli periplasmic space (Schulman, H., and Kennedy, E. P. (1979) J. Bacteriol. 137, 686-688), are composed of 8-10 units of glucose, the sole sugar, in beta 1 leads to 2 and beta 1 leads to 6 linkages (Schneider, J. E., Reinhold, V., Rumley, M. K., and Kennedy, E. P. (1979) J. Biol. Chem. 254, 10135-10138). Oligosaccharides in this family are variously substituted with succinyl ester residues, as well as with sn-1-phosphoglycerol and phosphoethanolamine, both derived from membrane phospholipids. These negatively charged oligosaccharides may function in cellular osmoregulation since their synthesis is under osmotic control (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1092-1095). We now report initial characterization of an enzyme catalyzing transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to synthetic beta-glucoside acceptors. The products are sn-1,2-diglyceride and beta-glucoside-6-phosphoglycerol. Localized in the inner membrane, the transferase has a requirement for divalent cations, of which manganese is most effective, and a pH optimum of 8.9 in vitro.     

Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans by Rhizobium meliloti ndv mutants.

The periplasmic cyclic beta-(1,2)-glucans of Rhizobium spp. are believed to provide functions during hypoosmotic adaptation and legume nodulation. In Rhizobium meliloti, cyclic beta-(1,2)-glucans are synthesized at highest levels when cells are grown at low osmolarity, and a considerable fraction (> or = 35%) of these glucans may become substituted with phosphoglycerol moieties. Thus far, two chromosomally encoded proteins, NdvA and NdvB, have been shown to function during cyclic beta-(1,2)-glucan biosynthesis; however, the precise roles for these proteins remain unclear. In the present study, we show that R. meliloti mutants lacking up to one-third of the downstream region of ndvB synthesize cyclic beta-(1,2)-glucans similar to those produced by wild-type cells with respect to size and phosphoglycerol substituent profile. In contrast, no phosphoglycerol substituents were detected on the cyclic beta-(1,2)-glucans synthesized by an R. meliloti ndvA mutant.     

Appearance of monoglyceride and triglyceride in the cell envelope of Escherichia coli mutants defective in diglyceride kinase

Diglyceride kinase mutants of Escherichia coli contain about 50- to 100-fold more 1,2-diglyceride than wild type cells. We now report that monoglyceride and triglyceride also accumulate in these strains. In mutant RZ60 (dgk-6) these compounds represent about 1 and 0.2%, respectively, of the total lipid fraction, while diglyceride represents 5-8% under most conditions. Monoglyceride accumulates predominantly in the outer membrane, while triglyceride builds up together with diglyceride in the cytoplasmic membrane. Under typical growth conditions about two-thirds of the diglyceride in E. coli arises in conjunction with synthesis of the membrane-derived oligosaccharides (Raetz, C.R.H., and Newman, K.F. (1979) J. Bacteriol. 137, 860-868). Inhibition of membrane-derived oligosaccharides (MDO) synthesis also curtails the accumulation of monoglyceride and triglyceride. However, there appears to be at least one other MDO-independent source of diglyceride and related metabolites. Since MDO synthesis is suppressed by high osmolarity (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. U.S. A. 79, 1092-1095), we have examined the effects of osmolarity on diglyceride accumulation in RZ60 (dgk-6). As expected, if MDO synthesis and diglyceride formation are coupled, the diglyceride level in RZ60 is higher at low osmolarity, while at high osmolarity the level of diglyceride is reduced to that observed in double mutants defective both in MDO synthesis and diglyceride kinase. Since dgk mutants do not grow at very low osmolarity, we have isolated several spontaneous phenotypic revertants that do. One class regains diglyceride kinase and has low diglyceride levels under all conditions. The other class remains defective in diglyceride kinase but tolerates higher diglyceride levels which amount to 13% of the total lipid during maximal induction of MDO synthesis at low osmolarity.     

Biosynthesis of membrane-derived oligosaccharides. Membrane-bound glucosyltransferase system from Escherichia coli requires polyprenyl phosphate.

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, are now recognized to be a family of closely related substances with important functions in osmotic adaptation and cell signaling. The synthesis of the beta-1,2-glucan backbone of MDO is catalyzed by a membrane-bound glucosyltransferase system previously shown to require UDP-glucose and (surprisingly) acyl carrier protein (Therisod, H., Weissborn, A. C., and Kennedy, E. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7236-7240). In the present study, no glucan intermediates bound to acyl carrier protein or to UDP could detected. The enzyme system, however, was found to be strongly inhibited by bacitracin and by amphomycin. Because the two antibiotics function by forming specific complexes with polyprenyl phosphates, their inhibitory effect suggests a prenol requirement for MDO biosynthesis. Furthermore, the activity of the glucosyltransferase was greatly stimulated by the addition of polyprenyl phosphates such as decaprenyl-P and dihydroheptaprenyl-P, but not by farnesyl-P. The same membrane preparations carry out the synthesis of polyprenyl-P-glucose, which is also stimulated by added polyprenyl-P, including farnesyl-P, the most active of those tested. Pulse chase experiments, however, indicate that the endogenous pool of polyprenyl-P-glucose cannot be an obligate intermediate in the MDO glucosyltransferase system.     

Structural studies of the membrane-derived oligosaccharides of Escherichia coli.

Membrane-derived oligosaccharides are a novel class of glucose-containing oligosaccharides found in the cell envelope of Escherichia coli and other Gram-negative organisms (Schulman, H., AND Kennedy, E.P. (1979) J. Bacteriol. 137, 686-688). Previous work has shown that these oligosaccharides contain sn-1-glycero-P and smaller amounts of phosphoethanolamine, derived from membrane phospholipids, attached to position 6 of certain of the glucose residues. The structure of the parent oligosaccharides (obtained by reduction with borohydride followed by alkaline hydrolysis) has now been studied. The oligosaccharide was permethylated, followed by hydrolysis and conversion of the products to methylated glucitol acetates, which were then analyzed and identified by gas-liquid chromatography and mass spectrometry. The membrane oligosaccharides contain 10 to 12 D-glucopyranoside residues/mol, linked solely by 1 yields 2 and 1 yields 6 bonds. They are highly branched structures, with four nonreducing termini per mol. Glucose units at the branch points are doubly substituted at positions 2 and 6. The low specific rotation of the oligosaccharide (+8.3 degrees) indicates that the glycosidic bonds are predominantly or entirely beta.     

Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane-derived oligosaccharides in Escherichia coli

Mutants of Escherichia coli defective in the mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mdoA locus has now been cloned into multicopy plasmids. A 5 kb DNA fragment is necessary to complement mdoA mutations. Cells harbouring the mdoA+ plasmid produced three to four times more MDO than wild-type cells. MDO overproduction did not affect the degree of MDO substitution with sn-1-phosphoglycerol residues. The biosynthesis of MDO remained under osmotic control in overproducing strains.     

A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti.

The periplasmic cyclic beta-1,2-glucans produced by bacteria within the Rhizobiaceae family provide functions during hypo-osmotic adaptation and plant infection. In Rhizobium meliloti, these molecules are highly modified with phosphoglycerol and succinyl substituents, and it is possible that the anionic character of these glucans is important for their functions. In the present study, we have used a thin-layer chromatographic screening method to identify a novel R. meliloti mutant specifically blocked in its ability to transfer phosphoglycerol substituents to the cyclic beta-1,2-glucan backbone. Further analysis revealed that the cyclic glucans produced by this mutant contained elevated levels of succinyl substituents. As a result, the overall anionic charge on the cyclic beta-1,2-glucans was found to be similar to that of wild-type cells. Despite this difference in cyclic beta-1,2-glucan structure, the mutant was shown to effectively nodulate alfalfa and to grow as well as wild-type cells in hypo-osmotic media.     

Properties of Escherichia coli mutants lacking membrane-derived oligosaccharides

Membrane-derived oligosaccharides (MDO) consist of branched substituted beta-glucan chains and are present in the periplasmic space of Escherichia coli and other gram-negative bacteria. A procedure for the isolation of mutants defective in MDO synthesis is described. Their phenotype was compared with a mdoA mutant previously identified, and they are mapped in the mdoA region. Mutants lacking MDO showed imparied chemotaxis on tryptone swarm plates, a reduced number of flagella, and an enhanced expression of the OmpC porin. Revertants able to form swarm rings again had regained the ability to synthesize MDO and showed the wild-type porin pattern. A second group of chemotactic revertants were mutated in the ompB gene region involved in osmoregulation, and they were still devoid of MDO. These findings provide evidence for a link between MDO biosynthesis and other functions of E. coli related to its adaptation to the environment.     

Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes.

Cofactor-independent phosphoglycerate mutase (iPGM) has been previously identified as a member of the alkaline phosphatase (AlkP) superfamily of enzymes, based on the conservation of the predicted metal-binding residues. Structural alignment of iPGM with AlkP and cerebroside sulfatase confirmed that all these enzymes have a common core structure and revealed similarly located conserved Ser (in iPGM and AlkP) or Cys (in sulfatases) residues in their active sites. In AlkP, this Ser residue is phosphorylated during catalysis, whereas in sulfatases the active site Cys residues are modified to formylglycine and sulfatated. Similarly located Thr residue forms a phosphoenzyme intermediate in one more enzyme of the AlkP superfamily, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1 (autotaxin). Using structure-based sequence alignment, we identified homologous Ser, Thr, or Cys residues in other enzymes of the AlkP superfamily, such as phosphopentomutase, phosphoglycerol transferase, phosphonoacetate hydrolase, and GPI-anchoring enzymes (glycosylphosphatidylinositol phosphoethanolamine transferases) MCD4, GPI7, and GPI13. We predict that catalytical cycles of all the enzymes of AlkP superfamily include phosphoenzyme (or sulfoenzyme) intermediates.     

Phosphoglycerol substituents present on the cyclic beta-1,2-glucans of Rhizobium meliloti 1021 are derived from phosphatidylglycerol.

The synthesis of periplasmic cyclic beta-1,2-glucans is a property unique to species of the family Rhizobiaceae. For this reason, it is generally believed that these molecules may play an important role in the plant infection process. In the present study, we determined that the cyclic beta-1,2-glucans produced by Rhizobium meliloti 1021 were predominantly anionic in character and contained both phosphoglycerol and succinic acid substituents. In addition, we demonstrated that phosphatidylglycerol was the source of the phosphoglycerol substituents present on these oligosaccharides and that greater than 60% of the total phospholipid turnover in this organism involved this substitution reaction.     

Identification of UDP-glucose as an intermediate in the biosynthesis of the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides of Escherichia coli constitute a closely related family of oligosaccharides containing approximately 9 glucose units variously substituted with sn-glycero-1-phosphate and phosphoethanolamine residues derived from the head groups of membrane phospholipids, and also with succinate in O-ester linkage (Kennedy, E.P., Rumley, M.K., Schulman, H., and van Golder, L.M.G. (1976) J. Biol. Chem. 251, 4208-4213). Studies with mutant strains defective in the synthesis of various nucleoside diphosphate sugars have now revealed that UDP-glucose is an essential intermediate in the biosynthesis of these oligosaccharides. Mutants unable to synthesize UDP-glucose do not contain significant amounts of the membrane-derived oligosaccharides. In contrast, a strain unable to synthesize ADP-glucose, the glucosyl donor for glycogen synthesis in E. coli, contained normal amounts of the membrane-derived oligosaccharides, although with a somewhat different pattern of distribution of the various subspecies. In confirmation of these genetic studies, pulse-label isotope tracer studies have been carried out with glucose of high specific activity, under conditions in which UDP-glucose comprises a large fraction of the total radioactivity in the low molecular weight pool. Subsequent "chase" experiments clearly revealed the conversion of UDP-glucose to the higher molecular weight membrane-derived oligosaccharides.     

Resistance to the antimicrobial peptide polymyxin requires myristoylation of Escherichia coli and Salmonella typhimurium lipid A

Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrA(C) strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3'-acyloxyacyl-linked myristate (C(14)) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group.     

Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in beta-subunit of F1-ATPase of Escherichia coli

Previous studies in which dicyclohexylcarbodiimide (DCCD) was used to inactivate F1-ATPase enzymes have suggested that two glutamate residues in the beta-subunit are essential for catalysis. In the Escherichia coli F1-ATPase, these are residues beta-Glu-181 and beta-Glu-192. Oligonucleotide-directed mutagenesis was used to change these residues to beta-Gln-181 and beta-Gln-192. The beta-Gln-181 mutation produced strong impairment of oxidative phosphorylation in vivo and also of ATPase and ATP-driven proton-pumping activities in membranes assayed in vitro. A low level of each activity was detected and an F1-ATPase appeared to be assembled normally on the membranes. Therefore, it is suggested that the carboxyl side chain at residue beta-181 is important, although not absolutely required, for catalysis in both directions on E. coli F1-ATPase. The beta-Gln-192 mutation produced partial inhibition of oxidative phosphorylation in vivo and membrane ATPase activity was reduced by 78%. These results contrast with the complete or near-complete inactivation seen when E. coli F1-ATPase is reacted with DCCD and imply that DCCD-inactivation is attributable more to the attachment of the bulky DCCD molecule than to the derivatization of the carboxyl side chain of residue beta-Glu-192. M. Ohtsubo and colleagues (Biochem. Biophys. Res. Commun. (1987) 146, 705-710) described mutagenesis of the F1-beta-subunit of thermophilic bacterium PS3. Mutations (Glu----Gln) of the residues homologous to Glu-181 and Glu-192 of E. coli F1-beta-subunit both caused total inhibition of ATPase activity. Therefore, there was a marked difference in results obtained when the same residues were modified in the PS3 and E. coli F1-beta-subunits.